Next Article in Journal
PssJ Is a Terminal Galactosyltransferase Involved in the Assembly of the Exopolysaccharide Subunit in Rhizobium leguminosarum bv. Trifolii
Next Article in Special Issue
A New Take on Prion Protein Dynamics in Cellular Trafficking
Previous Article in Journal
Plant-Derived Smoke Affects Biochemical Mechanism on Plant Growth and Seed Germination
Previous Article in Special Issue
The Effects of Immune System Modulation on Prion Disease Susceptibility and Pathogenesis
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
IJMS
Volume 21
Issue 20
10.3390/ijms21207765
ijms-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles thumb_up
...
Endorse textsms
...
Comment
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Review

Microglia in Prion Diseases: Angels or Demons?

by
Caterina Peggion
1,*,
Roberto Stella
2,
Paolo Lorenzon
3,
Enzo Spisni
4,
Alessandro Bertoli
1,5,6 and
Maria Lina Massimino
6,*
1
Department of Biomedical Sciences, University of Padova, 35131 Padova, Italy
2
Food Safety Division, Department of Chemistry, Istituto Zooprofilattico Sperimentale delle Venezie, 35020 Legnaro (PD), Italy
3
Department of Integrative Medical Biology (IMB), Umeå University, 901 87 Umeå, Sweden
4
Department of Biological, Geological, and Environmental Sciences, University of Bologna, 40126 Bologna, Italy
5
Padova Neuroscience Center, University of Padova, 35131 Padova, Italy
6
CNR Neuroscience Institute, Department of Biomedical Science, University of Padova, 35131 Padova, Italy
*
Authors to whom correspondence should be addressed.
Int. J. Mol. Sci. 2020, 21(20), 7765; https://doi.org/10.3390/ijms21207765
Submission received: 23 September 2020 / Revised: 16 October 2020 / Accepted: 16 October 2020 / Published: 20 October 2020
(This article belongs to the Special Issue Prions and Prion Diseases)
Browse Figures
Review Reports Versions Notes

Abstract

:
Prion diseases are rare transmissible neurodegenerative disorders caused by the accumulation of a misfolded isoform (PrPSc) of the cellular prion protein (PrPC) in the central nervous system (CNS). Neuropathological hallmarks of prion diseases are neuronal loss, astrogliosis, and enhanced microglial proliferation and activation. As immune cells of the CNS, microglia participate both in the maintenance of the normal brain physiology and in driving the neuroinflammatory response to acute or chronic (e.g., neurodegenerative disorders) insults. Microglia involvement in prion diseases, however, is far from being clearly understood. During this review, we summarize and discuss controversial findings, both in patient and animal models, suggesting a neuroprotective role of microglia in prion disease pathogenesis and progression, or—conversely—a microglia-mediated exacerbation of neurotoxicity in later stages of disease. We also will consider the active participation of PrPC in microglial functions, by discussing previous reports, but also by presenting unpublished results that support a role for PrPC in cytokine secretion by activated primary microglia.

1. Introduction—The Prion Protein, Prions and Prion Diseases

The cellular prion protein, PrPC, is a glycosylphosphatidylinositol (GPI)-anchored protein, residing in the outer leaflet of the plasma membrane (PM), expressed in several cell types and particularly abundant in the central nervous system (CNS) and immune cells [1].
PrPC is primarily renowned for being the precursor of prions, the proteinaceous infectious agents lacking nucleic acids that cause invariably fatal neurodegenerative disorders named transmissible spongiform encephalopathies (TSEs) or prion diseases in humans and other mammalian species [2,3,4]. Prion diseases are rare disorders, affecting about one or two people per million per year world-wide, nevertheless they attract remarkable attention due to the unique biology of the transmissible agent. Prions form upon the conversion of PrPC into an insoluble, protease-resistant conformer (PrPSc) that is the major, if not unique, component of the infectious particle. Although all TSEs share the presence of PrPSc aggregate deposition, they present with a variety of etiological and neuropathological traits, affecting different CNS areas, and causing various clinical manifestations.
The great majority of human prion diseases arise sporadically (e.g., sporadic Creutzfeldt–Jakob disease (CJD)), but a significant percentage (~10%) is of genetic origin, due to the autosomal dominant transmission of inherited mutations in the PrPC-coding gene (e.g., genetic CJD, Gerstmann–Sträussler–Scheinker syndrome and fatal familial insomnia) and about 5% of all cases develop on infectious grounds (e.g., Kuru, iatrogenic CJD and a new variant form of CJD that was transmitted to humans through the consumption of meat from bovine spongiform encephalopathy-affected cattle) [5].
The classical diagnostic triad of TSEs comprises spongiform vacuolation of the cerebral grey matter, neuronal loss, and chronic neuroinflammation [6]. Regarding the latter aspect, activation and proliferation of microglia and astrocytes have been recognized as obligatory features of the disease, regardless of the TSE form (reviewed in [7,8,9]). However, the determination of the precise role of glia in prion diseases and other neurodegenerative diseases, which are commonly associated with chronic neuroinflammation, is still a matter of debate [10,11,12,13,14]. Taking this context, it is worth remarking that the cellular mechanisms leading to brain damage in TSEs are far from being understood, and it is not yet clear if they rely on a gain in the toxicity of PrPSc aggregates or a loss of function of PrPC (due to its continuous conversion into PrPSc), or both. Concerning the loss of function hypothesis, the understanding of PrPC physiological function would be of paramount importance. While both the high evolutionary conservation and the almost ubiquitous expression of PrPC would suggest fundamental roles for the protein, and in spite of decades of extensive research, a comprehensive view of PrPC function, however, is still missing.
The possible involvement of PrPC in microglia pathophysiology and the role of microglia in PrPSc infection and propagation during prion disease are the major topic of this review. Particularly, we will discuss previous controversial results on the role of PrPC on microglial activation and cytokine release, and also provide our unpublished data.

2. Microglia Function in Health and Prion Diseases

2.1. Microglia Origin and Function in the Healthy Brain

Microglia belong to the glial system of non-neuronal cells and represent the resident immune population of the CNS. Although it has been studied for decades, the developmental origin of microglia remained debated for a long time [15]. Recent studies confirmed the prediction of the founder of the microglia field, Pio del Rio–Hortega [16], indicating that microglial cells are derived from c-Kit+ erythromyeloid precursors in the yolk sac that seed the CNS rudiment from the cephalic mesenchyme very early during embryogenesis and continuing until the blood–brain barrier is formed, following a gradual process of differentiation into highly specialized immune cells in the brain [17,18]. After such a developmental origin, self-renewal is the only source of new microglial cells in the healthy brain, which is regulated also by astrocytes and neurons through the activation of the microglial colony-stimulating factor (CSF)-1 tyrosine kinase receptor (CSF-1R) by its ligands CSF-1 and interleukin (IL)-34 [19].
Microglial cells play a key role in the maintenance of brain homeostasis from early development to adulthood, including neurodevelopment, synaptic pruning, neuronal circuit maturation [20,21], and the impairment of microglial functions which can lead to severe pathological outcomes.
Under healthy conditions, microglial cells are characterized by a small cell soma and numerous branching processes involved in the clearance of metabolic products and apoptotic cells or cell debris [22]. Key surface receptors (i.e., cluster of differentiation (CD) 45, CD14, and CD11b/CD18 (Macrophage-1 antigen)) maintain microglial cells in a resting but highly dynamic state [23] that is favored by the interaction with neurons, for example through the formation of a molecular complex between the neuronal transmembrane glycoprotein CD200 and its receptor CD200R present in the plasma membrane of microglial cells [24], or between the neuronal chemokine C-X3-C motif chemokine (CX3C) ligand 1 (CX3CL1, also named Fractalkine) and its receptor CX3CR1, expressed solely by microglial cells [25].

2.2. Microglia Involvement in Neuropathology

Under pathological circumstances, such as brain injury, microbial infection, or neurodegeneration, microglial activation leads to morphological changes, up-regulation of surface receptors, secretion of a multitude of cytokines, chemokines, and reactive oxygen species (ROS) [26], and the acquisition of a phagocytic phenotype [27]. Most in vitro and in vivo studies used the bacterial cell wall endotoxin lipopolysaccharide (LPS) (reviewed in [28,29,30]) or pro-inflammatory cytokines (e.g., interferon-γ (IFNγ) and tumor necrosis factor-α (TNF-α)) to achieve microglial activation [31].
Activated microglia could exist in a range of activation states that span from two opposite phenotypes, the pro-inflammatory (M1) and the anti-inflammatory (M2) phenotype, with the phenotype of activated microglia falling somewhere along this spectrum, depending on incoming signals. A precise balance between M1 and M2 is of paramount importance for the resolution of inflammation. Indeed, the M1 phase is characterized by the secretion of pro-inflammatory mediators, such as TNF-α, IL-1β, IL-6, nitric oxide (NO), ROS, chemokines (e.g., macrophage inflammatory protein 1α, monocyte chemoattractant protein-1 (MCP-1) and the IFN inducible protein 10) and neurotoxins. When such a strong pro-inflammatory response protracts over time (e.g., under chronic conditions such as neurodegenerative disorders), the sustained release of inflammatory mediators and oxidative and nitrosative stress may exacerbate neuronal damage [13,32,33], a circumstance that may well occur in prion diseases (see below, [9]). Conversely, in the M2 response, microglia exert numerous beneficial effects through the release of anti-inflammatory factors (e.g., IL-4, IL-10, transforming growth factor-β (TGF-β), insulin-like growth factor 1 and brain-derived neurotrophic factor) [34,35], protecting against brain damage.

2.3. Microglial Proliferation/Activation in Prion Diseases

An active involvement of microglial activation and cytokine signaling in prion diseases has been suggested by a wealth of observations reporting different microglial responses in human CJD [36,37], in bovine spongiform encephalopathies [38], in scrapie [39], in animal models of the diseases (i.e., rodents [40,41,42] or sheep [43] infected with experimental prion strains, or prion mimetics, see below) and in cultured microglial cells exposed to PrPSc [40,44].
Both in patient and in animal models of prion diseases, PrPSc aggregates have been found inside and around microglial cells, inducing activation that correlates temporally with the onset and development of clinical and molecular signs of the disease [40,45,46,47,48,49,50,51,52,53].
The huge experimental effort, however, failed to precisely define the microglial role in prion disease progression (for a comprehensive review, see [7]). It is well-accepted, on one hand, that microglial activation begins in the early stages of disease, preceding neuronal loss and spongiform neurodegeneration, which would suggest that microglial activation is a cause rather than a consequence of neuronal demise (reviewed in [9]). A wealth of evidence, on the other hand, supports the hypothesis that microglia play a neuroprotective role in prion disease-related neuronal damage [7].
One of the most exploited models for studying prion diseases is that involving the use of mice infected with different prion strains, which recapitulated most pathological features of TSEs. Using this experimental setting, the involvement of microglia in prion diseases was principally investigated in mice in which microglial activation and cytokine signalling was altered either genetically or pharmacologically [54,55,56].
Probably due to the variety of the experimental paradigms, however, such approaches provided conflicting conclusions on the role of microglia in prion disease pathogenesis, as discussed below.
Some studies showed a beneficial role of microglia activation in prion diseases. They demonstrated, for example, that Toll-like receptors (TLRs) are involved in prion-induced microglial activation, exploiting a protective function during disease pathogenesis [7] and suggesting that TLR signalling controls the progression of prion disease, as also indicated by the finding that a loss-of-function mutation in TLR4 decreased survival after prion infection [57].
Conversely, however, it also has been suggested that the chronic activation of microglia might have a detrimental effect in the defence against prions. Particularly, a key role in the sustained maintenance of activated microglia in prion disease is retained by the CSF-1R signalling pathway. Indeed, a reduction of proliferating microglia was observed in PrPSc-infected mice treated with the selective inhibitor of CSF-1R, GW2580, with a consequent slowdown in neuronal damage and disease progression [56].
Furthermore, the expression of the microglial GPI-anchored protein CD14, a TLR co-receptor involved in microglia activation, significantly increases in mice infected with different prion strains, as demonstrated by large-scale transcriptomic studies [58,59]. Interestingly, PrPSc-infected CD14 knock-out (KO) mice survived longer and expressed more anti-inflammatory cytokines (such as IL-10 and TGF-β [54], IL-13 [60]) and less pro-inflammatory IL-1β than PrPSc-infected wild-type (WT) mice [54], suggesting a harmful role for CD14-mediated signalling in prion pathogenesis (Figure 1).
Another key issue is the involvement of astroglial cells in prion replication, and propagation to neighboring cells (i.e., neurons) and throughout the CNS, which has been proposed to occur in prion diseases [34,61] and other neurodegenerative disorders associated with prion-like proteins [62]. While the direct participation of astrocytes and neurons in PrPSc replication and spread is well recognized [43,63,64,65,66,67,68,69], the role of microglia in such processes is still debated [34]. The low basal levels of PrPC in microglia may suggest that these cells unlikely support the PrPC-to-PrPSc transition and act as foci of prion propagation. Nonetheless, the increase of PrPC levels in microglia under inflammatory conditions, such as those occurring during prion infection or—experimentally—upon microglia activation by LPS (see below), may enhance the capability of microglial cells to favor prion replication and spreading [42,70].
Taking this context, it also is worth noting that in other neurodegenerative proteinopathies, sharing with prion diseases the molecular mechanisms of propagation of aberrant protein conformers [71,72], the contribution of microglia in cell-to-cell transmission of proteinaceous neuropathogens has been proposed. As examples, this is the case for α-synuclein in Parkinson’s disease models [73], and of the tau protein in Alzheimer’s disease-related tauopathies [74,75,76].

2.4. Cytokines/Chemokines Signalling Alteration in Prion Diseases

Another important and widely studied issue related to microglial response in prion diseases concerns the role of cytokines and chemokines. As previously described, both pro- and anti-inflammatory cytokines are increased in the CNS in response to prion infection. These molecules are produced by activated microglia and astrocytes, since leukocytes from the periphery do not infiltrate the CNS in prion diseases [9], and exert different functions in the inflammatory response to brain injury, as described above.
Regarding whole brain or isolated microglial cells of PrPSc-infected mice, an upregulation of anti-inflammatory cytokines and chemokines (principally in the early stages of prion infection) [10,41,77], or of pro-inflammatory ones [78,79,80,81,82,83,84,85,86,87] have been observed. The different results obtained in such studies may be attributable to several factors, including the used prion strains, the stage of disease, different mouse genetic backgrounds, or techniques applied for the analyte detection.
Among the pro-inflammatory cytokines, IL-1 seems to play a prominent detrimental role in prion-associated neuroinflammation, since knocking-out the IL-1 receptor 1 (IL-1R1) prolongs prion incubation times and delays disease progression in scrapie infected IL-1R1 KO [88,89].
The role of anti-inflammatory cytokines in prion diseases mainly has been investigated by means of IL-4, IL-10, and IL-13 KO mice. Among these cytokines, the only one playing a protective role in prion diseases is IL-10, as suggested by the finding that prion-inoculated IL-10 KO mice have a faster onset and progression of the disease. IL-10 absence also favors TNF-α expression, thereby promoting a sustained pro-inflammatory response [90]. However, conflicting results were obtained in IL-10 KO mice with a different genetic backgrounds [89], supporting the idea that the IL-10-neuroprotective effect is strongly dependent on the genetic context (Figure 2).
Also, numerous chemokines (e.g., MCP-1, Regulated upon Activation, Normal T cell Expressed, and Secreted (RANTES), C-X-C motif ligand (CXCL)3, CXCL9, CXCL10) and chemokine receptors (CCR) (e.g., CCR1, CCR5 and CCR3) are up-regulated in prion diseases, but their role in the pathology remains unclear since different studies provided conflicting results [7]. Signalling pathways activated by CXCL9 and CXCL10 seem to directly contribute to prion disease progression, as suggested by the finding that scrapie infected-CXCR3 KO mice (lacking the CXCL9 and CXCL10 receptor), although exhibiting exacerbated astrocytosis and accelerated accumulation of PrPSc, showed reduced microglia activation and pro-inflammatory factor secretions, and survived longer compared to scrapie-infected WT mice [91]. To contrast, prion infected-CCR1 KO mice (lacking the RANTES receptor) showed a worsened prion disease course and a lower survival rate with respect to infected WT mice [92].
Considering this, animal models provided conflicting results also for the involvement of the CX3CL1/CX3CR1 signalling axis in prion neuropathology. While Grizenkova et al., [55] suggested that CX3CR1 (a receptor for the CX3CL1 chemokine) activation is neuroprotective, a subsequent study demonstrated no contribution of the receptor to disease progression [93].

2.5. Microglial Activation by the Prion Mimetic PrP106-126 Neurotoxic Peptide

Considering the above reported controversial results provided by animal models, it is worth discussing a simpler experimental model that has been widely employed to study the microglia contribution to prion-related neurodegenerative processes. We specifically refer to the use of the fibrillogenic and neurotoxic peptide 106-126 derived from the human prion protein sequence (PrP106-126) [94], characterized by its intrinsic capability to form fibrils in vitro [95] and to induce a PrP-dependent neuronal cell death [96] by the activation of apoptotic cell death pathways [97]. Interestingly, the PrP106-126 neurotoxic effect requires the presence of microglia [98] and was demonstrated to be independent from de novo generation of PrPSc [99].
Particularly the PrP106-126 peptide, or other longer peptides containing the PrP106-126 sequence, induced the activation and proliferation of immortalized microglial cells [100,101,102] and primary microglia cultures [103,104,105,106,107,108,109,110,111], and stimulated astroglial proliferation [112,113]. Furthermore, such fibrillogenic peptides induced the secretion of different pro-inflammatory cytokines (i.e., TNF-α, [105]; RANTES, Granulocyte-CSF, and IL-12, [100]; IL-1β, IL-6, [104,106]), and caused an increase of NO synthase and NO release that appears to be essential for the mediation of neurotoxicity [98,105], as demonstrated by the neuronal death induced in neurons co-cultured with microglia treated with the PrP106-126 peptide [106] (Figure 2).
Taken together, the above summarized findings—based on the use of PrP-derived peptides—support the contention that microglial cells contribute to prion-related neurodegenerative processes by producing pro-inflammatory cytokines and oxidative stress, which are recognized as mediators of neuronal death. Nonetheless, it is worth underlining that some doubts on the relevance and validity of results obtained with prion protein-derived peptides were forwarded, since such studies often disregarded the non-infectious nature of PrP106-126, its absence in clinical cases, or its spontaneous production in in vivo experimental settings [7].

3. A possible Role for PrPC in Neuroinflammation

Until now, we have presented and discussed previous studies aimed at relating microglia activation and cytokine signalling in prion neuropathology in animal and cell models challenged with prions or prion surrogates. However, in line with the loss-of-function hypothesis in prion diseases, it also is plausible to consider that PrPC-to-PrPSc conversion and the consequent irreversible recruitment of the protein into newly formed prions may severely perturb physiologic PrPC functions in microglia and/or microglia-neuron crosstalk. The most reliable and exploited model for investigating such a hypothesis is the use of PrP-KO mice.
Although no phenotypic disturbances were originally recognized in PrP-KO mice [114,115], further investigation revealed numerous subtle phenotypes in different PrPC-deficient mice, many of which were exacerbated upon stressful conditions [116,117]. These models suggested pleiotropic functions for PrPC [1,116], among which was the involvement in the inflammatory response both in the CNS and peripheral extra-neural tissues [118,119,120,121,122,123,124]. The protective role of PrPC in the inflammatory response also was suggested by the higher expression of the protein in the so-called immuno-privileged sites, such as the CNS, eyes, placenta, fetus and testicles [125]. Despite controversial data, it is conceivable that PrPC is involved also in the regulation of phagocytosis, a fundamental process for both the resolution of inflammation and the immune response against pathogens [126,127,128,129].
Interestingly, PrPC seems to regulate the expression and/or the secretion of pro- or anti-inflammatory cytokines after systemic LPS administration in vivo, although the involved signalling pathways and mechanisms are largely unknown [121,123,124,130]. Recently, a new scenario emerged demonstrating that PrPC increases TNF-α production through the stimulation of pro-TNF-α cleavage (as demonstrated in serotoninergic neurons [131] and muscle cells [132]) by the activation of the TNF-α converting enzyme (TACE), a member of the A-Disintegrin-and-Metalloproteinase family, via its coupling to nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. Alternately, by activating the same NADPH oxidase-TACE axis and by modulating TACE localization in the plasma membrane, PrPC also may promote the shedding of transmembrane TNF-α receptors, thereby protecting against excessive pro-inflammatory TNF-α response [133].
Unfortunately, even such studies aimed at elucidating PrPC function in microglial cells using PrP-KO models provided contradictory data. Indeed, while early studies on immortalized microglial cells suggested a key role for PrPC in microglial activation and regulation of the inflammatory response [134,135], more recent data, obtained in murine primary microglial cultures stimulated with LPS, reported no difference in cell morphology, microglial marker expressions and cytokine production between cells expression, or not, PrPC [136].
Viewing this puzzling context, we analyzed the influence of PrPC in microglial function and cytokine production using a mouse model with a different genetic background with respect to those previously employed. Starting from cortical microglial primary cultures from 2–3 post-natal day transgenic (Tg) mice expressing (PrP-Tg) or not PrPC (PrP-KO), we induced microglia activation by adding LPS to the culture medium. Interestingly, LPS-treated (for 96 h) PrP-Tg microglial cells expressed higher PrPC amounts compared to non-treated cells (Figure 3A), as already observed in other cell models [130,137,138]. This result corroborated the hypothesis of a key role of PrPC in the microglial activation process.
Using the same experimental setting, we evaluated the secretion of pro- (IL-1β, IFN-γ, IL-6, TNF-α) and anti-inflammatory (IL-10) cytokines at 24 h and 96 h after LPS-activation of microglia expression, or not, PrPC. We chose this restricted set of cytokines because they were found to be de-regulated in CJD patients and prion infected mice [7], suggesting their involvement in prion neuropathology.
Shown in Figure 3B, our results demonstrated that, in our experimental paradigm (PrP-KO and PrP-expressing mice with an almost pure FVB genetic background, [139]), the secretion of both pro- and anti-inflammatory cytokines was higher in PrP-expressing microglia after 24 h of LPS treatment, while after prolonged inflammatory stimulus, only pro-inflammatory cytokine (IL-1β, IFN-γ, IL-6, and TNF-α) release was stimulated by the presence of PrPC (for experimental details, see the legend to Figure 3).
Such findings are in agreement with previous data obtained from immortalized microglia-like cell line, expressing or not PrPC, in which it was shown that PrPC expression alters TNF-α and IL-1β [134,135] or TGF-β and IL-10 [134] secretion. However, our results are in contrast with data obtained by Pinheiro et al., [136], reporting that genetic ablation of PrPC did not affect LPS-induced production of pro-inflammatory (TNF-α, IL-6, IL-1β) and anti-inflammatory (IL-10) cytokines.
We conceive that the discrepancy between our observations and the previously reported results may originate from the different genetic background of the mouse models (FVB, our experiments; B10.129Ola, Pinheiro and co-workers). It has been suggested, for example, that a polymorphism in the SIRPA gene, encoding the signal regulatory protein (Sirp) α, can be responsible for many phenotypes often associated to PrPC ablation in different mouse genetic backgrounds [140]. To the best of our knowledge, however, the SIRPA genotype was never analysed in the PrP-KO FVB strain that we have employed in our studies, thus further investigation is needed to understand the possible role of Sirpα, or a Sirpα-PrPC cross-talk, in microglial activation and cytokine production.

4. Concluding Remarks

As described in this review, microglia are likely to play a relevant role in prion neuropathology, but—in light of the conflicting data accumulated over time—it is yet to be clearly understood if microglia play as a beneficial actor in, or an enhancer of, prion-related neurodegeneration.
One of the most accredited pictures emerging from the above discussed reports envisages that—in the first stages of the disease—microglial cells act as a suppressor of neurotoxicity induced by PrPSc deposits by facilitating their removal and clearance and by secreting anti-inflammatory factors that prevent neuronal loss. However, in an advanced stage of disease, microglia would no longer be able to contrast the effects of prolonged PrPSc accumulation and neurotoxic signalling, which would rather elicit the switching of microglia to a sustained/chronic pro-inflammatory response, thus worsening brain damage.
Taking this view, it also is worth considering that the depletion of functional PrPC in microglia and microglia-neuron crosstalk may exacerbate the effects of microglia misregulation, thereby contributing to disease progression. Also in this case, however, past literature and the here-presented original data offer a panel of conflicting data, thus highlighting once more the need for a deeper understanding of the physiological PrPC function in different cellular environments and signalling pathways.
It is our opinion that a unifying definition of microglia role in prion disease pathogenesis is crucial for both getting major insights into the neurodegenerative processes and developing suited therapeutic strategies. Particularly, a prompt stimulation of the anti-inflammatory properties of microglia in the first stages of prion diseases may represent an optimal target to ameliorate disease progression and the quality of life of patients.

Author Contributions

Conceptualization, C.P., M.L.M., A.B.; Methodology C.P., M.L.M., E.S.; Formal analysis, R.S., E.S.; Investigation, C.P., P.L., E.S., M.L.M.; writing—original draft preparation, C.P., M.L.M.; writing—review and editing, R.S., C.P., M.L.M., P.L., A.B.; Supervision, A.B. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the University of Padova, grant number BIRD202151/20 to A.B.

Acknowledgments

Authors acknowledge the Medical Research Council (MRC) Prion Unit, London UK, for kindly providing the mouse strains.

Conflicts of Interest

The authors declare no conflict of interest.

Abbreviations

CCRchemokine receptors
CDcluster of differentiation
CJDCruetzfeldt-Jacob disease
CNScentral nervous system
CSFcolony-stimulating factor
CSF-1RCSF-1 receptor
CXCLC-X-C motif ligand
CX3Cchemokine C-X3-C motif chemokine
CX3CL1CX3C ligand 1
CX3CR1CX3C receptor 1
ELISAenzyme-linked immunosorbent assay
GPIglycosylphosphatidylinositol
IFNγinterferon-γ
ILinterleukin
IL-1R1IL-1 receptor 1
KOknock-out
LPSlipopolysaccharide
MCP-1monocyte chemoattractant protein-1
NADPHnicotinamide adenine dinucleotide phosphate
NOnitric oxide
PMplasma membrane
PrPCcellular prion protein
PrPScPrP scrapie
RANTESRegulated upon Activation, Normal T cell Expressed, and Secreted
ROSreactive oxygen species
SEMstandard error of the mean
Sirpsignal regulatory protein
TACETNF-α converting enzyme
Tgtransgenic
TGF-βtransforming growth factor-β
TLRtoll-like receptor
TNF-αtumor necrosis factor-α
TSEstransmissible spongiform encephalopathies
WBWestern blot
WTwild-type

References

  1. Castle, A.R.; Gill, A.C. Physiological Functions of the Cellular Prion Protein. Front. Mol. Biosci. 2017, 4, 19. [Google Scholar] [CrossRef] [Green Version]
  2. Prusiner, S.B. Prions. Sci. Am. 1984, 251, 50–59. [Google Scholar] [CrossRef] [PubMed]
  3. Prusiner, S.B. Prions. Proc. Natl. Acad. Sci. USA 1998, 95, 13363–13383. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  4. Prusiner, S.B. A Unifying Role for Prions in Neurodegenerative Diseases. Science 2012, 336, 1511–1513. [Google Scholar] [CrossRef] [Green Version]
  5. Imran, M.; Mahmood, S. An overview of human prion diseases. Virol. J. 2011, 8, 559. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  6. DeArmond, S.J.; Prusiner, S.B. Etiology and pathogenesis of prion diseases. Am. J. Pathol. 1995, 146, 785–811. [Google Scholar] [PubMed]
  7. Aguzzi, A.; Zhu, C. Microglia in prion diseases. J. Clin. Investig. 2017, 127, 3230–3239. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  8. Carroll, J.A.; Race, B.; Williams, K.; Striebel, J.; Chesebro, B. Microglia Are Critical in Host Defense against Prion Disease. J. Virol. 2018, 92, e00549-18. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  9. Obst, J.; Simon, E.; Mancuso, R.; Gomez-Nicola, D. The Role of Microglia in Prion Diseases: A Paradigm of Functional Diversity. Front. Aging Neurosci. 2017, 9, 207. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  10. Perry, V.H.; Cunningham, C.; Boche, D. Atypical inflammation in the central nervous system in prion disease. Curr. Opin. Neurol. 2002, 15, 349–354. [Google Scholar] [CrossRef]
  11. Schwartz, M.; Baruch, K. The resolution of neuroinflammation in neurodegeneration: Leukocyte recruitment via the choroid plexus. EMBO J. 2014, 33, 7–22. [Google Scholar] [CrossRef]
  12. Gomez-Nicola, D.; Perry, V.H. Microglial dynamics and role in the healthy and diseased brain: A paradigm of functional plasticity. Neuroscientist 2015, 21, 169–184. [Google Scholar] [CrossRef] [Green Version]
  13. Heneka, M.T.; McManus, R.M.; Latz, E. Inflammasome signalling in brain function and neurodegenerative disease. Nat. Rev. Neurosci. 2018, 19, 610–621. [Google Scholar] [CrossRef] [PubMed]
  14. Streit, W.J.; Xue, Q.S. Human CNS immune senescence and neurodegeneration. Curr. Opin. Immunol. 2014, 29, 93–96. [Google Scholar] [CrossRef]
  15. Ginhoux, F.; Garel, S. The mysterious origins of microglia. Nat. Neurosci. 2018, 21, 897–899. [Google Scholar] [CrossRef] [PubMed]
  16. Rio-Hortega, P. The microglia. Lancet 1939, 233, 1023–1026. [Google Scholar] [CrossRef]
  17. Ginhoux, F.; Prinz, M. Origin of microglia: Current concepts and past controversies. Cold Spring Harb. Perspect. Biol. 2015, 7, a020537. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  18. Matcovitch-Natan, O.; Winter, D.R.; Giladi, A.; Vargas Aguilar, S.; Spinrad, A.; Sarrazin, S.; Ben-Yehuda, H.; David, E.; Zelada González, F.; Perrin, P.; et al. Microglia development follows a stepwise program to regulate brain homeostasis. Science 2016, 353, aad8670. [Google Scholar] [CrossRef]
  19. Chitu, V.; Gokhan, Ş.; Nandi, S.; Mehler, M.F.; Stanley, E.R. Emerging Roles for CSF-1 Receptor and its Ligands in the Nervous System. Trends Neurosci. 2016, 39, 378–393. [Google Scholar] [CrossRef] [Green Version]
  20. Paolicelli, R.C.; Bolasco, G.; Pagani, F.; Maggi, L.; Scianni, M.; Panzanelli, P.; Giustetto, M.; Ferreira, T.A.; Guiducci, E.; Dumas, L.; et al. Synaptic pruning by microglia is necessary for normal brain development. Science 2011, 333, 1456–1458. [Google Scholar] [CrossRef] [Green Version]
  21. Ikegami, A.; Haruwaka, K.; Wake, H. Microglia: Lifelong modulator of neural circuits. Neuropathology 2019, 39, 173–180. [Google Scholar] [CrossRef] [Green Version]
  22. Raivich, G. Like cops on the beat: The active role of resting microglia. Trends Neurosci. 2005, 28, 571–573. [Google Scholar] [CrossRef] [PubMed]
  23. Korzhevskii, D.E.; Kirik, O. V Brain Microglia and Microglial Markers. Neurosci. Behav. Physiol. 2016, 46, 284–290. [Google Scholar] [CrossRef]
  24. Shrivastava, K.; Gonzalez, P.; Acarin, L. The immune inhibitory complex CD200/CD200R is developmentally regulated in the mouse brain. J. Comp. Neurol. 2012, 520, 2657–2675. [Google Scholar] [CrossRef] [PubMed]
  25. Wolf, Y.; Yona, S.; Kim, K.W.; Jung, S. Microglia, seen from the CX3CR1 angle. Front. Cell. Neurosci. 2013, 7, 26. [Google Scholar] [CrossRef] [Green Version]
  26. Kim, S.U.; de Vellis, J. Microglia in health and disease. J. Neurosci. Res. 2005, 81, 302–313. [Google Scholar] [CrossRef]
  27. Kreutzberg, G.W. Microglia: A sensor for pathological events in the CNS. Trends Neurosci. 1996, 19, 312–318. [Google Scholar] [CrossRef]
  28. Lund, S.; Christensen, K.V.; Hedtjärn, M.; Mortensen, A.L.; Hagberg, H.; Falsig, J.; Hasseldam, H.; Schrattenholz, A.; Pörzgen, P.; Leist, M. The dynamics of the LPS triggered inflammatory response of murine microglia under different culture and in vivo conditions. J. Neuroimmunol. 2006, 180, 71–87. [Google Scholar] [CrossRef] [Green Version]
  29. Andersson, P.B.; Perry, V.H.; Gordon, S. The acute inflammatory response to lipopolysaccharide in cns parenchyma differs from that in other body tissues. Neuroscience 1992, 48, 169–186. [Google Scholar] [CrossRef] [PubMed]
  30. Hoogland, I.C.M.; Houbolt, C.; van Westerloo, D.J.; van Gool, W.A.; van de Beek, D. Systemic inflammation and microglial activation: Systematic review of animal experiments. J. Neuroinflamm. 2015, 12, 114. [Google Scholar] [CrossRef] [Green Version]
  31. Lively, S.; Schlichter, L.C. Microglia Responses to Pro-inflammatory Stimuli (LPS, IFNγ+TNFα) and Reprogramming by Resolving Cytokines (IL-4, IL-10). Front. Cell. Neurosci. 2018, 12, 215. [Google Scholar] [CrossRef] [Green Version]
  32. Block, M.L.; Zecca, L.; Hong, J.S. Microglia-mediated neurotoxicity: Uncovering the molecular mechanisms. Nat. Rev. Neurosci. 2007, 8, 57–69. [Google Scholar] [CrossRef] [PubMed]
  33. Frank-Cannon, T.C.; Alto, L.T.; McAlpine, F.E.; Tansey, M.G. Does neuroinflammation fan the flame in neurodegenerative diseases? Mol. Neurodegener. 2009, 4, 47. [Google Scholar] [CrossRef] [Green Version]
  34. Carroll, J.A.; Chesebro, B. Neuroinflammation, Microglia, and Cell-Association during Prion Disease. Viruses 2019, 11, 65. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  35. Cherry, J.D.; Olschowka, J.A.; O’Banion, M.K. Neuroinflammation and M2 microglia: The good, the bad, and the inflamed. J. Neuroinflamm. 2014, 11, 98. [Google Scholar] [CrossRef] [Green Version]
  36. Sasaki, A.; Hirato, J.; Nakazato, Y. Immunohistochemical study of microglia in the Creutzfeldt-Jakob diseased brain. Acta Neuropathol. 1993, 86, 337–344. [Google Scholar] [CrossRef]
  37. Mühleisen, H.; Gehrmann, J.; Meyermann, R. Reactive microglia in Creutzfeldt-Jakob disease. Neuropathol. Appl. Neurobiol. 1995, 21, 505–517. [Google Scholar] [CrossRef]
  38. Vidal, E.; Márquez, M.; Tortosa, R.; Costa, C.; Serafín, A.; Pumarola, M. Immunohistochemical approach to the pathogenesis of bovine spongiform encephalopathy in its early stages. J. Virol. Methods 2006, 134, 15–29. [Google Scholar] [CrossRef] [PubMed]
  39. Vidal, E.; Acín, C.; Foradada, L.; Monzón, M.; Márquez, M.; Monleón, E.; Pumarola, M.; Badiola, J.J.; Bolea, R. Immunohistochemical Characterisation of Classical Scrapie Neuropathology in Sheep. J. Comp. Pathol. 2009, 141, 135–146. [Google Scholar] [CrossRef]
  40. Giese, A.; Brown, D.R.; Groschup, M.H.; Feldmann, C.; Haist, I.; Kretzschmar, H.A. Role of microglia in neuronal cell death in prion disease. Brain Pathol. 1998, 8, 449–457. [Google Scholar] [CrossRef]
  41. Baker, C.A.; Lu, Z.Y.; Zaitsev, I.; Manuelidis, L. Microglial activation varies in different models of Creutzfeldt-Jakob disease. J. Virol. 1999, 73, 5089–5097. [Google Scholar] [CrossRef] [Green Version]
  42. Baker, C.A.; Martin, D.; Manuelidis, L. Microglia from Creutzfeldt-Jakob disease-infected brains are infectious and show specific mRNA activation profiles. J. Virol. 2002, 76, 10905–10913. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  43. González, L.; Martin, S.; Begara-McGorum, I.; Hunter, N.; Houston, F.; Simmons, M.; Jeffrey, M. Effects of agent strain and host genotype on PrP accumulation in the brain of sheep naturally and experimentally affected with scrapie. J. Comp. Pathol. 2002, 126, 17–29. [Google Scholar] [CrossRef]
  44. Srivastava, S.; Katorcha, E.; Makarava, N.; Barrett, J.P.; Loane, D.J.; Baskakov, I. V Inflammatory response of microglia to prions is controlled by sialylation of PrPSc. Sci. Rep. 2018, 8, 11326. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  45. Miyazono, M.; Kitamoto, T.; Iwaki, T.; Tateishi, J. Colocalization of prion protein and β protein in the same amyloid plaques in patients with Gerstmann-Sträussler Syndrome. Acta Neuropathol. 1992, 83, 333–339. [Google Scholar] [CrossRef]
  46. Barcikowska, M.; Liberski, P.P.; Boellaard, J.W.; Brown, P.; Gajdusek, D.C.; Budka, H. Microglia is a component of the prion protein amyloid plaque in the Gerstmann-Sträussler-Scheinker syndrome. Acta Neuropathol. 1993, 85, 623–627. [Google Scholar] [CrossRef]
  47. Guiroy, D.C.; Wakayama, I.; Liberski, P.P.; Gajdusek, D.C. Relationship of microglia and scrapie amyloid-immunoreactive plaques in kuru, Creutzfeldt-Jakob disease and Gerstmann-Sträußler syndrome. Acta Neuropathol. 1994, 87, 526–530. [Google Scholar] [CrossRef] [PubMed]
  48. Van Everbroeck, B.; Dobbeleir, I.; De Waele, M.; De Leenheir, E.; Lübke, U.; Martin, J.J.; Cras, P. Extracellular protein deposition correlates with glial activation and oxidative stress in Creutzfeldt-Jakob and Alzheimer’s disease. Acta Neuropathol. 2004, 108, 194–200. [Google Scholar] [CrossRef]
  49. Williams, A.; Lucassen, P.J.; Ritchie, D.; Bruce, M. PrP deposition, microglial activation, and neuronal apoptosis in murine scrapie. Exp. Neurol. 1997, 144, 433–438. [Google Scholar] [CrossRef]
  50. Puoti, G.; Giaccone, G.; Mangieri, M.; Limido, L.; Fociani, P.; Zerbi, P.; Suardi, S.; Rossi, G.; Iussich, S.; Capobianco, R.; et al. Sporadic Creutzfeldt-Jakob disease: The extent of microglia activation is dependent on the biochemical type of PrPSc. J. Neuropathol. Exp. Neurol. 2005, 64, 902–909. [Google Scholar] [CrossRef] [Green Version]
  51. Kercher, L.; Favara, C.; Striebel, J.F.; LaCasse, R.; Chesebro, B. Prion protein expression differences in microglia and astroglia influence scrapie-induced neurodegeneration in the retina and brain of transgenic mice. J. Virol. 2007, 81, 10340–10351. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  52. West Greenlee, M.H.; Lind, M.; Kokemuller, R.; Mammadova, N.; Kondru, N.; Manne, S.; Smith, J.; Kanthasamy, A.; Greenlee, J. Temporal Resolution of Misfolded Prion Protein Transport, Accumulation, Glial Activation, and Neuronal Death in the Retinas of Mice Inoculated with Scrapie. Am. J. Pathol. 2016, 186, 2302–2309. [Google Scholar] [CrossRef] [Green Version]
  53. Vincenti, J.E.; Murphy, L.; Grabert, K.; McColl, B.W.; Cancellotti, E.; Freeman, T.C.; Manson, J.C. Defining the Microglia Response during the Time Course of Chronic Neurodegeneration. J. Virol. 2016, 90, 3003–3017. [Google Scholar] [CrossRef] [Green Version]
  54. Sakai, K.; Hasebe, R.; Takahashi, Y.; Song, C.H.; Suzuki, A.; Yamasaki, T.; Horiuchi, M. Absence of CD14 Delays Progression of Prion Diseases Accompanied by Increased Microglial Activation. J. Virol. 2013, 87, 13433–13445. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  55. Grizenkova, J.; Akhtar, S.; Brandner, S.; Collinge, J.; Lloyd, S.E. Microglial Cx3cr1knockout reduces prion disease incubation time in mice. BMC Neurosci. 2014, 15, 44. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  56. Gómez-Nicola, D.; Fransen, N.L.; Suzzi, S.; Perry, V.H. Regulation of Microglial Proliferation during Chronic Neurodegeneration. J. Neurosci. 2013, 33, 2481–2493. [Google Scholar] [CrossRef] [PubMed]
  57. Spinner, D.S.; Cho, I.S.; Park, S.Y.; Kim, J.I.; Meeker, H.C.; Ye, X.; LaFauci, G.; Kerr, D.J.; Flory, M.J.; Kim, B.S.; et al. Accelerated Prion Disease Pathogenesis in Toll-Like Receptor 4 Signaling-Mutant Mice. J. Virol. 2008, 82, 10701–10708. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  58. Hwang, D.; Lee, I.Y.; Yoo, H.; Gehlenborg, N.; Cho, J.H.; Petritis, B.; Baxter, D.; Pitstick, R.; Young, R.; Spicer, D.; et al. A systems approach to prion disease. Mol. Syst. Biol. 2009, 5, 252. [Google Scholar] [CrossRef] [PubMed]
  59. Xiang, W.; Windl, O.; Wünsch, G.; Dugas, M.; Kohlmann, A.; Dierkes, N.; Westner, I.M.; Kretzschmar, H.A. Identification of Differentially Expressed Genes in Scrapie-Infected Mouse Brains by Using Global Gene Expression Technology. J. Virol. 2004, 78, 11051–11060. [Google Scholar] [CrossRef] [Green Version]
  60. Hasebe, R.; Suzuki, A.; Yamasaki, T.; Horiuchi, M. Temporary upregulation of anti-inflammatory cytokine IL-13 expression in the brains of CD14 deficient mice in the early stage of prion infection. Biochem. Biophys. Res. Commun. 2014, 454, 125–130. [Google Scholar] [CrossRef] [Green Version]
  61. Mabbott, N.A. Immunology of Prion Protein and Prions. Prog. Mol. Biol. Transl. Sci. 2017, 150, 203–240. [Google Scholar] [CrossRef] [PubMed]
  62. Goedert, M.; Clavaguera, F.; Tolnay, M. The propagation of prion-like protein inclusions in neurodegenerative diseases. Trends Neurosci. 2010, 33, 317–325. [Google Scholar] [CrossRef]
  63. Victoria, G.S.; Arkhipenko, A.; Zhu, S.; Syan, S.; Zurzolo, C. Astrocyte-to-neuron intercellular prion transfer is mediated by cell-cell contact. Sci. Rep. 2016, 6, 20762. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  64. Carroll, J.A.; Striebel, J.F.; Rangel, A.; Woods, T.; Phillips, K.; Peterson, K.E.; Race, B.; Chesebro, B. Prion Strain Differences in Accumulation of PrPSc on Neurons and Glia Are Associated with Similar Expression Profiles of Neuroinflammatory Genes: Comparison of Three Prion Strains. PLOS Pathog. 2016, 12, e1005551. [Google Scholar] [CrossRef]
  65. Sisó, S.; Chianini, F.; Eaton, S.L.; Witz, J.; Hamilton, S.; Martin, S.; Finlayson, J.; Pang, Y.; Stewart, P.; Steele, P.; et al. Disease phenotype in sheep after infection with cloned murine scrapie strains. Prion 2012, 6, 174–183. [Google Scholar] [CrossRef] [PubMed]
  66. Hilton, K.J.; Cunningham, C.; Reynolds, R.A.; Perry, V.H. Early Hippocampal Synaptic Loss Precedes Neuronal Loss and Associates with Early Behavioural Deficits in Three Distinct Strains of Prion Disease. PLoS ONE 2013, 8, e68062. [Google Scholar] [CrossRef] [Green Version]
  67. Cronier, S.; Laude, H.; Peyrin, J.M. Prions can infect primary cultured neurons and astrocytes and promote neuronal cell death. Proc. Natl. Acad. Sci. USA 2004, 101, 12271–12276. [Google Scholar] [CrossRef] [Green Version]
  68. Hannaoui, S.; Maatouk, L.; Privat, N.; Levavasseur, E.; Faucheux, B.A.; Haïk, S. Prion propagation and toxicity occur in vitro with two-phase kinetics specific to strain and neuronal type. J. Virol. 2013, 87, 2535–2548. [Google Scholar] [CrossRef] [Green Version]
  69. Tahir, W.; Abdulrahman, B.; Abdelaziz, D.H.; Thapa, S.; Walia, R.; Schätzl, H.M. An astrocyte cell line that differentially propagates murine prions. J. Biol. Chem. 2020, 295, 11572–11583. [Google Scholar] [CrossRef]
  70. Muñoz-Gutiérrez, J.F.; Schneider, D.A.; Baszler, T.V.; Greenlee, J.J.; Nicholson, E.M.; Stanton, J.B. hTERT-immortalized ovine microglia propagate natural scrapie isolates. Virus Res. 2015, 198, 35–43. [Google Scholar] [CrossRef]
  71. Peggion, C.; Sorgato, M.C.; Bertoli, A. Prions and prion-like pathogens in neurodegenerative disorders. Pathogens 2014, 3, 149–163. [Google Scholar] [CrossRef] [Green Version]
  72. Ugalde, C.L.; Finkelstein, D.I.; Lawson, V.A.; Hill, A.F. Pathogenic mechanisms of prion protein, amyloid-β and α-synuclein misfolding: The prion concept and neurotoxicity of protein oligomers. J. Neurochem. 2016, 139, 162–180. [Google Scholar] [CrossRef]
  73. George, S.; Rey, N.L.; Tyson, T.; Esquibel, C.; Meyerdirk, L.; Schulz, E.; Pierce, S.; Burmeister, A.R.; Madaj, Z.; Steiner, J.A.; et al. Microglia affect α-synuclein cell-to-cell transfer in a mouse model of Parkinson’s disease. Mol. Neurodegener. 2019, 14, 34. [Google Scholar] [CrossRef]
  74. Asai, H.; Ikezu, S.; Tsunoda, S.; Medalla, M.; Luebke, J.; Haydar, T.; Wolozin, B.; Butovsky, O.; Kügler, S.; Ikezu, T. Depletion of microglia and inhibition of exosome synthesis halt tau propagation. Nat. Neurosci. 2015, 18, 1584–1593. [Google Scholar] [CrossRef] [PubMed]
  75. Španić, E.; Langer Horvat, L.; Hof, P.R.; Šimić, G. Role of Microglial Cells in Alzheimer’s Disease Tau Propagation. Front. Aging Neurosci. 2019, 11, 271. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  76. Simon, E.; Obst, J.; Gomez-Nicola, D. The Evolving Dialogue of Microglia and Neurons in Alzheimer’s Disease: Microglia as Necessary Transducers of Pathology. Neuroscience 2019, 405, 24–34. [Google Scholar] [CrossRef] [PubMed]
  77. Cunningham, C.; Boche, D.; Perry, V.H. Transforming growth factor beta1, the dominant cytokine in murine prion disease: Influence on inflammatory cytokine synthesis and alteration of vascular extracellular matrix. Neuropathol. Appl. Neurobiol. 2002, 28, 107–119. [Google Scholar] [CrossRef] [PubMed]
  78. Campbell, I.L.; Eddleston, M.; Kemper, P.; Oldstone, M.B.; Hobbs, M. V Activation of cerebral cytokine gene expression and its correlation with onset of reactive astrocyte and acute-phase response gene expression in scrapie. J. Virol. 1994, 68, 2383–2387. [Google Scholar] [CrossRef] [Green Version]
  79. Williams, A.E.; van Dam, A.M.; Man-A-Hing, W.K.; Berkenbosch, F.; Eikelenboom, P.; Fraser, H. Cytokines, prostaglandins and lipocortin-1 are present in the brains of scrapie-infected mice. Brain Res. 1994, 654, 200–206. [Google Scholar] [CrossRef]
  80. Kordek, R.; Nerurkar, V.R.; Liberski, P.P.; Isaacson, S.; Yanagihara, R.; Gajdusek, D.C. Heightened expression of tumor necrosis factor alpha, interleukin 1 alpha, and glial fibrillary acidic protein in experimental Creutzfeldt-Jakob disease in mice. Proc. Natl. Acad. Sci. USA 1996, 93, 9754–9758. [Google Scholar] [CrossRef] [Green Version]
  81. Brown, A.R.; Webb, J.; Rebus, S.; Walker, R.; Williams, A.; Fazakerley, J.K. Inducible cytokine gene expression in the brain in the ME7/CV mouse model of scrapie is highly restricted, is at a strikingly low level relative to the degree of gliosis and occurs only late in disease. J. Gen. Virol. 2003, 84, 2605–2611. [Google Scholar] [CrossRef]
  82. Cunningham, C.; Wilcockson, D.C.; Campion, S.; Lunnon, K.; Perry, V.H. Central and systemic endotoxin challenges exacerbate the local inflammatory response and increase neuronal death during chronic neurodegeneration. J. Neurosci. 2005, 25, 9275–9284. [Google Scholar] [CrossRef] [Green Version]
  83. Kim, J.I.; Ju, W.K.; Choi, J.H.; Kim, J.; Choi, E.K.; Carp, R.I.; Wisniewski, H.M.; Kim, Y.S. Expression of cytokine genes and increased nuclear factor-kappa B activity in the brains of scrapie-infected mice. Mol. Brain Res. 1999, 73, 17–27. [Google Scholar] [CrossRef]
  84. Lee, H.P.; Jun, Y.C.; Choi, J.K.; Kim, J.I.; Carp, R.I.; Kim, Y.S. The expression of RANTES and chemokine receptors in the brains of scrapie-infected mice. J. Neuroimmunol. 2005, 158, 26–33. [Google Scholar] [CrossRef]
  85. Tribouillard-Tanvier, D.; Striebel, J.F.; Peterson, K.E.; Chesebro, B. Analysis of protein levels of 24 cytokines in scrapie agent-infected brain and glial cell cultures from mice differing in prion protein expression levels. J. Virol. 2009, 83, 11244–11253. [Google Scholar] [CrossRef] [Green Version]
  86. Baker, C.A.; Manuelidis, L. Unique inflammatory RNA profiles of microglia in Creutzfeldt-Jakob disease. Proc. Natl. Acad. Sci. USA 2003, 100, 675–679. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  87. Riemer, C.; Neidhold, S.; Burwinkel, M.; Schwarz, A.; Schultz, J.; Krätzschmar, J.; Mönning, U.; Baier, M. Gene expression profiling of scrapie-infected brain tissue. Biochem. Biophys. Res. Commun. 2004, 323, 556–564. [Google Scholar] [CrossRef] [PubMed]
  88. Schultz, J.; Schwarz, A.; Neidhold, S.; Burwinkel, M.; Riemer, C.; Simon, D.; Kopf, M.; Otto, M.; Baier, M. Role of interleukin-1 in prion disease-associated astrocyte activation. Am. J. Pathol. 2004, 165, 671–678. [Google Scholar] [CrossRef] [Green Version]
  89. Tamgüney, G.; Giles, K.; Glidden, D.V.; Lessard, P.; Wille, H.; Tremblay, P.; Groth, D.F.; Yehiely, F.; Korth, C.; Moore, R.C.; et al. Genes contributing to prion pathogenesis. J. Gen. Virol. 2008, 89, 1777–1788. [Google Scholar] [CrossRef]
  90. Thackray, A.M.; McKenzie, A.N.; Klein, M.A.; Lauder, A.; Bujdoso, R. Accelerated prion disease in the absence of interleukin-10. J. Virol. 2004, 78, 13697–13707. [Google Scholar] [CrossRef] [Green Version]
  91. Riemer, C.; Schultz, J.; Burwinkel, M.; Schwarz, A.; Mok, S.W.F.; Gültner, S.; Bamme, T.; Norley, S.; van Landeghem, F.; Lu, B.; et al. Accelerated prion replication in, but prolonged survival times of, prion-infected CXCR3-/- mice. J. Virol. 2008, 82, 12464–12471. [Google Scholar] [CrossRef] [Green Version]
  92. LaCasse, R.A.; Striebel, J.F.; Favara, C.; Kercher, L.; Chesebro, B. Role of Erk1/2 activation in prion disease pathogenesis: Absence of CCR1 leads to increased Erk1/2 activation and accelerated disease progression. J. Neuroimmunol. 2008, 196, 16–26. [Google Scholar] [CrossRef] [Green Version]
  93. Striebel, J.F.; Race, B.; Carroll, J.A.; Phillips, K.; Chesebro, B. Knockout of fractalkine receptor Cx3cr1 does not alter disease or microglial activation in prion-infected mice. J. Gen. Virol. 2016, 97, 1481–1487. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  94. Forloni, G.; Chiesa, R.; Bugiani, O.; Salmona, M.; Tagliavini, F. Review: PrP 106-126-25 years after. Neuropathol. Appl. Neurobiol. 2019, 45, 430–440. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  95. Tagliavini, F.; Prelli, F.; Verga, L.; Giaccone, G.; Sarma, R.; Gorevic, P.; Ghetti, B.; Passerini, F.; Ghibaudi, E.; Forloni, G. Synthetic peptides homologous to prion protein residues 106-147 form amyloid-like fibrils in vitro. Proc. Natl. Acad. Sci. USA 1993, 90, 9678–9682. [Google Scholar] [CrossRef] [Green Version]
  96. Brown, D.R.; Herms, J.; Kretzschmar, H.A. Mouse cortical cells lacking cellular PrP survive in culture with a neurotoxic PrP fragment. Neuroreport 1994, 5, 2057–2060. [Google Scholar] [CrossRef] [PubMed]
  97. Forloni, G.; Angeretti, N.; Chiesa, R.; Monzani, E.; Salmona, M.; Bugiani, O.; Tagliavini, F. Neurotoxicity of a prion protein fragment. Nature 1993, 362, 543–546. [Google Scholar] [CrossRef]
  98. Brown, D.R.; Schmidt, B.; Kretzschmar, H.A. Role of microglia and host prion protein in neurotoxicity of a prion protein fragment. Nature 1996, 380, 345–347. [Google Scholar] [CrossRef]
  99. Fioriti, L.; Quaglio, E.; Massignan, T.; Colombo, L.; Stewart, R.S.; Salmona, M.; Harris, D.A.; Forloni, G.; Chiesa, R. The neurotoxicity of prion protein (PrP) peptide 106-126 is independent of the expression level of PrP and is not mediated by abnormal PrP species. Mol. Cell. Neurosci. 2005, 28, 165–176. [Google Scholar] [CrossRef]
  100. Thellung, S.; Corsaro, A.; Villa, V.; Venezia, V.; Nizzari, M.; Bisaglia, M.; Russo, C.; Schettini, G.; Aceto, A.; Florio, T. Amino-Terminally Truncated Prion Protein PrP90-231 Induces Microglial Activation in Vitro. Ann. N. Y. Acad. Sci. 2007, 1096, 258–270. [Google Scholar] [CrossRef]
  101. Bai, Y.; Li, Y.; Wang, G.; Zhou, X.; Zhao, D. PrP 106-126 altered PrP mRNA gene expression in mouse microglia BV-2 cells. Virol. Sin. 2010, 25, 440–444. [Google Scholar] [CrossRef]
  102. Chang, J.; Yang, L.; Kouadir, M.; Peng, Y.; Zhang, S.; Shi, F.; Zhou, X.; Yin, X.; Zhao, D. Antibody-mediated inhibition of integrin α5β1 blocks neurotoxic prion peptide PrP106-126-induced activation of BV2 microglia. J. Mol. Neurosci. 2012, 48, 248–252. [Google Scholar] [CrossRef]
  103. Brown, D.R. Prion protein-overexpressing cells show altered response to a neurotoxic prion protein peptide. J. Neurosci. Res. 1998, 54, 331–340. [Google Scholar] [CrossRef]
  104. Peyrin, J.M.; Lasmézas, C.I.; Haïk, S.; Tagliavini, F.; Salmona, M.; Williams, A.; Richie, D.; Deslys, J.P.; Dormont, D. Microglial cells respond to amyloidogenic PrP peptide by the production of inflammatory cytokines. Neuroreport 1999, 10, 723–729. [Google Scholar] [CrossRef]
  105. Fabrizi, C.; Silei, V.; Menegazzi, M.; Salmona, M.; Bugiani, O.; Tagliavini, F.; Suzuki, H.; Lauro, G.M. The stimulation of inducible nitric-oxide synthase by the prion protein fragment 106--126 in human microglia is tumor necrosis factor-alpha-dependent and involves p38 mitogen-activated protein kinase. J. Biol. Chem. 2001, 276, 25692–25696. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  106. Garção, P.; Oliveira, C.R.; Agostinho, P. Comparative study of microglia activation induced by amyloid-beta and prion peptides: Role in neurodegeneration. J. Neurosci. Res. 2006, 84, 182–193. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  107. Yang, L.; Zhou, X.; Yang, J.; Yin, X.; Han, L.; Zhao, D. Aspirin inhibits cytotoxicity of prion peptide PrP106-126 to neuronal cells associated with microglia activation in vitro. J. Neuroimmunol. 2008, 199, 10–17. [Google Scholar] [CrossRef] [PubMed]
  108. Herms, J.W.; Madlung, A.; Brown, D.R.; Kretzschmar, H.A. Increase of intracellular free Ca2+ in microglia activated by prion protein fragment. Glia 1997, 21, 253–257. [Google Scholar] [CrossRef]
  109. Kouadir, M.; Yang, L.; Tan, R.; Shi, F.; Lu, Y.; Zhang, S.; Yin, X.; Zhou, X.; Zhao, D. CD36 participates in PrP(106-126)-induced activation of microglia. PLoS ONE 2012, 7, e30756. [Google Scholar] [CrossRef]
  110. Tu, J.; Yang, L.; Zhou, X.; Qi, K.; Wang, J.; Kouadir, M.; Xu, L.; Yin, X.; Zhao, D. PrP106-126 and Aβ 1-42 peptides induce BV-2 microglia chemotaxis and proliferation. J. Mol. Neurosci. 2014, 52, 107–116. [Google Scholar] [CrossRef]
  111. Wang, J.; Zhao, D.; Pan, B.; Fu, Y.; Shi, F.; Kouadir, M.; Yang, L.; Yin, X.; Zhou, X. Toll-like receptor 2 deficiency shifts PrP106-126-induced microglial activation from a neurotoxic to a neuroprotective phenotype. J. Mol. Neurosci. 2015, 55, 880–890. [Google Scholar] [CrossRef]
  112. Forloni, G.; Del Bo, R.; Angeretti, N.; Chiesa, R.; Smiroldo, S.; Doni, R.; Ghibaudi, E.; Salmona, M.; Porro, M.; Verga, L. A neurotoxic prion protein fragment induces rat astroglial proliferation and hypertrophy. Eur. J. Neurosci. 1994, 6, 1415–1422. [Google Scholar] [CrossRef] [PubMed]
  113. Brown, D.R.; Schmidt, B.; Kretzschmar, H.A. A prion protein fragment primes type 1 astrocytes to proliferation signals from microglia. Neurobiol. Dis. 1998, 4, 410–422. [Google Scholar] [CrossRef] [Green Version]
  114. Bueler, H.; Fischer, M.; Lang, Y.; Bluethmann, H.; Lipp, H.P.; DeArmond, S.J.; Prusiner, S.B.; Aguet, M.; Weissmann, C. Normal development and behaviour of mice lacking the neuronal cell-surface PrP protein. Nature 1992, 356, 577–582. [Google Scholar] [CrossRef]
  115. Manson, J.C.; Clarke, A.; Hooper, M.L.; Aitchison, L.; McConnell, I.; Hope, J. 129/Ola mice carrying a null mutation in PrP that abolishes mRNA production are developmentally normal. Neurodegener. Dis. 1994, 8, 121–127. [Google Scholar] [CrossRef] [PubMed]
  116. Wulf, M.A.; Senatore, A.; Aguzzi, A. The biological function of the cellular prion protein: An update. BMC Biol. 2017, 15, 34. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  117. Massimino, M.L.; Peggion, C.; Loro, F.; Stella, R.; Megighian, A.; Scorzeto, M.; Blaauw, B.; Toniolo, L.; Sorgato, M.C.; Reggiani, C.; et al. Age-dependent neuromuscular impairment in prion protein knockout mice. Muscle Nerve 2016, 53, 269–279. [Google Scholar] [CrossRef]
  118. Linden, R.; Martins, V.R.; Prado, M.A.M.; Cammarota, M.; Izquierdo, I.; Brentani, R.R. Physiology of the prion protein. Physiol. Rev. 2008, 88, 673–728. [Google Scholar] [CrossRef]
  119. Ballerini, C.; Gourdain, P.; Bachy, V.; Blanchard, N.; Levavasseur, E.; Grégoire, S.; Fontes, P.; Aucouturier, P.; Hivroz, C.; Carnaud, C. Functional Implication of Cellular Prion Protein in Antigen-Driven Interactions between T Cells and Dendritic Cells. J. Immunol. 2006, 176, 7254–7262. [Google Scholar] [CrossRef]
  120. Hu, W.; Nessler, S.; Hemmer, B.; Eagar, T.N.; Kane, L.P.; Leliveld, S.R.; Müller-Schiffmann, A.; Gocke, A.R.; Lovett-Racke, A.; Ben, L.H.; et al. Pharmacological prion protein silencing accelerates central nervous system autoimmune disease via T cell receptor signalling. Brain 2010, 133, 375–388. [Google Scholar] [CrossRef] [Green Version]
  121. Martin, G.R.; Keenan, C.M.; Sharkey, K.A.; Jirik, F.R. Endogenous prion protein attenuates experimentally induced colitis. Am. J. Pathol. 2011, 179, 2290–2301. [Google Scholar] [CrossRef]
  122. Gourdain, P.; Ballerini, C.; Nicot, A.B.; Carnaud, C. Exacerbation of experimental autoimmune encephalomyelitis in prion protein (PrPc)-null mice: Evidence for a critical role of the central nervous system. J. Neuroinflamm. 2012, 9, 25. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  123. Tsutsui, S.; Hahn, J.N.; Johnson, T.A.; Ali, Z.; Jirik, F.R. Absence of the cellular prion protein exacerbates and prolongs neuroinflammation in experimental autoimmune encephalomyelitis. Am. J. Pathol. 2008, 173, 1029–1041. [Google Scholar] [CrossRef] [Green Version]
  124. Liu, J.; Zhao, D.; Liu, C.; Ding, T.; Yang, L.; Yin, X.; Zhou, X. Prion protein participates in the protection of mice from lipopolysaccharide infection by regulating the inflammatory process. J. Mol. Neurosci. 2015, 55, 279–287. [Google Scholar] [CrossRef] [PubMed]
  125. Bakkebø, M.K.; Mouillet-Richard, S.; Espenes, A.; Goldmann, W.; Tatzelt, J.; Tranulis, M.A. The Cellular Prion Protein: A Player in Immunological Quiescence. Front. Immunol. 2015, 6, 450. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  126. De Almeida, C.J.G.; Chiarini, L.B.; Pereira da Silva, J.; Silva, P.M.R.E.; Martins, M.A.; Linden, R. The cellular prion protein modulates phagocytosis and inflammatory response. J. Leukoc. Biol. 2005, 77, 238–246. [Google Scholar] [CrossRef]
  127. Nitta, K.; Sakudo, A.; Masuyama, J.; Xue, G.; Sugiura, K.; Onodera, T. Role of cellular prion proteins in the function of macrophages and dendritic cells. Protein Pept. Lett. 2009, 16, 239–246. [Google Scholar] [CrossRef]
  128. Uraki, R.; Sakudo, A.; Ando, S.; Kitani, H.; Onodera, T. Enhancement of phagocytotic activity by prion protein in PrP-deficient macrophage cells. Int. J. Mol. Med. 2010, 26, 527–532. [Google Scholar] [CrossRef] [PubMed]
  129. Jeon, J.W.; Park, B.C.; Jung, J.G.; Jang, Y.S.; Shin, E.C.; Park, Y.W. The Soluble Form of the Cellular Prion Protein Enhances Phagocytic Activity and Cytokine Production by Human Monocytes Via Activation of ERK and NF-kappaB. Immune Netw. 2013, 13, 148–156. [Google Scholar] [CrossRef]
  130. Salvesen, Ø.; Reiten, M.R.; Espenes, A.; Bakkebø, M.K.; Tranulis, M.A.; Ersdal, C. LPS-induced systemic inflammation reveals an immunomodulatory role for the prion protein at the blood-brain interface. J. Neuroinflamm. 2017, 14, 106. [Google Scholar] [CrossRef] [Green Version]
  131. Pradines, E.; Loubet, D.; Mouillet-Richard, S.; Manivet, P.; Launay, J.M.; Kellermann, O.; Schneider, B. Cellular prion protein coupling to TACE-dependent TNF-α shedding controls neurotransmitter catabolism in neuronal cells. J. Neurochem. 2009, 110, 912–923. [Google Scholar] [CrossRef] [PubMed]
  132. Stella, R.; Massimino, M.L.; Sandri, M.; Sorgato, M.C.; Bertoli, A. Cellular prion protein promotes regeneration of adult muscle tissue. Mol. Cell. Biol. 2010, 30, 4864–4876. [Google Scholar] [CrossRef] [Green Version]
  133. Ezpeleta, J.; Boudet-Devaud, F.; Pietri, M.; Baudry, A.; Baudouin, V.; Alleaume-Butaux, A.; Dagoneau, N.; Kellermann, O.; Launay, J.M.; Schneider, B. Protective role of cellular prion protein against TNFα-mediated inflammation through TACE α-secretase. Sci. Rep. 2017, 7, 7671. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  134. Ding, T.; Zhou, X.; Kouadir, M.; Shi, F.; Yang, Y.; Liu, J.; Wang, M.; Yin, X.; Yang, L.; Zhao, D. Cellular prion protein participates in the regulation of inflammatory response and apoptosis in BV2 microglia during infection with Mycobacterium bovis. J. Mol. Neurosci. 2013, 51, 118–126. [Google Scholar] [CrossRef] [PubMed]
  135. Shi, F.; Yang, L.; Kouadir, M.; Yang, Y.; Ding, T.; Wang, J.; Zhou, X.; Yin, X.; Zhao, D. Prion protein participates in the regulation of classical and alternative activation of BV2 microglia. J. Neurochem. 2013, 124, 168–174. [Google Scholar] [CrossRef] [PubMed]
  136. Pinheiro, L.P.; Linden, R.; Mariante, R.M. Activation and function of murine primary microglia in the absence of the prion protein. J. Neuroimmunol. 2015, 286, 25–32. [Google Scholar] [CrossRef] [Green Version]
  137. Mariante, R.M.; Nóbrega, A.; Martins, R.A.P.; Areal, R.B.; Bellio, M.; Linden, R. Neuroimmunoendocrine regulation of the prion protein in neutrophils. J. Biol. Chem. 2012, 287, 35506–35515. [Google Scholar] [CrossRef] [Green Version]
  138. Wang, M.; Zhao, D.; Yang, Y.; Liu, J.; Wang, J.; Yin, X.; Yang, L.; Zhou, X. The cellular prion protein negatively regulates phagocytosis and cytokine expression in murine bone marrow-derived macrophages. PLoS ONE 2014, 9, e102785. [Google Scholar] [CrossRef]
  139. Mallucci, G.R.; Ratté, S.; Asante, E.A.; Linehan, J.; Gowland, I.; Jefferys, J.G.R.; Collinge, J. Post-natal knockout of prion protein alters hippocampal CA1 properties, but does not result in neurodegeneration. EMBO J. 2002, 21, 202–210. [Google Scholar] [CrossRef]
  140. Nuvolone, M.; Kana, V.; Hutter, G.; Sakata, D.; Mortin-Toth, S.M.; Russo, G.; Danska, J.S.; Aguzzi, A. SIRPα polymorphisms, but not the prion protein, control phagocytosis of apoptotic cells. J. Exp. Med. 2013, 210, 2539–2552. [Google Scholar] [CrossRef]
  141. Saura, J.; Tusell, J.M.; Serratosa, J. High-Yield Isolation of Murine Microglia by Mild Trypsinization. Glia 2003, 44, 183–189. [Google Scholar] [CrossRef] [PubMed]
  142. Peggion, C.; Stella, R.; Chemello, F.; Massimino, M.L.; Arrigoni, G.; Cagnin, S.; Biancotto, G.; Franchin, C.; Sorgato, M.C.; Bertoli, A. The Prion Protein Regulates Synaptic Transmission by Controlling the Expression of Proteins Key to Synaptic Vesicle Recycling and Exocytosis. Mol. Neurobiol. 2019, 56, 3420–3436. [Google Scholar] [CrossRef] [PubMed]
  143. Vignali, D.A.A. Multiplexed particle-based flow cytometric assays. J. Immunol. Methods 2000, 243, 243–255. [Google Scholar] [CrossRef]
Ijms 21 07765 g001 550
Figure 1. The GPI-anchored cell surface TLR co-receptor CD14 is upregulated in microglial cells during prion infection, while CD14 genetic deletion blocks (T-arrow) CD14 upregulation and prolongs survival in prion infected knock-out mice (red × symbol).
Figure 1. The GPI-anchored cell surface TLR co-receptor CD14 is upregulated in microglial cells during prion infection, while CD14 genetic deletion blocks (T-arrow) CD14 upregulation and prolongs survival in prion infected knock-out mice (red × symbol).
Ijms 21 07765 g001
Ijms 21 07765 g002 550
Figure 2. Exposure to PrPSc or prion mimetics (e.g., the fibrillogenic 106-126 peptide) activates microglial cells promoting the secretion of either pro-inflammatory IL-1 (resulting in enhanced neurodegenerative cues), or anti-inflammatory IL-10 (exerting neuroprotective effects).
Figure 2. Exposure to PrPSc or prion mimetics (e.g., the fibrillogenic 106-126 peptide) activates microglial cells promoting the secretion of either pro-inflammatory IL-1 (resulting in enhanced neurodegenerative cues), or anti-inflammatory IL-10 (exerting neuroprotective effects).
Ijms 21 07765 g002
Ijms 21 07765 g003 550
Figure 3. (A) LPS treatment increased PrPC expression in primary murine microglial cells. Primary microglial cells were isolated from PrP-KO mice with an almost pure FVB genotype (strain F10), and PrP-Tg mice in which physiologic PrPC expression levels were rescued over the F10 genetic background (strain Tg46) [139]. Both mouse lines were kindly provided by the MRC Prion Unit at UCL, London, UK. Cell cultures were established and maintained as described previously. [141]. All procedures were performed in compliance with European and Italian (D.L. 26/2014) laws concerning the care and use of laboratory animals and were approved by the Italian Ministry of Health, and by the Ethical Committee of the University of Padova (Authorization n. 743/2017-PR). Cells were cultured for 96 h in the absence (–) or in the presence (+) of lipopolysaccharide (LPS) (500 ng/mL), then cells were lysed, and extracted proteins were subjected to Western blot (WB), as described previously [142]. The left panel shows a representative WB, out of three biological replicates (i.e., different primary cell cultures), using a mouse monoclonal antibody to PrPC (clone 8H4, Abcam). A mouse monoclonal anti-α-Tubulin antibody (clone B-5-1-2, Sigma–Aldrich) also was used to check for the loading of equal protein amounts. The right panel reports the densitometric analysis of PrPC immuno-reactive bands in control (–) or LPS-treated cells (+), normalized to the corresponding signal of α-Tubulin. Data were then normalized to PrPC amounts in control cells. Data show that LPS addition significantly increases PrPC expression levels in PrP-Tg-derived microglial cells. As expected, no PrPC reactive band was observed in PrP-KO samples, confirming the specificity of the immunosignal. Values are expressed as mean ± standard error of the mean (SEM), n = 3. Statistical analysis was based on unpaired two-tailed Student’s t-test. ** p-value < 0.01. (B) PrPC regulates the production of cytokines by primary murine microglial cells upon LPS treatment. Microglial cells (as in panel A) were maintained in culture in the presence of LPS (500 ng/mL) for 24 h or 96 h. The amounts of selected cytokines (IL-1β, IL-10, IFN-γ, IL-6 and TNF-α) released in the culture medium were quantified by a customized enzyme-linked immunosorbent assay (ELISA) detection kit. The assays were performed in 96-well filter plates by Multiplexed Luminex®-based immunoassay, as previously described [143], following the manufacturer’s instructions, and analyzed in the BioPlex 200 instrument (BioRad). The table reports the cytokine amounts (estimated from a standard curve using a fifth-order polynomial equation and adjusted for the dilution factor), normalized to the total protein content (determined by a Lowry assay kit (Sigma-Aldrich)) in the corresponding cell lysates and then subtracted for the values obtained in the absence of LPS. Values are expressed as mean ± SEM, n = 5 (biological cultures for each genotype). Statistical analysis was based on unpaired two-tailed Student’s t-test. * p-value < 0.05.
Figure 3. (A) LPS treatment increased PrPC expression in primary murine microglial cells. Primary microglial cells were isolated from PrP-KO mice with an almost pure FVB genotype (strain F10), and PrP-Tg mice in which physiologic PrPC expression levels were rescued over the F10 genetic background (strain Tg46) [139]. Both mouse lines were kindly provided by the MRC Prion Unit at UCL, London, UK. Cell cultures were established and maintained as described previously. [141]. All procedures were performed in compliance with European and Italian (D.L. 26/2014) laws concerning the care and use of laboratory animals and were approved by the Italian Ministry of Health, and by the Ethical Committee of the University of Padova (Authorization n. 743/2017-PR). Cells were cultured for 96 h in the absence (–) or in the presence (+) of lipopolysaccharide (LPS) (500 ng/mL), then cells were lysed, and extracted proteins were subjected to Western blot (WB), as described previously [142]. The left panel shows a representative WB, out of three biological replicates (i.e., different primary cell cultures), using a mouse monoclonal antibody to PrPC (clone 8H4, Abcam). A mouse monoclonal anti-α-Tubulin antibody (clone B-5-1-2, Sigma–Aldrich) also was used to check for the loading of equal protein amounts. The right panel reports the densitometric analysis of PrPC immuno-reactive bands in control (–) or LPS-treated cells (+), normalized to the corresponding signal of α-Tubulin. Data were then normalized to PrPC amounts in control cells. Data show that LPS addition significantly increases PrPC expression levels in PrP-Tg-derived microglial cells. As expected, no PrPC reactive band was observed in PrP-KO samples, confirming the specificity of the immunosignal. Values are expressed as mean ± standard error of the mean (SEM), n = 3. Statistical analysis was based on unpaired two-tailed Student’s t-test. ** p-value < 0.01. (B) PrPC regulates the production of cytokines by primary murine microglial cells upon LPS treatment. Microglial cells (as in panel A) were maintained in culture in the presence of LPS (500 ng/mL) for 24 h or 96 h. The amounts of selected cytokines (IL-1β, IL-10, IFN-γ, IL-6 and TNF-α) released in the culture medium were quantified by a customized enzyme-linked immunosorbent assay (ELISA) detection kit. The assays were performed in 96-well filter plates by Multiplexed Luminex®-based immunoassay, as previously described [143], following the manufacturer’s instructions, and analyzed in the BioPlex 200 instrument (BioRad). The table reports the cytokine amounts (estimated from a standard curve using a fifth-order polynomial equation and adjusted for the dilution factor), normalized to the total protein content (determined by a Lowry assay kit (Sigma-Aldrich)) in the corresponding cell lysates and then subtracted for the values obtained in the absence of LPS. Values are expressed as mean ± SEM, n = 5 (biological cultures for each genotype). Statistical analysis was based on unpaired two-tailed Student’s t-test. * p-value < 0.05.
Ijms 21 07765 g003
Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Share and Cite

MDPI and ACS Style

Peggion, C.; Stella, R.; Lorenzon, P.; Spisni, E.; Bertoli, A.; Massimino, M.L. Microglia in Prion Diseases: Angels or Demons? Int. J. Mol. Sci. 2020, 21, 7765. https://doi.org/10.3390/ijms21207765

AMA Style

Peggion C, Stella R, Lorenzon P, Spisni E, Bertoli A, Massimino ML. Microglia in Prion Diseases: Angels or Demons? International Journal of Molecular Sciences. 2020; 21(20):7765. https://doi.org/10.3390/ijms21207765

Chicago/Turabian Style

Peggion, Caterina, Roberto Stella, Paolo Lorenzon, Enzo Spisni, Alessandro Bertoli, and Maria Lina Massimino. 2020. "Microglia in Prion Diseases: Angels or Demons?" International Journal of Molecular Sciences 21, no. 20: 7765. https://doi.org/10.3390/ijms21207765

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Metrics

Back to TopTop