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

International Journal of Molecular Sciences

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

Aušrine˙ Areškeviˇciut¯ e˙ 1,* , Thomas Litman 2 , Helle Broholm 1, Linea C. Melchior 1, Pia R. Nielsen 3, Alison Green 4, Jens O. Eriksen 3, Colin Smith 4 and Eva L. Lund 1

1 Department of Pathology, Danish Reference Center for Prion Diseases, Copenhagen University Hospital, 2100 Copenhagen, Denmark; [email protected] (H.B.); [email protected] (L.C.M.); [email protected] (E.L.L.) 2 Department of Immunology and Microbiology, University of Copenhagen, 2200 Copenhagen, Denmark; [email protected] 3 Department of Surgical Pathology, Zealand University Hospital, 4000 Roskilde, Denmark; [email protected] (P.R.N.); [email protected] (J.O.E.) 4 National Creutzfeldt–Jakob Disease Research and Surveillance Unit, University of Edinburgh, Edinburgh EH8 9AB, UK; [email protected] (A.G.); [email protected] (C.S.) * Correspondence: [email protected]

Abstract: 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

Citation: Areškeviˇciut¯ e,˙ A.; Litman, analysis was performed using NanoString nCounter technology with human neuroinflammation T.; Broholm, H.; Melchior, L.C.; panel+. The observed gene expression patterns were regionally and sub-regionally distinct, sug- Nielsen, P.R.; Green, A.; Eriksen, J.O.; gesting a variable neuroinflammatory response. Interestingly, the observed differences could not be Smith, C.; Lund, E.L. Regional explained by the molecular subtypes of sporadic Creutzfeldt–Jakob disease. Furthermore, analyses Differences in Neuroinflammation- of canonical pathways and upstream regulators based on differentially expressed genes indicated Associated Gene Expression in the an overlap between biological processes taking place in different brain regions. This suggests that Brain of Sporadic Creutzfeldt–Jakob even smaller-scale spatial data reflecting subtle changes in brain cells’ functional heterogeneity and Disease Patients. Int. J. Mol. Sci. 2021, their immediate pathologic microenvironments are needed to explain the observed differential gene 22, 140. https://dx.doi.org/ expression in a greater detail. 10.3390/ijms22010140

Keywords: prion disease; Creutzfeldt–Jakob disease; neuroinﬂammation; microglia; immune re- Received: 27 November 2020 sponse; gene expression analysis; brain microenvironment; regional pathogenesis; biomarkers Accepted: 22 December 2020 Published: 25 December 2020

Publisher’s Note: MDPI stays neu- tral with regard to jurisdictional claims 1. Introduction in published maps and institutional Prion diseases, which are caused by misfolded cellular prion proteins termed pri- afﬁliations. ons and denoted PrPSc, are a group of infectious and invariably fatal neurodegenerative diseases that can be acquired (<1%), genetic (∼15%), and sporadic (85%) [1–4]. Spo- radic Creutzfeldt–Jakob disease (sCJD), the most common of all PrPSc prion diseases, has 14 subtypes that are deﬁned by the methionine/valine polymorphism at codon 129 in Copyright: © 2020 by the authors. Li- the prion protein gene (PRNP), a type of present protease K-resistant PrPSc fragments censee MDPI, Basel, Switzerland. This (type 1 = 21 kDa, type 2 = 19 kDa), and distinct phenotypic features such as the appearance article is an open access article distributed under the terms and conditions of the and distribution of Kuru plaques and pronounced cortical or thalamic changes [5–9]. C Sc Creative Commons Attribution (CC BY) The templated misfolding of cellular prion protein (PrP ) into PrP is central to the license (https://creativecommons.org/ disease pathogenesis [10]. However, neurodegeneration is a result of multiple complex licenses/by/4.0/). molecular processes occurring in different brain regions simultaneously. In sCJD, a highly

Int. J. Mol. Sci. 2021, 22, 140. https://dx.doi.org/10.3390/ijms22010140 https://www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2021, 22, 140 2 of 17

variable selective regional vulnerability, although poorly understood, can be observed in different disease subtypes [11–13]. It has become clear that several brain region-specific factors must play a role. Specific cell types, with their intrinsic firing properties and metabolic activity, varying concen- trations of metal ions, and differences in toxicity restricting molecular machines such as protein-folding and quality-control systems, are considered to be involved in region selection for the establishment of neurodegeneration [12]. As has been shown by many studies of prion disease animal models, neuroinflammation is an inseparable part of neurodegeneration [14–16]. Several studies investigating gene expression in various prion disease models indicate that upregulated genes are pre- dominantly expressed by activated microglia, suggesting their major role in the regulation of inflammation, metabolism, respiratory stress, and possibly other functions [17–19]. Microglia are brain-resident macrophages constantly surveilling their microenvironment to ensure the elimination of brain cell function-disturbing events [20]. Upon the detection of abnormal activity, depending on the stimuli, microglia initiate different re- sponses to stabilize the microenvironment [21]. Accumulating evidence suggests that microglia also play a role in synaptic organization, the control of neuronal excitability, phagocytic debris removal, trophic support for brain protection and repair, and the induction of neurotoxic reactive astrocytes killing neurons and mature oligodendrocytes [19,22,23]. However, surprisingly little is known about regional microglia-induced neuroinflammation patterns even in the most common human prion diseases [24–26]. Understanding the differences in potentially pathogenic molecular events taking place in different brain regions is essential for the further deciphering of complex molecular networks, their interactions, and their potential use for novel therapeutic strategies. Thus, the current study was designed to provide a comprehensive overview of the regional neuroinflammatory differences in sCJD brain samples from deceased patients using an RNA amplification-free approach that allowed direct counting of RNAs of interest present in the sample. RNA amplification-free solution was provided by NanoString technologies, which also offered a 770-gene panel designed to represent the core processes of neuroinflammation—namely, immunity and inflammation, neurobiology and neuropathology, and metabolism and stress [27].

2. Results 2.1. Regional Differences in Gene Expression Based on the 265 most variably expressed genes across all the samples included in the study (Supplementary material S1a, a list of the 265 genes), the following main patterns were observed: (1) primary separation between the frontal cortex (FC) and cerebellum (CB) samples, and (2) secondary separation of sCJD from control tissue samples (Figure1a,b). Due to major differences in gene expression proﬁles between FC and CB, as illustrated in the heatmap (Figure1a), these tissues were treated separately in the subsequent analyses. The gene expression signature of sCJD CB sample FFCJD_CB-20 suggested that it was an FC sample, and thus it was removed from further analyses.

2.2. Top Differentially Expressed Genes (DEGs) The neuroinflammatory gene analysis revealed several significantly differentially expressed and functionally interesting genes. We were most interested in identifying Differentially Expressed Genes (DEGs) that would be (1) common to both brain regions in sCJD samples as compared to controls; (2) unique to each brain region in sCJD samples as compared to controls; and (3) unique to a brain region regardless of sample type—i.e., sCJD or control. The three groups of DEGs identified following these comparison criteria were named as follows: (1) disease-specific, brain region non-exclusive DEGs; (2) disease-specific, brain region exclusive DEGs; and (3) disease non-specific, brain region exclusive DEGs. Table1 provides an overview of the top genes that belong to each group of interest. Int. J. Mol. Sci. 2021, 21, x FOR PEER REVIEW 3 of 19 Int. J. Mol. Sci. 2021, 22, 140 3 of 17

Figure 1.1. ((a)) AA heatmapheatmap andand unsupervisedunsupervised two-waytwo-way hierarchical clusteringclustering based onon thethe 265265 mostmost variable genes indicate severalseveral clustersclusters andand sub-clusters: sub-clusters: yellow yellow frames— frames— sporadic sporadic Creutzfeldt–Jakob Creutzfeldt–Jakob disease disease (sCJD) (sCJD separation) separation from from control control tissues tis- (CT)sues and(CT) sCJD and sCJD frontal frontal cortex cortex (FC) ( separationFC) separation from from cerebellum cerebellum (CB); (CB magenta); magenta lines—sub-clusters lines—sub-clusters within within sCJD sCJD FC andFC and CB samples.CB samples (b). Principal(b) Principal component component analysis analysis indicating indicating a clear a separationclear separation between between CJD and CJD CT and clusters, CT clusters, as well as FCwell and as CBFC clusters;and CB clu C1—sub-clustersters; C1—sub 1;-cluster C2—sub-cluster 1; C2—sub 2;-cluster Dx—diagnosis. 2; Dx—diagnosis.

The gene expression signature of sCJD CB sample FFCJD_CB-20 suggested that it was an FC sample, and thus it was removed from further analyses. Due to major differences in gene expression profiles between FC and CB, as illustrated in the heatmap (Figure 1a), these tissues were treated separately in the subsequent analyses.

2.2. Top Differentially Expressed Genes (DEGs) Int. J. Mol. Sci. 2021, 21, x FOR PEER REVIEW 4 of 19 Int. J. Mol. Sci. 2021, 21, x FOR PEER REVIEW 4 of 19

Int. J. Mol. Sci. 2021, 21, x FOR PEER REVIEW 4 of 19

The neuroinflammatory gene analysis revealed several significantly differentially ex- Int. J. Mol. Sci. 2021, 21, x FOR PEER REVIEWThe neuroinflammatory gene analysis revealed several significantly differentially4 of ex- 19 pressed and functionally interesting genes. We were most interested in identifying Differ- pressed and functionally interesting genes. We were most interested in identifying Differ- entiallyThe Expressed neuroinflammatory Genes (DEGs gene) that analysis would revealed be (1) common several significantlyto both brain differentially regions in sCJD ex- entially Expressed Genes (DEGs) that would be (1) common to both brain regions in sCJD samplespressed asand compared functionally to controls interesting; (2) uniquegenes. We to each were brain most region interested in sCJD in identifying samples as Differ- com- samplesThe neuroinflammatoryas compared to controls gene; (analysis2) unique revealed to each severalbrain region significantly in sCJD differentially samples as com- ex- paredentially to Expressedcontrols; and Genes (3) (uniqueDEGs) thatto a wouldbrain region be (1) commonregardless to of both sample brain type regions—i.e., in sCJDsCJD pressedpared to and controls functionally; and (3) interesting unique to genes. a brain We region were regardlessmost interested of sample in identifying type—i.e., Differ- sCJD orsamples control. as The compared three groups to controls of DEGs; (2) uniqueidentified to each following brain regionthese comparison in sCJD samples criteria as werecom- entiallyor control. Expressed The three Genes groups (DEGs of DEGs) that wouldidentified be ( following1) common these to both comparison brain regions criteria in sCJDwere namedpared toas controlsfollows:; (and1) disease (3) unique-specific, to a brain region nonregardless-exclusive of sample DEGs; (type2) disease—i.e., -sCJDspe- samplesnamed as as follows: compared (1) to disease controls-specific,; (2) unique brain to region each brainnon-exclusive region in DEGssCJD samples; (2) disease as com--specific,or control. brain Theregion three exclusive groups DEGsof DEGs; and identified (3) disease following non-specific, these comparison brain region criteria exclusive were paredcific, brainto controls region; and exclusive (3) unique DEGs to; aand brain (3) regiondisease regardless non-specific, of sample brain regiontype— i.e.,exclusive sCJD DEGs.named Table as follows: 1 provides (1) disease an overview-specific, of thebrain top region genes nonthat- exclusivebelong to eachDEGs group; (2) disease of interest.-spe- orDEGs. control. Table The 1 providesthree groups an overview of DEGs of identified the top genes following that belong these comparison to each group criteria of interest. were cific, brain region exclusive DEGs; and (3) disease non-specific, brain region exclusive Table 1. Summary of differentnamedially asexpress follows:ed genes (1) disease, their m-olecularspecific, function brain region, and their non associated-exclusive biological DEGs; (processes.2) disease -spe- Table 1. Summary of differentDEGs.ially Table express 1 edprovides genes, their an overview molecular of function the top, and genes their that associated belong tobiological each group processes. of interest. Table 1. Summary of differentcific,ial brainly express regioned genes exclusive, their mDEGsolecular; and function (3) disease, and their non associated-specific, biological brain region processes. exclusive Int. J. Mol. Sci. 2021Brain, 22 , 140 4 of 17 Brain DEGs. Table 1 provides an overview of the top genes4 that belong to each group of4 interest. BrainTable 1. SummaryGene of differentGeneial lyE xpressionexpressed genes, theirMolecular molecular F functionunction, and4 their associatedBiological biological Process processes. 4 Region Gene Gene Expression Molecular Function 4 Biological Process 4 Region BrainTable 1. Summary of differentially expressed genes, their molecular function, and theirAcute associated-phase biological response; processes. cellular Gene Gene Expression Molecular Function 4 Acute-phaseBiological response; Process cellular 4 Table 1. Summary of differentially expressed genes, their molecularThe SERPINA3 function, protein and their inhibits associated protein biological metabolic processes. process; endo- RBrainegion The SERPINA3 protein inhibits protein metabolic process; endo- † The SERPINA3 protein inhibits4 protein metabolic process; 4endo- SERPINA3Gene †, Gene Expression serine Molecularproteases by Function binding to plasmic Biologicalreticulum toProcess Golgi vesicle - Brain SERPINA3 , serine proteases by binding to plasmicAcute-phase reticulum response; to Golgi cellular vesicle - Region SERPINA3Gene †, Gene Expression serineMolecular proteases Function by binding4 to plasmicBiological reticulum Process 4 to Golgi vesicle- Region Serpin Family A them,The SERPINA3 and thus inducing protein inhibits an irre- mediatedprotein metabolic transport; process; neutrophil endo- Serpin Family A them, and thus inducing an irre- Acutemediated-phase transport; response; neutrophil cellular SERPINA3Member 3 †, versibleserine proteases conformational by binding change; toAcute-phase degranulaplasmic response; reticulumtion; post cellular to-translational Golgi vesicle - Member 3 versibleTheThe SERPINA3 SERPINA3 conformational proteinprotein inhibitschange; degranulaprotein metaboliction; post process;-translational endo- Member 3 identicalversible conformationalprotein binding. change; protein proteindegranula metabolic modification;tion; process; post-translational blood coagu- Serpin Family† A identicalthem,inhibits and serine protein thus proteases inducing binding. by an irre- proteinmediated modification; transport; neutrophil blood coagu- SERPINA3SERPINA3†, , seidenticalrine proteases protein by binding. binding toendoplasmic plasmicprotein reticulum modification;reticulum to Golgito Golgi blood vesicle coagu-- binding to them, and thus lation. SerpinMember Family A3 versible conformational change;vesicle-mediated lation.degranula tion; transport; post-translational Serpin Family A them,inducing and an thus irreversible inducing an irre- mediatedlation. transport; neutrophil Member 3 identical protein binding. neutrophilNegativeprotein degranulation; modification; regulation of blood apoptotic coagu- Member 3 versibleconformational conformational change; change; degranulaNegative regulationtion; post-translational of apoptotic Negatively regulates cytokinepost-translational processlation. and protein inflammatory re- identicalNegativelyidentical protein regulate binding. binding.s cytokine proteinprocess modification; and inflammatory blood re- coagu- SOCS3, signal transduction; 1-phosphati-modiﬁcation;sponseNegative, bloodof regulationreceptor coagulation. signaling of apoptotic path- SOCS3, signal transduction; 1-phosphati-Negativelation.sponse regulation , of receptor of apoptotic signaling path- Suppressor of dylinositolNegativelyNegatively- regulates3regulate-kinase sregulator cytokine ac- wayprocess via JAKand -iSTATnflammatory, and of re-tyro- Suppressor of dylinositol-3-kinase regulatorprocess ac- wayNegative and via inﬂammatory JAK regulation-STAT, and of apoptotic of tyro- CytokineSuppressor Signal- of tivity;dylinositolcytokine phosphotyrosine signal-3-kinase transduction; regulator residue ac- sineway phosphorylation via JAK-STAT, and of STAT of tyro- pro- CytokineSOCS3, Signal- tivity;signal phosphotyrosinetransduction; 1-phosphati- residueresponse, sinesponse ofphosphorylation receptor, of receptor signaling signaling of STAT p pro-ath- CytokineSOCS3, Signal- Negativelytivity;1-phosphatidylinositol-3- phosphotyrosine regulates cytokine residue processsine phosphorylation and inflammatory of STAT re- pro- Suppressoring 3 of binding;dylinositol protei-3-kinasen kinase regulator inhibitorpathway ac- tein;way via positivevia JAK-STAT, JAK -regulationSTAT and, and of of of cell tyro- dif- SuppressorSOCS3,ing 3 of signalbinding;kinase transduction; regulator protein activity; kinase 1-phosphati- inhibitor sponsetein; positive, of receptor regulation signaling of cell path- dif- activity. tyrosineferentiation; phosphorylation post-translational of STAT 1 Cytokine Signal- activity.tivity; phosphotyrosine residue ferentiation;sine phosphorylation post-translational of STAT pro- 1 CytokineSuppressor Signaling of 3 dylinositolphosphotyrosine-3-kinase residue regulator ac- way via JAK-STAT, and of tyro- activity. protein;ferentiation; positive regulation post-translational of cell 1 ing 3 binding;binding; proteinprotein kinase kinase inhibitor prtein;otein positive modification. regulation of cell dif-

1 Frontal Cytokine Signal- tivity; phosphotyrosine residuedifferentiation; sine phosphorylation post-translational of STAT pro- Frontal inhibitor activity. Cellprotein adhesion; modification. cellular protein Frontal activity. ferentiation; post-translational Frontal1 Cortex ing 3 binding; protein kinase inhibitorprotein Celltein; modification. adhesion; positive regulation cellular protein of cell dif- Cortex Probably important to cell-matrix metabolicCell adhesion; process; cellular inflammatory protein CortexCortex † Probably important to cell-matrixCell adhesion; metabolicprotein modification. cellular process; protein inflammatory & Cere- SPP1 †, activity.Probably important to ferentiation; post-translational 1 &Frontal Cere- SPP1 , Probably important to cell-matrix metabolic process; inflammatory & Cere-& Cere- SPP1† †, interaction; cytokine activity; metabolicex- response;Cell process;adhesion; positive inflammatory cellular regulation protein of bellumCortex SecretedSPP1 ,Phos- interaction;cell-matrix interaction; cytokine activity; ex- prresponse;otein modification. positive regulation of bellumFrontal tracellular matrix binding; integ-response;transcription, positive regulation DNA-templated; of Group 1 DEGs Secreted Probablycytokine activity;important to cell-matrix metabolic process; inflammatory bellum Secreted Phos-† tracellular matrix binding; integ- transcription, DNA-templated; Group 1 DEGs & Cere- phoproteinSPP1 , 1 tracellular matrix binding; integ-transcription,Celltranscription, adhesion; DNA-templated; DNAcellular-templated; protein Group 1 DEGs Group 1 DEGs CortexPhosphoprotein 1 rinextracellular binding. matrix binding; post-translational protein modifi- phoprotein 1 rinProbablyinteraction; binding. important cytokine to activity; cell-matrixpost-translational ex- post metabolicresponse;-translational positiveprocess; protein protein regulationinflammatory modifi- of bellum Secreted Phos-† rinintegrin binding. binding. post-translational protein modifi- & Cere- SPP1 , tracellular matrix binding; integ-modification.cation.transcription, DNA-templated; Group 1 DEGs phoprotein 1 interaction; cytokine activity; ex- response;cation. positive regulation of bellum Secreted Phos- CellrinCell-surface binding.-surface receptor that that plays a post-translational protein modifi- tracellularCell-surface matrix receptor binding; that plays integ- a transcription, DNA-templated; Group 1 DEGs phoprotein 1 roleplays in acell role-cell in cell-cell interactions, cell Positivecation. regulation of heterotypic rolerin binding. in cell-cell interactions, cell Positivepost-translational regulation protein of heterotypic modifi- adhesionroleinteractions, in cell and-cell cell migration, interactions, adhesion helping cellPositive cellPositive regulation-cell adhesion; regulation of heterotypic cell of migration;heterotypic CD44 †, adhesionCelland- migration,surface and receptor migration, helping that helping playscell-cell a cell adhesion;-cell adhesion; cell migration; cell migration; CD44CD44†, †, adhesion and migration, helping cation.cell-cell adhesion; cell migration; CD44 †, themrolethem in to to cell sense sense-cell andand interactions, respond respond to cellextracellular extracellularPositive matrix regulation matrix disassembly; ofdisassembly; heterotypic CD44CD44 Molecule Molecule Cellthem-surface to sense receptor and respond that plays to a extracellular matrix disassembly; CD44 Molecule changesadhesionto changes in and the in the migration,tissue tissue microenvi- helpingnegative negativecell regulation-cell adhesion;regulation of apoptotic cell of apoptoticmigration; CD44 †, rolechangesmicroenvironment; in cell in- cellthe interactions,tissue collagen microenvi- cellprocess; Positivenegative inflammatory regulation regulation response. of of heterotypic apoptotic ronment;them to sense collagen and andrespond hyalu- to process;extracellular inflammatory matrix disassembly; response. CD44 Molecule adhesionronment;and hyaluronic andcollagen migration, acid and hyalu- helping cellprocess;-cell adhesion; inflammatory cell migration; response. CD44 †, ronicchanges acid in binding. the tissue microenvi- negative regulation of apoptotic Int. J. Mol. Sci. 2021, 21, x FOR PEER REVIEW themronicbinding. toacid sense binding. and respond to extracellular matrix disassembly;5 of 19 CD44 Molecule ronment; collagen and hyalu- Immunity;process; inflammatory innate immunity; response. mast Int. J. Mol. Sci. 2021, 21, x FOR PEER REVIEW changes in the tissue microenvi-Immunity;Immunity;negative innate regulation innate immunity; immunity; of mast apoptotic mast5 of 19 Int. J. Mol. Sci. 2021, 21, x FOR PEER REVIEW Immunity; innate immunity; mast5 of 19 FCER1GFCER1G,, IgEronicIgE-binding-binding acid binding. protein; receptor;cell activation;cell activation; phagocytosis, phagocytosis, en- FCER1G, ronment;IgE-binding collagen protein; and receptor; hyalu- process;cell activation; inflammatory phagocytosis, response. en- Fc FragmentFc Fragment of IgE of identicalreceptor; protein identical binding; protein IgGengulfment; gulfment;Immunity; positive positive innate regulation immunity;regulation of mastof ReceptorFc Fragment Ig of ronicidenticalbinding; acidIgG protein binding. binding. binding; IgGinterleukin-10, gulfment; -6, positive and -4 regulation of IgE FCER1G,Receptor Ig binding.IgE-binding protein; receptor; interleukinApoptoticcell activation; process;-10, -phagocytosis,6, and cell - 4differentia- produc- en- IgE Receptor Ig binding. production.ApoptoticImmunity;interleukin process; innate-10, -6, immunity; andcell -differentia-4 produc- mast Fc Fragment of identical protein binding; IgG tion.tion;Apoptoticgulfment; membrane positiveprocess; organization; regulationcell differentia- ofpro- FCER1G, IgE-binding protein; receptor;Apoptotic tion;celltion. activation; process;membrane cell phagocytosis, organization; en- pro- IgE Receptor Ig Adapterbinding. protein; cargo receptordifferentiation; teintion;interleukin transport; membrane membrane-10, negati - 6,organization; andve - 4regulation produc- pro- Fc FrDAB2,agment of identical protein binding; IgG gulfment; positive regulation of Adapteractivity;Adapter clathrinprotein; cargoadaptorcargo receptor activity;organization; teinof protein transport; protein binding transport;negati andve protein regulation lo- DABDAB2, Adaptor Adapter protein; cargo receptor teintion. transport; negative regulation IgEDAB2 ReceptorDAB2,, Ig activity;binding.receptor clathrin activity; clathrinadaptor activity;negative ofinterleukin protein regulation binding-10, of - protein6, andand -protein4 produc- lo- DAB Adaptor proteinactivity; C clathrin-terminus adaptor binding; activity; calizationof protein tobinding plasma and membrane; protein lo- DABDABProtein Adaptor Adaptor 2 adaptor activity; protein bindingtion. and protein localization proteinSMAD binding.C-terminus binding; calizationpositive regulation to plasma of membrane; cell migra- ProteinProtein 2 2 C-terminus binding; SMAD to plasma membrane; positive Protein 2 SMAD binding. positive regulation of cell migra- SMADbinding. binding. regulationtionpositive and of cell regulationearly migration endosome of and cell to migra- late 2 earlytionendosome endosome and early to transport. late endosome endosome to late transport.endosomeCell adhesion transport. mediated by integ- Frontal ITGB5, A receptor for fibronectin; integ-Cell adhesion mediated by A receptor for fibronectin; Cellrin; celladhesion migration; mediated extracellular by integ- Cortex IntegrinITGB5ITGB5, Subunit, Arin receptor binding; for signaling fibronectin; receptor integ-integrin; cell migration; ITGB5, Aintegrin receptor binding; for fibronectin; signaling integrin;matrix cell organization; migration; extracellular stress fiber IntegrinIntegrin Subunit Subunit rin binding; signaling receptorextracellular rin; cell matrix migration; organization; extracellular IntegrinBeta Subunit 5 activity;rinreceptor binding; virus activity; signaling receptor virus receptor activity. matrix organization; stress fiber Group 2 DEGs Beta 5 stressassembly;matrix fiber assembly; organization; host- host-virusvirus stressinteraction. fiber Beta 5 activity;receptor virus activity. receptor activity. GRAP *, interaction.assembly; host-virus interaction. GRAPGRAP*, *, Frontal GrowthGRAP Factor *, Couples signals from Growth Factor Couples signals from receptorCell-cell Cell signaling;-cell signaling; Ras protein Ras protein sig- FrontalCortex ReceptorGrowth -FactorBound receptor and cytoplasmic FrontalReceptor-Bound Growth Factor Candouples cytoplasmic signals from tyrosine receptor kinasessignal Cellnal transduction; transduction;-cell signaling; sensory sensory Ras protein percep- sig- Cortex Receptor-Bound Ctyrosineouples kinasessignals tofrom the Rasreceptor Cell-cell signaling; Ras protein sig- CortexProtein ReceptorProtein 2-Related -2Bound-Re- and cytoplasmic tyrosine kinasesperception nal transduction; of sound. sensory percep- toandsignaling the cytoplasmic Ras pathway.signaling tyrosine pathway. kinases tionnal transduction; of sound. sensory percep- AdaptorlatedProtein Adaptor Protein 2-Re- to the Ras signaling pathway. tion of sound. latedlatedProtein AdaptorAdaptor Protein Serine/threonine-protein kinase; TGFBR1,Protein Serine/threoninereceptor; activin -binding;protein kinase;ATP TransformingTGFBR1, Apoptosis; differentiation; growth receptor;binding; metalactivin ion binding; binding; ATP GrowthTransforming Factor Apoptosis;regulation. differentiation; growth 2 Transforming binding;SMAD binding; metal ion transforming binding; Apoptosis; differentiation; growth

Growth Factor binding; metal ion binding; regulation. 2 Beta Receptor 1 Growth Factor regulation. 2 SMADgrowth binding;factor beta transforming binding. Beta Receptor 1 BetaTNFRSF10B Receptor *, 1 growthReceptor factor for the beta cytotoxic binding. ligand growth factor beta binding. Apoptosis; cellular response to TumorTNFRSF10B Necrosis *, ReceptorTNFSF10/TRAIL for the cytotoxic mediating ligand TNFRSF10B *, Receptor for the cytotoxic ligand Apoptosis;mechanical cellularstimulus; response leukocyte to TumorFactor ReceptorNecrosis TNFSF10/TRAILapoptosis; promotes mediating the activa- Apoptosis; cellular response to Tumor Necrosis TNFSF10/TRAIL mediating mechanicalmigration; response stimulus; to leukocyte endoplas- Group 2 DEGs FactorSuperfamily, Receptor apoptosis;tion of NF -promoteskappa-B; essentialthe activa- for mechanical stimulus; leukocyte Factor Receptor apoptosis; promotes the activa- migration;mic reticulum response stress. to endoplas- Group 2 DEGs Superfamily, tion of NF-kappa-B; essential for migration; response to endoplas- Group 2 DEGs Superfamily,Member 10b ERtion stress of NF-induced-kappa- B;apoptosis. essential for mic reticulum stress. Member 10b ER stress-inducedinduced apoptosis.apoptosis. Cellular response to hormone G-protein coupled receptor; Cellularstimulus; response glucose homeostasis;to hormone ADRA2A *, Cellular response to hormone Gtransducer;-protein coupled protein receptor; heterodimeri- stimulus;positive regulation glucose homeostasis; of cell migra- AdrenoceptorADRA2A *, transducer;zation activity; protein protein heterodimeri- homodi- positivetion, cell regulationpopulation of proliferation, cell migra- AdrenoceptorAlpha 2A zationmerization activity; activity. protein homodi- tioncytokine,, cellcell populationproduction;population proliferation,proliferation, Rho protein Alpha 2A merization activity. cytokinesignal transduction. production; Rho protein signalChemical transduction. synaptic transmission; G-protein coupled receptor; signal transduction. GRM2, Chemicalglutamate synaptic homeostasis transmission; and secre- Gtransducer;-protein coupled may mediate receptor; sup- Chemical synaptic transmission; GlutamateGRM2, G-protein coupled receptor; glutamatetion; G protein homeostasis-coupled and glutamate secre- GRM2, transducer;pression of neurotransmissionmay mediate sup- or glutamate homeostasis and secre- Cere- MetabotropicGlutamate transducer; may mediate sup- tion;receptor G protein signaling-coupled pathway; glutamate regu- Glutamate pressionmay be involved of neurotransmission in synaptogene- or tion; G protein-coupled glutamate Cere- Metabotropic pression of neurotransmission or receptor signaling pathway; regu- bellumCere- MetabotropicReceptor 2 may be involved in synaptogene- lationreceptor of synapticsignaling transmission, pathway; regu- sismay or be synaptic involved stabilization. in synaptogene- bellum Receptor 2 lationglutamatergic.lation ofof synapticsynaptic transmission,transmission, sis or synaptic stabilization. glutamatergic.Cell cycle; cell division; host-virus Cellinteraction; cycle; cell cerebral division; cortex host cell-virus mi- Hydrolase; in neurons, involved Cell cycle; cell division; host-virus RHOA, interaction;gration; cellular cerebral response cortex to cell chem- mi- Hydrolase;in the inhibition in neurons, of the initialinvolved interaction; cerebral cortex cell mi- RasRHOA, Homolog Hydrolase; in neurons, involved gration;okine and cellular cytokine response stimuli; to fore-chem- RHOA, inspine the growth.inhibition Upon of the activation initial by gration; cellular response to chem- FamilyRas Homolog Member in the inhibition of the initial okinebrain radialand cytokine glial cell stimuli; differentia- fore- Ras Homolog spineCaMKII, growth. modulates Upon dendriticactivation by okine and cytokine stimuli; fore- Family Member spine growth. Upon activation bybrain radial glial cell differentia- FamilyA Member CaMKII, modulates dendritic tion;brain neuron radial glialapoptosis, cell differentia- morpho- spineCaMKII, structural modulates plasticity. dendritic A tion;genesis, neuron proliferation, apoptosis, and morpho- differ- spine structural plasticity. genesis,entiation. proliferation, and differentiation. Int. J. Mol. Sci. 2021, 21, x FOR PEER REVIEW 5 of 19

Int. J. Mol. Sci. 2021, 21, x FOR PEER REVIEW 5 of 19

Apoptotic process; cell differentiation;Apoptotic membrane process; organization; cell differentia- pro- Apoptotic process; cell differentia- Adapter protein; cargo receptor teinApoptotiction; transport; membrane process; negati organization; cellve differentia-regulation pro- DAB2, tion; membrane organization; pro- activity;Adapter clathrin protein; adaptor cargo receptor activity; oftion;tein protein transport; membrane binding negati organization; andve protein regulation pro- lo- DABDAB2, Adaptor Adapter protein; cargo receptor tein transport; negative regulation DAB2, proteinAdapteractivity; C clathrinprotein;-terminus adaptorcargo binding; receptor activity; calization teinof protein transport; tobinding plasma negati and membrane;ve proteinregulation lo- DABProteinDAB2, Adaptor 2 activity; clathrin adaptor activity; of protein binding and protein lo- DAB Adaptor SMADactivity;protein binding. Cclathrin-terminus adaptor binding; activity; positiveofcalization protein regulation tobinding plasma andof membrane; cell protein migra- lo- DABProtein Adaptor 2 protein C-terminus binding; calization to plasma membrane; Protein 2 proteinSMAD binding.C-terminus binding; tioncalizationpositive and earlyregulation to plasma endosome of membrane; cell to migra- late Protein 2 SMAD binding. positive regulation of cell migra- SMAD binding. endosomepositivetion and regulationearly transport. endosome of cell tomigra- late tion and early endosome to late Celltionendosome adhesionand early transport. mediatedendosome by to integ- late ITGB5, A receptor for fibronectin; integ- endosome transport. rin;endosomeCell cell adhesion migration; transport. mediated extracellular by integ- IntegrinITGB5, Subunit rinA receptor binding; for signaling fibronectin; receptor integ- Cell adhesion mediated by integ- ITGB5, A receptor for fibronectin; integ- matrixCellrin; celladhesion organization; migration; mediated extracellular stress by fiberinteg- IntegrinITGB5,Beta Subunit 5 activity;Arin receptor binding; virus for signaling receptorfibronectin; receptor activity. integrin; cell migration; extracellular Integrin Subunit rin binding; signaling receptor assembly;rin;matrix cell organization; migration; host-virus extracellular interaction.stress fiber IntegrinBeta Subunit 5 rinactivity; binding; virus signaling receptor receptor activity. matrix organization; stress fiber GRAPBeta 5 *, activity; virus receptor activity. matrixassembly; organization; host-virus stressinteraction. fiber Beta 5 activity; virus receptor activity. assembly; host-virus interaction. Frontal GrowthGRAP Factor *, assembly; host-virus interaction. Int. J. Mol. Sci. 2021, 22, 140 GRAP *, Couples signals from receptor Cell-cell signaling; Ras5 ofprotein 17 sig- CortexFrontal ReceptorGrowthGRAP- FactorBound *, Frontal Growth Factor andCouples cytoplasmic signals fromtyrosine receptor kinases nalCell transduction;-cell signaling; sensory Ras protein percep- sig- Frontal GrowthProtein Factor2-Re- Cortex Receptor-Bound toCouples the Ras signals signaling from pathway. receptor tionCell -ofcell sound. signaling; Ras protein sig- Cortex Receptor-Bound and cytoplasmic tyrosine kinases nal transduction; sensory percep- Cortex ReceptorlatedProtein Adaptor- 2Bound-Re- and cytoplasmic tyrosine kinases nal transduction; sensory percep- Protein 2-Re- Tableandto 1. Cont.the cytoplasmic Ras signaling tyrosine pathway. kinases naltion transduction; of sound. sensory percep- latedProteinProtein Adaptor 2-Re- to the Ras signaling pathway. tion of sound. lated Adaptor to the Ras signaling pathway. tion of sound. Brain latedProtein Adaptor Serine/threonine-protein4 kinase; 4 TGFBR1,GeneProtein Gene Expression Molecular Function Biological Process Region Protein receptor;Serine/threonine activin binding;-protein kinase;ATP TransformingTGFBR1, Serine/threonineSerine/threonine-protein-protein kinase; Apoptosis; differentiation; growth TGFBR1, binding;Serine/threoninereceptor; metal activin ion -binding;protein binding; kinase; ATP GrowthTGFBR1TGFBR1, Factor, kinase; receptor; activin regulation. 2 Transforming Apoptosis; differentiation; growth SMADreceptor; binding; activin transforming binding; ATP TransformingTransforming binding;binding; ATPmetal binding; ion binding; metal Apoptosis;Apoptosis; differentiation; differentiation; growth growth BetaGrowth Receptor Factor 1 regulation. 2 growthbinding; factor metal beta ion binding.binding;

Growth Factor Beta ion binding; SMAD binding; regulation. Growth Factor SMAD binding; transforming regulation.

2 Growth Factor regulation.

2 Growth Factor regulation. 2 Beta Receptor 1 TNFRSF10BReceptor 1 *, ReceptorSMADtransforming binding; for the growth transformingcytotoxic factor ligand Beta Receptor 1 growth factor beta binding. Beta Receptor 1 growthbeta binding. factor beta binding. Apoptosis; cellular response to Frontal TumorTNFRSF10B Necrosis *, TNFSF10/TRAILgrowthReceptor factor for the beta cytotoxicmediating binding. ligand Cortex TNFRSF10B *, ReceptorReceptor for thethecytotoxic cytotoxic ligand mechanicalApoptosis; cellularstimulus; response leukocyte to FactorTumorTNFRSF10B Receptor Necrosis *, apoptosis;ReceptorTNFSF10/TRAIL for promotes the cytotoxic mediating the activa- ligand Apoptosis; cellular response to TNFRSF10B *, ligand TNFSF10/TRAIL migration;Apoptosis;mechanical response cellular stimulus; response to leukocyte endoplas- to Group 2 DEGs Tumor Necrosis TNFSF10/TRAIL mediating Apoptosis; cellular response to TumorTumorFactorSuperfamily, Necrosis ReceptorNecrosis tionTNFSF10/TRAILapoptosis;mediating of NF- apoptosis;kappapromotes -mediatingB; essential the activa- for mechanical stimulus; leukocyte Factor Receptor apoptosis; promotes the activa-mechanicalmicmechanicalmigration; reticulum stimulus; response stimulus; stress. leukocyte to leukocyte endoplas- Group 2 DEGs FactorFactor Receptor Receptor apoptosis;promotes the promotes activation the of activa- MemberSuperfamily, 10b ERtion stress of NF-induced-kappa- B;apoptosis. essential migration; for migration; response response to to endoplas- Group 2 DEGs migration; response to endoplas- Group 2 DEGs mic reticulum stress. Group 2 DEGs Superfamily,Superfamily, tionNF-kappa-B; of NF-kappa essential-B; essential for for Superfamily,Member 10b tionER stress of NF-induced-kappa-B; apoptosis. essential endoplasmicfor Cellularmic reticulum reticulum response stress. stress. to hormone Member 10b ER stress-induced mic reticulum stress. Member 10b ER stress-induced apoptosis. Gapoptosis.-protein coupled receptor; stimulus;Cellular response glucose homeostasis;to hormone ADRA2A *, Cellular response to hormone transducer;G-protein coupled protein receptor;heterodimeri- CellularpositiveCellularstimulus; response regulationresponse glucose to hormone tohomeostasis; of hormone cell migra- AdrenoceptorADRA2A *, G-protein coupled receptor; zationG-protein activity; coupled protein receptor; homodi- stimulus;tionstimulus;, glucose cell population glucose homeostasis; homeostasis; proliferation, ADRA2AADRA2A*, *, transducer;transducer; proteinprotein heterodimeri-positive regulation of cell migra- AdrenoceptorADRA2AAlpha 2A *, transducer; protein heterodimeri-positivepositive regulation regulation of cell of cell migra- AdrenoceptorAdrenoceptor Alpha merizationtransducer;zationheterodimerization activity; activity. protein protein activity; heterodimeri- homodi- cytokinepositivetion, cell regulation production;population of proliferation,Rho cell proteinmigra- AdrenoceptorAlpha 2A zation activity; protein homodi-migration,tion, cell populationpopulation proliferation, Alpha2A 2A zationmerizationprotein activity; homodimerization activity. protein homodi- signaltioncytokine, cell transduction. populationproduction; proliferation, Rho protein Alpha 2A merization activity. proliferation,cytokine cytokine production; production; Rho protein merizationactivity. activity. Chemicalcytokinesignal transduction. production; synaptic transmission; Rho protein G-protein coupled receptor; Rho protein signal transduction. GRM2, G-protein coupled receptor; glutamatesignal transduction. homeostasis and secre- ChemicalChemical synaptic synaptic transmission; transmission; transducer;Gtransducer;-protein coupled may mediatemediate receptor; sup- Chemical synaptic transmission; GlutamateGRM2GRM2,, G-protein coupled receptor; glutamatetion;Chemicalglutamate homeostasisG protein synaptic homeostasis-coupled and transmission; andglutamate secre- 2 G-protein coupled receptor; pressionGtransducer;suppression-protein of coupled neurotransmission may of mediate receptor; sup- or Cere- MetabotropicGlutamateGRM2, secretion;receptorglutamate G protein-coupled signaling homeostasis pathway; and secre-regu- Glutamate maytransducer;neurotransmission be involved may mediatein or synaptogene- may sup- tion; G protein-coupled glutamate MetabotropicGlutamate pression of neurotransmissionglutamate or tion; receptorG protein signaling-coupled glutamate bellumCere- MetabotropicReceptorGlutamate 2 pressionbe involved of neurotransmission in or lationtion;receptor G of protein synapticsignaling-coupled transmission, pathway; glutamate regu- Cere- MetabotropicReceptor 2 sispressionmay or besynaptic involved of neurotransmission stabilization. in synaptogene- pathway; or receptor regulation signaling of synaptic pathway; regu- Cere- Metabotropic synaptogenesis or synaptic glutamatergic.receptor signaling pathway; regu- bellum Receptor 2 may be involved in synaptogene-transmission,lation of glutamatergic. synaptic transmission, bellum Receptor 2 sisstabilization. or synaptic stabilization. lation of synaptic transmission, bellum Receptor 2 sis or synaptic stabilization. Celllationglutamatergic. cycle; of synaptic cell division; transmission, host-virus sis or synaptic stabilization. Cell cycle; cell division; host-virus Group 2 DEGs glutamatergic. interaction;glutamatergic.Cell cycle; cell cerebral division; cortex host cell-virus mi- Hydrolase;Hydrolase; in neurons,neurons, involvedinteraction; Cell cycle; cerebral cell cortex division; cell host-virus RHOA, involved in the inhibition of migration;gration;Cellinteraction; cycle; cellular cellular cell cerebral response division; response cortex to host to cell chem--virus mi- RHOA, inHydrolase; the inhibition in neurons, of the initial involved interaction; cerebral cortex cell mi- RasRHOA, Homolog Hydrolase;the initial spine in neurons, growth. involvedchemokine okineinteraction;gration; andand cellular cytokinecytokine cerebral response stimuli; stimuli; cortex to cellfore- chem- mi- Cere-Int. J. Mol.Ras Sci. Homolog2021, 21, x FOR Family PEER REVIEW spineHydrolase;in the growth. inhibition in neurons,Upon of the activation initialinvolved by 6 of 19 Int. J. Mol. Sci. 2021,RHOA, 21, x FOR PEER REVIEW Upon activation by CaMKII, forebraingration; radial cellular glial cell response to chem-6 of 19 bellum FamilyMemberRas Homolog Member A in the inhibition of the initial brainokine radial and cytokine glial cell stimuli; differentia- fore- Ras Homolog CaMKII,inspinemodulates the growth.inhibition modulates dendritic Upon of the spinedendritic activation initial differentiation; byokine and neuron cytokine apoptosis, stimuli; fore- FamilyRas HomologA Member tion;okinebrain neuron radialand cytokine glialapoptosis, cell stimuli; differentia- morpho- fore- spinespinestructural structuralgrowth. plasticity. Upon plasticity. activation morphogenesis, by proliferation, and Family Member CaMKII, modulates dendritic brain radial glial cell differentia- A CaMKII, modulates dendriticdifferentiation. genesis,tion; neuron proliferation, apoptosis, and morpho- differ- A CaMKII,spine structural modulates plasticity. dendritic tion;Astrocyte neuron differentiation; apoptosis, morpho- central A spine structural plasticity. Astrocyteentiation.tion;Astrocytegenesis,differentiation; neuron proliferation, differentiation; apoptosis, central and morpho- central differ- S100B, spineCalcium structural-dependent plasticity. protein genesis,nervous proliferation,system development; and differ- in- S100B, CalciumCalcium-dependent-dependent protein protein nervousgenesis,nervousentiation. system proliferation,system development; development; and differ- in- S100S100B Calcium, binding;binding; identical protein protein bind-innateentiation.nate immune immuneresponse; response; regulation

S100 Calcium binding; identical protein bind- nate immune response; regulation 2

S100 Calcium binding; protein regulation of cell shape; positive 2 Binding Protein ing; protein homodimerization of cell shape; positive regulation of Binding ProteinB B activity;homodimerization tau protein activity; binding. regulationcell population of cell population proliferation and B activity;tau protein tau binding. protein binding. proliferationcell population and apoptotic proliferation and apoptotic process. Cere- process.apoptotic process. bellum bellum TerminatesTerminates thethe action action of of GABA GABA by its high afﬁnity SLC6A1, by its high affinity sodium-de-NeurotransmitterNeurotransmitter transport; transport; neu- SLC6A1SLC6A1,, bysodium-dependent its high affinity reuptakesodium-de- Neurotransmitter transport; neu- Group 2 DEGs Solute Carrier pendent reuptake into presynap- rotransmitter reuptake; synapse

Group 2 DEGs neurotransmitter reuptake; SoluteSolute Carrier Carrier pendentinto presynaptic reuptake terminals; into presynap- rotransmitter reuptake; synapse synapse organization; transport FamilyFamily 6 Member 6 Mem- 1 ticidentical terminals; protein identical binding; protein organization; transport across across blood-brain barrier. ber 1 binding;metal ion metal binding; ion binding; neu- blood-brain barrier. neurotransmitter binding. rotransmitter binding. ASB2, ASB2, Mediates the ubiquitination and Intracellular signal transduction; Ankyrin Repeat Mediates the ubiquitination and Intracellular signal transduction; Ankyrin Repeat subsequent proteasomal degra- post-translational protein modifi- and SOCS Box subsequent proteasomal degra- post-translational protein modifi- and SOCS Box dation of target proteins. cation. dation of target proteins. cation. Containing 2 Transcriptional activator or re- pressor; plays a role in differenti- DLX1, ation of interneurons, in the de- Cell differentiation; transcription; Distal-Less velopment of the ventral fore- transcription regulation; develop- Frontal Distal-Less velopment of the ventral fore- transcription regulation; develop- Frontal Homeobox 1 brain and diencephalic subdivi- mental protein. Homeobox 1 brain and diencephalic subdivi- mental protein. Cortex sions, in craniofacial patterning and morphogenesis. Acts as a messenger during syn- Nervous system development;

Acts as a messenger during syn- Nervous system development; 3

3 aptic development and remodel- positive regulation of long-term NRGN, ing; calmodulin binding; phos- synaptic potentiation; postsynap- Neurogranin phatidic acid binding; phosphati- tic modulation of chemical synap-

dylinositol-3,4,5-trisphosphate tic transmission; signal transduc- binding. tion; telencephalon development. Group 3 DEGs Adaptive immune response; brain Group 3 DEGs Transcriptional activator; plays a Adaptive immune response; brain Transcriptional activator; plays a development; cell fate specifica- role in brain development being development; cell fate specifica- role in brain development being tion; cerebral cortex neuron differ- EOMES, required for the specification and tion; cerebral cortex neuron differ- EOMES, required for the specification and entiation; cerebral cortex regionali- Eomesodermin the proliferation of the interme- entiation; cerebral cortex regionali- Eomesodermin the proliferation of the interme- zation; stem cell population diate progenitor cells and their zation; stem cell population diate progenitor cells and their maintenance; interferon-gamma Cere- progeny in the cerebral cortex. maintenance; interferon-gamma Cere- progeny in the cerebral cortex. production. production. bellum Probably transports thyroxine from the bloodstream to the Cellular protein metabolic process; TTR, brain; hormone activity; identical extracellular matrix organization; Transthyretin protein binding; protein-contain- neutrophil degranulation; purine

ing complex binding; thyroid nucleobase metabolic process. hormone binding. 1 Disease-specific, brain region non-exclusive differentially expressed genes (DEGs). 2 Disease-specific, brain region exclu- 1 Disease-specific, brain region non-exclusive differentially expressed genes (DEGs). 2 Disease-specific, brain region exclusive DEGs. 3 Disease non-specific, brain region exclusive DEGs. 4 Information gathered from http://uniprot.org. * Gene, the sive DEGs. 3 Disease non-specific, brain region exclusive DEGs. 4 Information gathered from http://uniprot.org. * Gene, the expression of which is highly differential also between the sub-clusters of the given sCJD brain region. † Gene, the expres- expression of which is highly differential also between the sub-clusters of the given sCJD brain region. † Gene, the expression of which is highly differential also between the sub-clusters of the sCJD cerebellum, but not frontal cortex samples. sion of which is highly differential also between the sub-clusters of the sCJD cerebellum, but not frontal cortex samples.

2.3. Inter-Regionally Overlapping Canonical Pathways and Upstream Regulators Within the FC samples, 184 genes were identified as differentially expressed between the sCJD and control tissues (Supplementary material S1b, a list of the 184 DEGs); whereas, within the CB samples, we identified 88 DEGs (Supplementary material S1c, a Int.Int. J.J. Mol.Mol. Sci.Sci. 20212021,, 2121,, xx FORFOR PEERPEER REVIEWREVIEW 66 ofof 1919 Int. Int.Int. J.J.J. Mol.Mol.Mol. Sci.Sci.Sci. 20212021,,, 212121,,, xxx FORFORFOR PEERPEERPEER REVIEWREVIEWREVIEW 66 ofof 1919

AstrocyteAstrocyte differentiation;differentiation; centralcentral AstrocyteAstrocyte differentiation;differentiation; centralcentral S100B,S100B, CalciumCalcium--dependentdependent proteinprotein nervousnervous systemsystem development;development; in-in- S100B,S100B, CalciumCalcium--dependentdependent proteinprotein nervousnervous systemsystem development;development; in-in- S100S100 CalciumCalcium binding;binding; identicalidentical proteiproteinn bind-bind- natenate immuneimmune response;response; regulationregulation

S100S100 CalciumCalcium binding;binding; identicalidentical proteiproteinn bind-bind- natenate immuneimmune response;response; regulationregulation 2 2

2 Binding Protein ing; protein homodimerization of cell shape; positive regulation of 2 2 Binding Protein ing; protein homodimerization of cell shape; positive regulation of BindingBinding ProteinProtein ing;ing;ing; proteinproteinprotein homodimerizationhomodimerizationhomodimerization ofof cellcell shape;shape; positivepositive regulationregulation ofof BB activity;activity; tautau proteinprotein binding.binding. cellcell populationpopulation proliferationproliferation andand BB activity;activity; tautau proteinprotein binding.binding. cellcell populationpopulation proliferationproliferation andand Cere-Cere- apoptoticapoptotic process.process. Int. J. Mol. Sci. 2021Cere-Cere-22 apoptoticapoptotic process.process. bellumbellum, , 140 TerminatesTerminates thethe actionaction ofof GABAGABA 6 of 17 bellumbellum TerminatesTerminates thethe actionaction ofof GABAGABA SLC6A1,SLC6A1, byby itsits highhigh affinityaffinity sodiumsodium--de-de- NeurotransmitterNeurotransmitter transport;transport; neu-neu- SLC6A1,SLC6A1, byby itsits highhigh affinityaffinity sodiumsodium--de-de- NeurotransmitterNeurotransmitter transport;transport; neu-neu- Group 2 DEGs Group 2 DEGs SoluteSolute CarrierCarrier pendentpendent reuptakereuptake intointo presynap-presynap- rrotransmitterotransmitter reuptake;reuptake; synapsesynapse Group 2 DEGs Group 2 DEGs Group 2 DEGs SoluteSolute CarrierCarrier pendentpendent reuptakereuptake intointo presynap-presynap- rrotransmitterotransmitter reuptake;reuptake; synapsesynapse FamilyFamily 66 Mem-Mem- Tabletictic 1. Cont.terminals;terminals; identicalidentical proteinprotein organization;organization; transporttransport acrossacross FamilyFamily 66 Mem-Mem- tictic terminals;terminals; identicalidentical proteinprotein organization;organization; transporttransport acrossacross berber 11 binding;binding; metalmetal ionion binding;binding; neu-neu- bloodblood--brainbrain barrier.barrier. Brain berber 11 binding;binding; metalmetal ionion binding;binding;4 neu-neu- bloodblood--brainbrain barrier.barrier.4 Gene Gene Expression rotransmitterrotransmitterMolecular Functionbinding.binding. Biological Process Region rotransmitterrotransmitter binding.binding. ASB2ASB2,ASB2,, Mediates the ubiquitination ASB2,ASB2, MediatesMediates thethe ubiquitinationubiquitination andandIntracellular IntraIntracellularcellular signal transduction;signalsignal transduction;transduction; AnkyrinAnkyrinAnkyrin Repeat RepeatRepeat and MediatesMediatesand subsequent thethe ubiquitinationubiquitination andand IntraIntracellularcellular signalsignal transduction;transduction; AnkyrinAnkyrin RepeatRepeat subsequentsubsequent proteasomalproteasomal degra-degra-post-translationalpostpost--translationaltranslational protein proteinprotein modifi-modifi- andandSOCS SOCSSOCS Box BoxBox subsequentsubsequentproteasomal proteasomalproteasomal degradation of degra-degra- postpost--translationaltranslational proteinprotein modifi-modifi- andand SOCSSOCS BoxBox dationdation ofof targettarget proteins.proteins. modiﬁcation.cation.cation. ContainingContainingContaining 2 22 dationdationtarget of proteins.of targettarget proteins.proteins. cation.cation. ContainingContaining 22 TranscriptionalTranscriptionalTranscriptional activator activatoractivator or oror re-re- TranscriptionalTranscriptionalrepressor; plays activator aactivator role in oror re-re- pressor;pressor; playsplays aa rolerole inin differenti-differenti- pressor;pressor;differentiation playsplays a ofa rolerole inin differenti-differenti- DLX1,DLX1, ationation ofof interneurons,interneurons, inin thethe de-de- CellCell differentiation;differentiation; transcription;transcription; DLX1DLX1,DLX1,, ationationinterneurons, ofof interneurons,interneurons, in the inin thethe de-de-Cell differentiation;CellCell differentiation;differentiation; transcription; transcription;transcription; Distal-LessDistalDistal--LessLess velopmentvelopmentdevelopment ofof of thethe the ventralventral ventral fore-fore-transcriptiontranscriptiontranscription regulation; regulation;regulation; develop-develop- FrontalFrontal DistalDistal--LessLess velopmentvelopment ofof thethe ventralventral fore-fore- transcriptiontranscription regulation;regulation; develop-develop- FrontalFrontalFrontal HomeoboxHomeoboxHomeobox 1 11 brainbrainforebrain andand anddiencephalicdiencephalic diencephalic subdivi-subdivi-developmentalmentalmental protein.protein. protein. CortexCortexCortex HomeoboxHomeobox 11 brainbrain andand diencephalicdiencephalic subdivi-subdivi- mentalmental protein.protein. CortexCortex sions,sions,subdivisions, inin craniofacialcraniofacial in craniofacial patterningpatterning sions,sions,patterning inin craniofacialcraniofacial and patterningpatterning andand morphogenesis.morphogenesis. andandmorphogenesis. morphogenesis.morphogenesis. ActsActsActs asas as a a messenger messenger during duringduring syn-syn- NervousNervous systemsystem development;development;

ActsActs asas aa messengermessenger duringduring syn-syn-NervousNervousNervous system systemsystem development; development;development; 3 3

synaptic development and

3 aptic development and remodel- positive regulation of long-term 3 3 aptic development and remodel-positivepositive regulation regulation of long-term of long-term 3 apticapticremodeling; developmentdevelopment calmodulin andand remodel-remodel- positivepositive regulationregulation ofof longlong--termterm NRGN,NRGN, ing;ing; calmodulincalmodulin binding;binding; phphos-os-synapticsynapticsynaptic potentiation; potentiation;potentiation; postsynap-postsynap- NRGNNRGN,NRGN,, ing;ing;ing;binding; calmodulincalmodulincalmodulin phosphatidic binding;binding;binding; acid phphphos-os- synapticsynaptic potentiation;potentiation; postsynap-postsynap- Neurogranin phatidic acid binding; phosphati-postsynaptictic modulation modulation of chemical of synap- NeurograninNeurogranin phatidicbinding; acid binding; phosphati- tic modulation of chemical synap- NeurograninNeurogranin phatidicphatidic acidacid binding;binding; phosphati-phosphati-chemicaltictic modulationmodulation synaptic transmission; ofof chemicalchemical synap-synap- dylinositoldylinositolphosphatidylinositol-3,4,5---3,4,53,4,5--trisphosphatetrisphosphate tictic transmission;transmission; signalsignal transduc-transduc- dylinositoldylinositol--3,4,53,4,5--trisphosphatetrisphosphatesignal tictic transduction; transmission;transmission; signalsignal transduc-transduc- binding.binding.trisphosphate tion;tion; telencephalontelencephalon devdevelopment.elopment. binding.binding. telencephalontion;tion; telencephalontelencephalon development. devdevelopment.elopment. Group 3 DEGs

Group 3 DEGs binding. Group 3 DEGs AdaptiveAdaptive imimmunemune response;response; brainbrain Group 3 DEGs Group 3 DEGs Group 3 DEGs TranscriptionalTranscriptionalTranscriptional activator; activator;activator; playsplays aa AdaptiveAdaptive imimmunemune response;response; brainbrain TranscriptionalTranscriptional activator;activator; playsplaysAdaptive aa development;development; immune response; cellcell fatefate brain specifica-specifica- plays a role in brain development; cell fate specifica- rolerole inin brainbrain developmentdevelopment beingbeingdevelopment; development; cell fate cell fate specifica- roleroledevelopment inin brainbrain developmentdevelopment being required beingbeing tion;tion; cerebralcerebral cortexcortex neuronneuron differ-differ- EOMES,EOMES, requiredrequired forfor thethe specificationspecification specification; andand tion; tion; cerebralcerebral cerebral cortexcortex cortex neuronneuron differ-differ- EOMESEOMES,EOMES,, requiredrequiredfor the specification forfor thethe specificationspecification and the andand entiation;entiation; cerebralcerebral cortexcortex regionali-regionali- Eomesodermin the proliferation of the interme-neuronentiation;entiation; differentiation; cerebralcerebral cerebral cortexcortex regionali-regionali- EomesoderminEomesodermin theproliferation proliferation of the of the interme- EomesoderminEomesodermin thethe proliferationproliferation ofof thethe interme-interme-cortexzation;zation; regionalization; stemstem cellcell stem populationpopulation cell diatediateintermediate progenitorprogenitor progenitor cellscells andand theirtheir zation;zation; stemstem cellcell populationpopulation diatediate progenitorprogenitor cellscells andand theirtheirpopulation maintenance;maintenance; maintenance; interferoninterferon--gammagamma Cere-Cere- progenyprogenycells and inin their thethe progeny cerebralcerebral in cortex.cortex. maintenance;maintenance; interferoninterferon--gammagamma Cere-Cere- progenyprogeny inin thethe cerebralcerebral cortex.cortex.interferon-gamma production.production. production. Cere-bellumbellum the cerebral cortex. production.production. bellumbellumbellum ProbablyProbablyProbably transports transports thyroxinethyroxine ProbablyProbably transportstransports thyroxinethyroxine thyroxine from the fromfrom tthehe bloodstreambloodstream toto thethe CellularCellularCellular protein proteinprotein metabolic metabolicmetabolic process;process; fromfrombloodstream tthehe bloodstreambloodstream to the brain; toto thethe CellularCellular proteinprotein metabolicmetabolic process;process; TTR,TTR, brain;brain; hormonehormone activity;activity; identicalidenticalprocess; extracellularextracellular extracellular matrixmatrix matrix organization;organization; TTRTTR,TTR,, brain;brain;hormone hormonehormone activity; activity;activity; identical identicalidentical extracellularextracellular matrixmatrix organization;organization; organization; neutrophil TransthyretinTransthyretinTransthyretin proteinproteinprotein binding; binding; proteinprotein--contain-contain- neutrophilneutrophil degranulation;degranulation; purinepurine TransthyretinTransthyretin proteinprotein binding;binding; proteinprotein--contain-contain-degranulation;neutrophilneutrophil purine degranulation;degranulation; nucleobase purinepurine ingingprotein-containing complexcomplex binding;binding; complex thyroidthyroid nucleobasenucleobase metabometaboliclic process.process. inginging complexcomplexcomplex binding;binding;binding; thyroidthyroidthyroidmetabolic nucleobasenucleobase process. metabometabolicliclic process.process.process. hormonehormonebinding; thyroid binding.binding. hormone hormonehormone binding.binding. 11 DiseaseDisease--specific,specific, brainbrain regionregion nonnon--exclusiveexclusive differentiallydifferentiallybinding. expressedexpressed genesgenes ((DEGsDEGs)).. 22 DiseaseDisease--specific,specific, brainbrain regionregion exclu-exclu- 111 DiseaseDisease---specific,specific, brainbrain regionregion nonnon---exclusiveexclusive differentiallydifferentially expressedexpressed genesgenes ((DEGsDEGs)))... 222 DiseaseDisease---specific,specific, brainbrain regionregion exclu-exclu- 1 Disease-specific,sivesive DEGsDEGs brain.. 33 DiseaseDisease region non-exclusive nonnon--specific,specific, differentially brainbrain regionregion expressed exclusiveexclusive genes DEGsDEGs (DEGs)... 44 Information Information2 Disease-specific, gatheredgathered brain fromfrom region http://uniprot.orghttp://uniprot.org exclusive DEGs... ** GeneGene,, thethe 3 sivesive DEGsDEGs... 333 DiseaseDisease nonnon---specific,specific, brainbrain4 regionregion exclusiveexclusive DEGsDEGs... 444 Information InformationInformation gatheredgatheredgathered fromfromfrom http://uniprot.orghttp://uniprot.org... ** GeneGene,,, thethethe Disease non-specific,expressionexpression brainofof whichwhich region isis exclusivehighlyhighly differentialdifferential DEGs. Information alsoalso betweenbetween gathered thethe sub fromsub--clusters clustershttp://uniprot.org ofof thethe givengiven. * Gene, sCJDsCJD the brainbrain expression region.region. of †† Gene,Gene, which the isthe expres-expres- highly differentialexpressionexpression also of betweenof whichwhich the isis highly sub-clustershighly differentialdifferential of the given alsoalso sCJD betweenbetween brain region.thethe subsub†---clustersGene,clusters the ofof expression thethe givengiven of sCJDsCJD which brainbrain is highly region.region. differential ††† Gene, Gene, also thethe expres-expres- sionsion ofof whichwhich isis highlyhighly differentialdifferential alsoalso betweenbetween thethe subsub--clustersclusters ofof thethe sCJDsCJD cerebellum,cerebellum, butbut notnot frontalfrontal cortexcortex samples.samples. between thesionsion sub-clusters ofof whichwhich of isis the highlyhighly sCJD differentialdifferential cerebellum, butalsoalso not betweenbetween frontal cortex thethe subsub samples.---clustersclusters ofof thethethe sCJDsCJD cerebellum,cerebellum, butbut notnot frontalfrontal cortexcortex samples.samples. 2.3.2.3. InterInter--RegionallyRegionally OverlappingOverlapping CanonicalCanonical PathwaysPathways andand UpstreamUpstream RegulatorsRegulators 2.3. Inter-Regionally2.3.2.3. InterInter--RegionallyRegionally Overlapping OverlappingOverlapping Canonical CanonicalCanonical Pathways PathwaysPathways and Upstream andand UpstreamUpstream Regulators RegulatorsRegulators WithinWithin thethe FCFC samples,samples, 184184 genesgenes werewere identifiedidentified asas differentiallydifferentially expressedexpressed betweenbetween Within theWithinWithin FC samples, thethe FCFC samples,samples, 184 genes 184184 were genesgenes identified werewere identifiedidentified as differentially asas differentiallydifferentially expressed expressedexpressed between betweenbetween thethe sCJD sCJD and and control control tissues tissues ( (SupplementarySupplementary material material S1S1bb,, a a list list of of the the 184 184 DEGs DEGs);); the sCJDthethe and sCJD sCJD control and and tissues control control (Supplementary tissues tissues ( (SupplementarySupplementary material S1b, material material a list of theS1S1bb 184,,, a a a DEGs); list list list of of of whereas, the the the 184 184 184 DEGs DEGs DEGs);); whereaswhereas,, withinwithin thethe CBCB samples,samples, wewe identifiedidentified 8888 DEGsDEGs ((SupplementarySupplementary materialmaterial S1S1cc,, aa within thewhereaswhereas CB samples,,,, withinwithinwe thethe identified CBCB samples,samples, 88 DEGs wewe identifiedidentified (Supplementary 8888 DEGsDEGs material ((SupplementarySupplementary S1c, a list materialmaterial of the S1S1cc,,, aaa 88 DEGs). In total, 68 DEGs were found to be common between FC and CB (Figure 4a; Supplementary material S1d, a list of 68 DEGs). Interestingly, despite differences in the number of DEGs identified in the FC and CB sCJD disease signature, the effects of these gene sets appear very similar at the pathway level; a near-complete overlap between canonical pathways for the two contrasts (FC sCJD and CB sCJD) is observed, with the main difference being in the p-values, which reflect higher significance for FC due to the higher number of DEGs identified in this tissue. The most significantly enriched canonical pathways shared between FC and CB included the neuroinflammation signaling pathway, dendritic cell maturation, NF-κB sig- Int. J. Mol. Sci. 2021, 22, 140 7 of 17

Int. J. Mol. Sci. 2021, 21, x FOR PEER REVIEW 7 of 17 naling, acute phase response signaling, and Myc-mediated apoptosis signaling (Figure2 ). An overlap was also observed among upstream regulators identiﬁed in the FC and CB sCJD samples when compared to the control samples. The top inter-regionally common upstreamwhen compared regulators to th includede control samples. IFNG, TNF, The top TGFB1, inter-regionally IL-6, and common IL-1B (Figure upstream3). Lists regu- of the toplators 40 identiﬁed included IFNG, pathways TNF, andTGFB1, upstream IL-6, and regulators IL-1B (Figure are 3). provided Lists of the in top Figures 40 identified2 and3. pathways and upstream regulators are provided in Figures 2 and 3.

Neuroinflammation Signaling Pathway Dendritic Cell Maturation NF-κB Signaling Acute Phase Response Signaling Myc Mediated Apoptosis Signaling Death Receptor Signaling IL-6 Signaling Production of Nitric Oxide and Reactive Oxygen Species Natural Killer Cell Signaling CD28 Signaling in T Helper Cells B Cell Receptor Signaling Induction of Apoptosis by HIV1 PI3K Signaling in B Lymphocytes TNFR1 Signaling TWEAK Signaling Phagosome Formation Apoptosis Signaling Complement System iNOS Signaling

Regulation of IL-2 Expression in T Lymphocytes Pathways Role of NFAT in Regulation of the Immune Response Fcγ Receptor-mediated Phagocytosis in Macrophages

Canonical PTEN Signaling TREM1 Signaling Angiopoietin Signaling IL-12 Signaling and Production in Macrophages HIF1α Signaling STAT3 Pathway PEDF Signaling LPS-stimulated MAPK Signaling Granulocyte Adhesion and Diapedesis LXR/RXR Activation Necroptosis Signaling Pathway IL-10 Signaling IL-8 Signaling HMGB1 Signaling Molecular Mechanisms of Cancer Toll-like Receptor Signaling Inflammasome pathway Crosstalk between Dendritic Cells and Natural Killer Cells

0 5 10 15 20 25 -LogP

common CJDvsCT CB CJDvsCT FC CJDvsCT

FigureFigure 2. Top 2. 40Top canonical 40 canonical pathways pathways regulated regulated differently differently between the the sCJD sCJD and and control control samples samples but overlapping but overlapping between between the sCJD FC and CB. the sCJD FC and CB. Int. J. Mol. Sci. 2021, 22, 140 8 of 17 Int. J. Mol. Sci. 2021, 21, x FOR PEER REVIEW 9 of 19

IFNG TNF TGFB1 IL6 IL1B CSF1 IL10 APP STAT1 NFkB (complex) CSF2 MYD88 NR3C1 IL4 TLR4 AGT STAT3 APOE TP53 IL1 IL2 IL33 RELA Upstream Regulators Upstream TNFSF11 CEBPB JUN NFKB1 IL13 KRAS IL15 SPI1 IL1A PSEN1 CSF3 LDL GFI1 P38 MAPK KIT MAPK1 Il3

0.00 10.00 20.00 30.00 40.00 50.00 60.00 -LogP

common CJDvsCT CB CJDvsCT FC CJDvsCT

Figure 3.3. TopTop 4040 upstream upstream regulators regulators predicted predicted differently differently between between the the sCJD sCJD and and control control samples samples butoverlapping but overlapping between be- thetween sCJD the FC sCJD and FC CB. and CB.

Int. J. Mol. Sci. 2021, 22, 140 9 of 17

2.4. sCJD FC and CB-Exclusive DEGs Although the FC and CB sCJD signatures shared 68 DEGs, resulting in largely overlapping core molecular processes, each brain region also demonstrated unique single-gene expression patterns. The FC-exclusive-sCJD signature consisted of 116 DEGs, while the CB-exclusive-sCJD signature consisted of only 20 DEGs (Figure4a; Supplementary material S1e, lists of 116 and 20 DEGs). When the FC-exclusive-sCJD signature was applied to the CB samples, the sCJD and control tissue clusters were formed, confirming that the FC 116 Int. J. Mol. Sci. 2021, 21, x FOR PEER REVIEW 11 of 19 DEGs are sCJD-specific and that their expression profile differs between the two brain regions (Figure4b,c).

Figure 4. sCJDFigure FC 4. and sCJD CB FC speciﬁc and CB specific gene proﬁles.gene profiles. (a) (a Graphic) Graphic number visualization visualization of DEGs of DEGsthat are that exclusive are exclusiveand common and common to sCJD FC and CB samples. (b) Clustering of FC sCJD and control samples based on the 116 FC-associated gene signatures. to sCJD FC and(c) Incomplete CB samples. clustering (b) Clustering of the CB sCJD of and FC control sCJD andsamples control based sampleson the 116 basedFC-associated on the gene 116 signature FC-associateds. (d) Cluster- genes signature. (c) Incompleteing clustering of CB sCJD ofand the control CB sCJDsamples and based control on the samples 20 CB-associated based on gene the signature 116 FC-associateds. (e) Poor clustering genes of signature. FC sCJD and (d ) Clustering of CB sCJD andcontrol control samples samples based on based the 20 CB on-associated the 20 CB-associated gene signatures. genes Yellowsignature. frame indicates (e) clusters Poor clustering of the same type of FC samples; sCJD and control blue frame highlights the main clusters indicated by unsupervised hierarchical clustering; yellow arrow heads indicate samples basedsamples on the out 20of their CB-associated clusters. genes signature. Yellow frame indicates clusters of the same type samples; blue frame highlights the main clusters indicated by unsupervised hierarchical clustering; yellow arrow heads indicate samples out of their clusters. Int.Int. J. Mol. J. Mol. Sci. Sci.2021 2021, 22, ,21 140, x FOR PEER REVIEW 12 of10 19 of 17

AlthoughAlthough thethe sCJD CB CB-exclusive-exclusive signature signature consisted consisted of ofonly only 20 20DEGs, DEGs, they they clearly clearly clusteredclusteredthe the sCJDsCJD and control tissue tissue CB CB samples samples apart. apart. Interestingly, Interestingly, when when the theCB CBsigna- signa- tureture was was testedtested onon thethe FC samples, the the clustering clustering of of the the sCJD sCJD and and control control tissue tissue samples samples waswas rather rather poor,poor, confirmingconfirming that this 20 20 DEG DEG profile profile is is sCJD sCJD CB CB-specific-specific (Figure (Figure 4d,e).4d,e). However,However, when evaluating evaluating these these results, results, one one should should keep keep in mind in mind that thatthe analysis the analy- is based on and biased by a background of 800 pre-selected genes that are mainly neu- sis is based on and biased by a background of 800 pre-selected genes that are mainly roinflammatory. neuroinflammatory.

2.5.2.5. Sub-Regional Sub-Regional Differences:Differences: Variance Variance in in the the Strength Strength of ofNeuroinflammation Neuroinflammation Interestingly,Interestingly, underunder the sCJD sCJD cluster clusterss there there was was a aformation formation of oftwo two FC FC and and two two CB CB sub-clusters (Figure 1a). In FC, the gene expression analysis of the first sub-cluster (C1) sub-clusters (Figure1a). In FC, the gene expression analysis of the first sub-cluster (C1) versus the second sub-cluster (C2) revealed 181 DEGs which overlapped with the FC sCJD versus the second sub-cluster (C2) revealed 181 DEGs which overlapped with the FC versus control tissue DEG signature (Figure 5a) (Supplementary material S1f, a list of the sCJD versus control tissue DEG signature (Figure5a) (Supplementary material S1f, a list 181 DEGs). In CB, the difference in gene expression pattern between the two sub-clusters of the 181 DEGs). In CB, the difference in gene expression pattern between the two sub- was even more apparent because the C2 resembled the gene expression pattern seen in clusters was even more apparent because the C2 resembled the gene expression pattern the control tissues, which suggested that some sCJD patients may present with pro- seennounced in the cerebellar control tissues, inflammation which and suggested some seem that some to lack sCJD neuroinflammatory patients may present changes with pronounced(Figure 1a). The cerebellar gene expression inflammation analysis and ofsome the CB seem C1 versus to lack C2 neuroinflammatory revealed 50 DEGs (Figure changes (Figure5b; Supplementary1a). The gene material expression S1g, analysis a list of ofthe the 50 CBDEGs C1). versus C2 revealed 50 DEGs (Figure 5b; Supplementary material S1g, a list of the 50 DEGs).

Figure 5. Heatmap and unsupervised two-way hierarchical clustering based on (a) 181 DEG between FC sub-cluster 1 (C1) and sub-cluster 2 (C2). (b) 50 DEG between CB sub-cluster 1 (C1) and sub-cluster 2 (C2). CJD—sporadic Creutzfeldt–Jakob disease; FC—frontal cortex; CB—cerebellum. Int. J. Mol. Sci. 2021, 22, 140 11 of 17

Altogether, analyses of the DEGs involved in the FC and CB sub-clusters formation confirmed variance in the intensity of sub-regional inflammation and the existence of “strong” and “weak” neuroinflammation profiles.

3. Discussion The involvement and dysregulation of major biological processes such as immune system response, metabolism, developmental biology, and vesicle-mediated transport in prion disease pathogenesis have been confirmed by numerous transcriptomic studies using both human and animal tissues [14–16,18,19,24–26,28–31]. However, data on the neuroinflammatory events taking place in different brain regions of human sCJD are surprisingly scarce. Thus, we hypothesized that the expression of neuroinflammation-associated genes is different in distinct brain regions and aimed to identify disease- and brain region- specific genes as well as to provide an overview of the neuroinflammatory landscape in sCJD patients’ brains using an 800 gene expression panel designed by NanoString. To our knowledge, this is the first published study describing the expression of neuroinflammation- associated genes in human brain samples from the FC and CB of sCJD patients and age- and sex-matched normal controls using such panel. We generated data that provide an overview of differentially expressed genes, the most involved canonical pathways and upstream regulators, and their biological functions in sCJD patients as compared to controls; in FC compared to CB; and even the sub-clusters observed within each brain region. The data indicated neuroinflammatory differences in distinct brain regions and varying intensities of inflammation within the same brain regions of sCJD patients with different disease subtypes, which broadens our understanding of prion disease pathogenesis from the aspect of inflammation (Table1). Interestingly, the regional sub-clusters could not be explained by patients’ sex, age group, polymorphic codon 129 in the PRNP, or type of dominant PrPSc. Undoubtedly, a study with a larger number of sCJD cases with different subtypes would ensure firmer conclusions. Nevertheless, the current results imply that the impact of the sCJD subtype may not be the strongest or sole factor determining the strength of the neuroinflammatory gene expression profile. Curiously, a study by Makarava et al. performed with the NanoString neuroinflammation panel to investigate mice infected with 22L (astrocyte-associated) and ME7 (neuron- associated) PrPSc strains found that their established signature for prion disease-associated gene expression was independent of the brain region or prion cell tropism [32]. In our study, however, although the neuroinflammation may seem uniform, considering the overlap of canonical pathways and upstream regulators, we still see specific genes, the expression of which is regulated differently in different brain regions. It is important to look for changes at the single gene expression level to ensure that their unique and subtle roles in disease pathogenesis are not overlooked when multiple genes are pooled together in the enriched sets needed for pathway determination. Other independent research groups using PCR and immunohistochemistry-based gene and protein expression approaches for the investigation of human sCJD brain immunity also identified the presence of regional differences. For example, Llorens et al. investigated the expression of 25 selected inflammation- associated genes in the FC and CB of 30 sCJD patients with the MM 1 and VV 2 subtypes [25]. They observed that the upregulation of these genes was higher in the FC in sCJD MM 1 and in the CB in sCJD VV 2 and concluded that regional gene regulation differences depend on the patient’s genotype at codon 129 in the PRNP [25,26]. Furthermore, Franceschini et al. proposed that the PrPSc strain and polymorphic codon 129 also influence regional microglia activation, because their study of activated microglia distribution throughout the brain of different sCJD subtypes demonstrated that microgliosis, PrPSc deposition, and spongiform change were correlated but varied markedly among the sCJD subtypes [11]. Int. J. Mol. Sci. 2021, 22, 140 12 of 17

Importantly, the studies on human sCJD samples were in agreement with the notion that neuroinflammation in the brain of sCJD patients is not regionally uniform. However, to understand the key cellular and molecular differences between and within distinct brain regions presenting variable pathology, a larger and statistically higher-powered study is needed, preferably combining data on spatial cells’ distribution and their molecular signatures. Currently, scientific evidence implies that microglia are the key drivers of neuroinflammation in prion disease, and the pathway analysis based on the DEG sets we identified in our study supports this notion, as the most significantly involved pathways include neuroinflammation signaling and metabolic processes [19]. Nevertheless, our pathway analysis also suggests the importance of brain dendritic cells, providing new insight on the different cell types involved in the disease development (Figure2). Furthermore, in our study we identified regionally exclusive genes that are likely involved in the formation and development of different brain structures, such as FC and CB. However, we also provided a list of disease-associated top differentially expressed genes in the two brain regions as well as common DEGs. A few literature sources attempting to explain the role of these genes in prion or other neurodegenerative disease pathogenesis were found. For example, in the brain SERPINA3 is mainly expressed by astrocytes, and its deregu- lation has been linked to the pathogenesis of several diseases, including schizophrenia and Alzheimer’s [33–35]. SERPINA3 overexpression has previously been reported in the frontal and occipital cortices of various human prion diseases [36]. Several hypotheses considering the pathogenic effects of SERPINA3 overexpression in prion diseases have been proposed— e.g., that it may contribute to PrPSc formation or hamper PrPSc clearance [36]. However, conclusive explanations for the molecular mechanism of SERPINA3 upregulation and its role in disease development are still lacking. SOCS3 proteins are expressed by multiple immune cells where they reside in a cell’s cy- tosol and negatively regulate cytokines that signal through the JAK/STAT pathway. SOCS3 was one of the few identified genes that were suggested to be prion disease pathogenesis- specific [37]. In another RNA expression study with prion-infected mice models, SOCS3 was detectable early and regulated by the phosphorylation of specific STAT protein complexes [38]. FCER1G is a microglia-expressed hub gene associated with aging and neurodegeneration [39]. Changes in the FCER1G expression were also reported in mice infected with PrPSc [40,41]. The FCER1G is a part of larger molecular complexes and among its other functions is considered to selectively mediate IL-4 production by basophils, to prime T cells towards effector T-helper 2 subset, to form a functional signaling complex in myeloid cells, and to drive the maturation of antigen-presenting cells. CD44 expression is linked to reactive astrocyte heterogeneity observed in the brain during prion disease in mice, and it was suggested as a biomarker to enhance the identification of distinct prion agent strains [42]. CD44 antigen is a receptor for hyaluronic acid and can interact with SPP1 and other ligands, allowing it to participate in a wide variety of cellular functions, including lymphocyte activation, recirculation and homing, hematopoiesis, and tumor metastasis. Finally, the SPP1 encodes a cytokine known for the upregulation of interferon-gamma and interleukin-12 expression and the reduction in interleukin-10 production, leading to type I immunity characterized by intense phagocytic activity. SPP1 is a key factor regulating the degeneration and regeneration of injured nerves via the c-Fos, PKCα, and p-ERK/ERK pathways [43]. Moreover, SPP1 was found to be upregulated and act as an essential modulator of macrophage phenotypes and their ability to clear pathogenic beta-amyloid forms in mice models of Alzheimer’s disease [44,45]. However, its role in prion diseases has not yet been determined. The biological roles of these and other genes listed in Table1 seem highly relevant to prion disease pathogenesis and suggest the importance of various brain cells’ proliferation, Int. J. Mol. Sci. 2021, 22, 140 13 of 17

differentiation, migration, and trafficking characteristics. To elucidate the phenotypic and functional heterogeneity of brain cells in their various pathologic microenvironments observed in distinct brain regions as well as within the same brain region of different human sCJD subtypes, methods that enable the collection of multiplex molecular and spatial information, such as NanoString GeoMx digital spatial profiler or advanced mass spectrometry-based proteomics, could be of great value [46,47]. For the reproduction and research of specific brain microenvironments and cell subtypes within them as well as the investigation of the effectiveness of novel therapeutic compounds for disease treatment or prevention, humanized cerebral organoid platforms and advanced spatial transcriptomic techniques such as multiplexed error-robust fluorescence in situ hybridization could also benefit the future research of prion disease [48,49].

4. Materials and Methods 4.1. Samples Fresh frozen (FF) brain samples from two brain regions—namely, the FC and CB— were collected from 14 sCJD patients and 10 brain donors with no neuropathological changes. In total, 47 RNA samples were analyzed: 27 sCJDs and 20 normal controls. One commercial brain RNA sample included in the NanoString nCounter training kit (Lot. 5201A-0618, 80036) was used as a control and a reference sample for potential experiments in the future. Age- and sex-matched sCJD and healthy brain samples included seven male and female sCJD patients and ﬁve male and female healthy brain donors. Median age at death of sCJD patients was 73 years (range 60–77), and the median age at death of the healthy brain donors was 67.5 years (range 57–74). A maximum of 3 years difference was allowed when selecting age- and sex-matched healthy brain donors for sCJD patients. The included patients had the following sCJD subtypes: 5/14 were sCJD MM 1, 4/14 were VV 2, and 5/14 were single cases of different subtypes, including MV 1, MV 2K, MV 2K + C, MM 1 + 2, and MM 2C + 1. All the sCJD samples were from the Danish prion diseases cohort gathered at the Danish Reference Center for Prion Diseases. All the control tissue samples were received from Edinburgh Brain Bank, University of Edinburgh, Scotland (Supplementary material S3).

4.2. RNA Extraction and Evaluation Total RNA extraction from FF brain samples was performed using the RNeasy Mini Kit (Qiagen, Hilden, Germany) following the manufacturer’s recommendations. The Bioana- lyzer 2100 (Agilent Technologies, Santa Clara, CA, USA) and NanoDrop spectrophotometer (Thermo Fisher Scientiﬁc, Waltham, MA, USA) were used to evaluate the extracted RNA purity (wavelength absorbance ratio A260/280 ~2.0, and A260/230 ~2.0), concentration (ng/µL), and quality (DV300 ≥ 50%, the percentage of RNA fragments ≥300 nucleotides) or integrity (RIN > 4).

4.3. NanoString A commercially available NanoString 757 gene neuroinﬂammation panel comple- mented with an additional 30 genes of choice and 13 housekeeping genes was used to generate the expression data (Supplementary material S2, a list of genes in the panel). To our knowledge, this new panel with pre-selected genes associated with immunity and inﬂammation, neurobiology and neuropathology, and metabolism and stress has not previously been used in investigations of human prion diseases. The total RNA input for each sample extracted from fresh frozen tissues was 50 ng. All the included samples had an A260/280 absorbance ratio between 1.97 and 2.17, and at least 50% of all the RNAs present in each sample were equal to or longer than 300 base pairs; alternatively, the RNA integrity value (RIN) value had to exceed 4. Int. J. Mol. Sci. 2021, 22, 140 14 of 17

The nCounter XT CodeSet Gene Expression Panel assay and Prep Station were used for the direct hybridization of unique barcodes to target RNAs and their placement into the cartridges. A Digital Analyzer was used for the detection and the counting of hy- bridized probes, and nSolver software version 4.0 was used for data normalization. All the procedures were performed following the NanoString guidelines. Using nSolver Advanced Analysis, the quality of the experiment was assessed by hybridization effectiveness, which is measured by the expression of internal six positive and eight negative RNA control transcripts included in the CodeSet. The same six positive controls were used for the normalization of the run-to-run introduced count variability. The sample-to-sample variability was normalized using housekeeping genes selected by the geNorm algorithm. In total, 13 housekeeping genes were included in the neuroin- ﬂammation panel: AARS, ASB7, CCDC127, CNOT10, CSNK2A2, FAM104A, GUSB, LARS, MTO1, SUPT7L, TADA2B, TBP, XPNPEP1.

4.4. Statistical Analysis The genes that were differentially expressed between groups were identiﬁed by ANOVA (p < 0.05), and signiﬁcance was adjusted for multiple testing by estimating false discovery rate (FDR) [50]. For a gene to be considered differentially expressed in a paired analysis, the p value should be lower than 0.05 (p < 0.05) and its log2-fold change higher than 1 (log2FC > 1), unless indicated otherwise. All the data were log2-transformed prior to analysis and visualization in Qlucore Omics Explorer v.3.6 (Qlucore AB, Lund, Sweden), including principal component analysis, heat maps, and unsupervised hierarchical clustering. Functional analysis, including pathway, upstream regulator, and network analysis, was performed in Ingenuity Pathway Analysis (IPA, Qiagen, Redwood City, CA, USA).

5. Conclusions The current study presents the results of the first attempt to reveal molecular neuroinflammatory events occurring in different brain regions of sCJD patients using the NanoString 800 gene neuroinflammation panel+. Our preliminary data on regional brain microenvironments indicate distinct neuroinflammation patterns between the different brain regions and within the same brain region, implying the presence of immune cells with different activation statuses and molecular profiles that might be specific to certain co-factors in their immediate microenvironment. The current study presents an overview of the most involved neuroinflammation- associated pathways and their biological functions affecting different brain regions in sCJD. Furthermore, we identified several significantly deregulated and functionally interesting genes that are of value for further studies. Future research is needed for an extensive molecular characterization of different cell types in order to recognize their impact on the microenvironment and, hence, disease development. This knowledge is needed to improve the current diagnostic strategies and speed up the discovery of therapeutically targetable molecules.

Supplementary Materials: Supplementary materials can be found at https://www.mdpi.com/1422 -0067/22/1/140/s1. Author Contributions: Conceptualization, A.A., H.B., L.C.M., and E.L.L.; formal analysis and validation, A.A., T.L., and J.O.E.; investigation, A.A. and P.R.N.; resources, A.A., E.L.L., A.G., and C.S.; data curation, A.A.; writing—original draft preparation, A.A. and T.L.; writing—review and editing, H.B., L.C.M., P.R.N., A.G., J.O.E., C.S., and E.L.L.; visualization, A.A. and T.L.; supervision, E.L.L.; project administration, A.A. All authors have read and agreed to the published version of the manuscript. Funding: This research received no external funding. Institutional Review Board Statement: The study was conducted according to the guidelines of the Declaration of Helsinki, and approved by the Danish Ethics Committee (De Videnskabsetiske Komiteer, protocol code.: H-16029969, 2016-08-24). Int. J. Mol. Sci. 2021, 22, 140 15 of 17

Informed Consent Statement: Patient consent was waived due to inclusion of the samples donated for research. Data Availability Statement: Datasets related to this article are available in the Gene Expres- sion Omnibus (GEO, https://www.ncbi.nlm.nih.gov/geo/) with the following accession number: (GSE160208) [NCBI tracking system #21381188]. Acknowledgments: The authors thank Edinburgh Brain Bank, which is funded by the United Kingdom Medical Research Council, for providing healthy brain tissues used as controls. Conﬂicts of Interest: The authors declare no conﬂict of interest.

Abbreviations sCJD Sporadic Creutzfeldt–Jakob disease CT Control tissue (normal brain) FC Frontal cortex CB Cerebellum DEG(s) Differentially expressed gene(s)

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