Prion protein and Aβ-related synaptic toxicity impairment

Alzheimer's disease (AD), the most common neurodegenerative disorder, goes along with extracellular amyloid-β (Aβ) deposits. The cognitive decline observed during AD progression correlates with damaged spines, dendrites and synapses in hippocampus and cortex. Numerous studies have shown that Aβ oligomers, both synthetic and derived from cultures and AD brains, potently impair synaptic structure and functions. The cellular prion protein (PrPC) was proposed to mediate this effect. We report that ablation or overexpression of PrPC had no effect on the impairment of hippocampal synaptic plasticity in a transgenic model of AD. These findings challenge the role of PrPC as a mediator of Aβ toxicity

Engulfment of cerebral apoptotic bodies controls the course of prion disease in a mouse strain–dependent manner

Progressive accumulation of PrPSc, a hallmark of prion diseases, occurs when conversion of PrPC into PrPSc is faster than PrPSc clearance. Engulfment of apoptotic bodies by phagocytes is mediated by Mfge8 (milk fat globule epidermal growth factor 8). In this study, we show that brain Mfge8 is primarily produced by astrocytes. Mfge8 ablation induced accelerated prion disease and reduced clearance of cerebellar apoptotic bodies in vivo, as well as excessive PrPSc accumulation and increased prion titers in prion-infected C57BL/6 × 129Sv mice and organotypic cerebellar slices derived therefrom. These phenotypes correlated with the presence of 129Sv genomic markers in hybrid mice and were not observed in inbred C57BL/6 Mfge8−/− mice, suggesting the existence of additional strain-specific genetic modifiers. Because Mfge8 receptors are expressed by microglia and depletion of microglia increases PrPSc accumulation in organotypic cerebellar slices, we conclude that engulfment of apoptotic bodies by microglia may be an important pathway of prion clearance controlled by astrocyte-borne Mfge8

Prion disease in brain slice cultures

Prion diseases or transmissible spongiform encephalopathies (TSEs) are lethal infectious diseases affecting humans and a variety of animal species. The prevailing hypothesis suggests that the infectious disease-causing particle termed “the prion” consist of a misfolded form of the normal cellular prion protein (PrPC) that has been converted into a disease-associated form (PrPSc). It is believed that PrPSc, either as a monomer or as a highly order aggregate, catalyses the conversion of PrPC into PrPSc and thus becomes self-propagating (i.e. infectious). The molecular composition of PrPSc, the mechanism of conversion and how the conversion leads to pathology is not clear. None-the-less, all familiar TSEs display mutations within the Prnp locus and ablation of Prnp confers resistance to TSEs in mice. In the main part of the thesis I investigated the role of microglia in prion disease. In order to do so, I first established a novel assay allowing for prion replication in organotypic cerebellar slice cultures. These cultures are thin slices of living cerebellar brain tissue that can be kept alive for up to 26 weeks ex vivo. In prion-infected cultures prepared from mice overexpressing PrPC, PrPSc could be recovered as early as 3 weeks post-inoculation. I transmitted the prion-infected slices to a secondary prion transmission assay that allowed us to determine the amount of infectivity generated within the slices. The amount of infectivity recovered from organotypic cerebellar slices 5 weeks post-inoculation was equal to what could be recovered from a terminally scrapie-sick mouse 150 days post-inoculation. No infectivity was recovered from prion-exposed Prnpo/o slices. I could show that the localization and the replication properties of PrPSc in our cultures behaved similar to what is seen in vivo. Next, I utilized a transgenic mouse model (CD11b-HSVTK) that allowed for the conditional depletion of microglia. Microglia could be ablated from CD11b-HSVTK slices within 2 weeks with no obvious consequences for other neural cells populations. A 15-fold increase in prion infectivity was observed 30 days post-inoculation in prion-infected microglia-depleted slices relative to non-depleted slices. This increase in prion-infectivity could blocked by adding back exogenous macrophages to microglia-depleted tissue. In addition, depletion of microglia allowed for an infection of the tissue with an amount of prions that would normally not lead to an infection. Our results strongly suggests that microglia play a role in inhibiting prion replication and could represent an important central nervous system (CNS) anti-prion defense. For the second part of the thesis I developed a novel method to distinguish between the inoculum used to infect cells (input) and the newly synthesized prions that can be recovered from prion-infected cells (output). This technique allows for the study of prion-replication in non-diving cells without the interference of persisting residual inoculum, which could interfere with the interpretation of the experiment. I use homogenates of prion-infected C4/C4 mice that express a deletion mutant of the prion protein lacking the octa-repeat region (ΔPrP32-93). Brain homogenates from these mice can be used to infect cultures expressing the full-length prion protein. PrPSc is subsequently detected with an antibody recognizing a linear epitope in the octa-repeat region of PrPC. This epitope is absent in the inoculum, but present in the newly generated PrPSc, allowing for a specific detection of de novo replicated PrPSc. This allows for the investigation of which post-mitotic or slowly dividing cells can be prion-infected. I show with the technique that cerebellar granule neurons and astrocytes can be infected. I also show that I cannot infect microglia, most likely due to their low expression of PrPC. I am currently testing which other CNS or peripheral nervous system (PNS)-derived cells can support prion replication. Zusammenfassung Prionenerkrankungen oder transmissible spongiforme Enzephalopathien (TSE) sind tödlich verlaufende, übertragbare Erkrankungen, die eine Vielzahl verschiedener Spezies befallen können. Eine vorherrschende Hypothese besagt, dass das infektiöse, die Erkrankung auslösende Agens – auch als „Prion“ bezeichnet – aus fehlgefaltetem normalem zellulärem Prionprotein (PrPC) besteht, welches in die Erkrankungs- assoziierte Form (PrPSc) konvertiert worden ist. Man nimmt an, dass PrPSc, entweder als Monomer oder als Aggregat hoher Organisationsstufe, die Konversion von PrPC zu PrPSc katalysiert und auf diese Weise im infektiösen Sinne selbst-replizierend wird. Weder die molekulare Zusammensetzung von PrPSc und der Mechanismus der Konversion, noch der Zusammenhang zwischen der Umfaltung und den zu beobachtenden pathologischen Veränderungen sind bekannt. Allerdings weisen alle familiären Fälle von TSE Mutationen im Prnp Lokus auf und die genetische Ausschaltung („knockout“) von Prnp bewirkt in Mäusen eine Resistenz gegen TSE. Der Hauptteil der Doktorarbeit befasst sich mit der Rolle von Mikrogliazellen in Prionerkrankungen. Als Vorraussetzung hierfür musste zunächst ein neuer Assay entwickelt werden, um die Replikation der Prionen in organotypischen zerebellären Gewebskulturen („slice cultures“) zu untersuchen. Bei diesen Kulturen handelt es sich um dünne Schnitte aus lebendem zerebellärem Gewebe, welches ex vivo bis zu 26 Wochen am Leben erhalten werden kann. In prioneninfizierten slice cultures aus PrPC überexprimieren Mäusen, konnte PrPSc bereits 3 Wochen nach Inokulation nachgewiesen werden. Die mit Prionen infizierten slice cultures wurden nachfolgend in einem weiteren Prionen Transmissionsexperiment untersucht, welches erlaubt die Menge an in den Kulturen erzeugter Infektiosität zu bestimmen. Die Menge an Infektiosität, welche nach 5 Wochen in den slice cultures nachgewiesen werden konnte, entsprach derjenigen einer terminal an Scrapie erkrankten Maus 150 Tage nach Inokulation. Dagegen fand sich keine Infektiosität in Prion exponierten Kulturen aus Prnpo/o Mäusen. Es zeigte sich, dass PrPSc in den Kulturen eine ähnliche räumliche Verteilung und Replikationseigenschaften wie in der in vivo Situation aufwies. Im weiteren Verlauf wurde ein transgenes Mausmodell (CD11b-HSVTK) verwendet, in welchem eine getriggerte Depletion von Mikrogliazellen möglich ist. Mikrogliazellen konnten aus Kulturen von CD11b-HSVTK Mäusen innerhalb von zwei Wochen ohne offensichtliche negative Konsequenzen für andere (neuronale) Zellpopulationen depletiert werden. In infizierten Mikroglia-depletierten Kulturen konnte 30 Tage nach Inokulation – im Vergleich zu nicht depletierten Kulturen – ein 15-facher Anstieg der Prion-Infektiosität beobachtet werden. Der Anstieg der Prion- Infektiosität konnte durch Zugabe von exogenen Makrophagen aufgehoben werden. Zusätzlich konnten durch die Depletion von Mikrogliazellen die Kulturen schon mit einer Prionendosis infiziert werden, die normalerweise nicht für eine Infektion ausreichen würde. Die Ergebnisse legen nahe, dass Mikrogliazellen an einer Hemmung der Replikation von Prionen beteiligt sind und einen wichtigen Abwehrmechanismus des ZNS gegen Prionen darstellen. Im zweiten Teil meiner Doktorarbeit habe wurde eine neue Methode entwickelt, um zwischen dem für die Zellinfektion benutzten Inokulum (Input) und den neu synthetisierten Prionen, die in infizierten Zellen nachgewiesen werden können (Output), zu unterscheiden. Diese Technik erlaubt es, die Replikation von Prionen in postmitotischen Zellen ohne den Einfluss von noch vorhandenem restlichem Inokulum, welches die Interpretation des Experimentes beeinträchtigen könnte, zu untersuchen. Hierzu wurden Homogenate von Prionen infizierten C4/C4 Mäusen, welche eine Deletionsmutante des Prionproteins ohne die Oktarepeatregion (ΔPrP32- 93) exprimieren, verwendet. Hirnhomogenate von diesen Mäusen können zur Infektion von Kulturen benutzt werden, welche das Volllängen Prionprotein exprimieren. In der Folge kann PrPSc mit einem Antikörper detektiert werden , der gegen ein lineares Epitop in der Oktarepeatregion von PrPC gerichtet ist. Dieses Epitop fehlt im Inokulum, ist allerdings im neu entstandenen PrPSc enthalten, was einen spezifischen Nachweis von de novo erzeugtem PrPSc ermöglicht. Somit ist es möglich zu untersuchen, welche postmitotischen oder sich langsam teilenden Zellen mit Prionen infizierbar sind. Mit dieser Technik konnte gezeigt werden, dass Neuronen der Granularzellschicht des Kleinhirnkortex und Astrozyten mit Prionen infiziert werden können. Ausserdem wurde nachweisen, dass Mikrogliazellen nicht infizierbar sind, was am wahrscheinlichsten an den geringen Expressionsspiegeln von PrPC in diesem Zelltyp liegt. Gegenwärtig wird untersucht, welche weiteren Zellen des ZNS oder des peripheren Nervensystems (PNS) die Replikation von Prionen unterstützen können

Defined inflammatory states in astrocyte cultures: correlation with susceptibility towards CD95-driven apoptosis

A complete cytokine mix (CCM) or its individual components tumour necrosis factor-alpha (TNF-α), interleukin-1beta (IL-1β) and interferon-gamma (IFN-γ) were used to switch resting murine astrocytes to reactive states. The transformation process was characterized by differential up-regulation of interleukin-6 (IL-6), cyclooxygenase-2 (COX-2) and inducible nitric oxide synthetase (iNOS) mRNA and protein and a subsequent release of prostaglandin E2, nitric oxide (NO) and IL-6. Both CD95L and anti-CD95 antibodies triggered caspase activation followed by apoptotic death in fully pro-inflammatory astrocytes, whereas resting cells were totally resistant. Two other death-inducing ligands, TNF and TNF-related apoptosis-inducing ligand (TRAIL) did not induce apoptosis in reactive astrocytes. The switch in astrocyte sensitivity was accompanied by up-regulation of caspase-8 and CD95 as well as the capacity to recruit Fas-associated death domain (FADD) to the activated death receptor complex. Neither CD95-mediated death, nor other inflammatory parameters were affected by inhibition of iNOS or COX, respectively. Accordingly, IFN-γ was absolutely essential for up-regulation of iNOS, but not for the switch in apoptosis sensitivity. In contrast, p38 kinase activity was identified as an important controller of both the inflammatory reaction and apoptosis both in astrocytes stimulated with CCM and in glia exposed to TNF and IL-1 only

Modification of apoptosis-related genes and CD95 signaling in cytokine-treated astrocytes

Inflammatory activation of astrocytes with a complete cytokine mix consisting of tumor necrosis factor, interleukin-1 and interferon-gamma renders these otherwise resistant cells highly susceptible to cell death induction via the CD95 pathway. In dying cells, we observed several classical apoptotic features such as chromatin condensation and cytoplasmic blebbing. These events were however quickly followed by a rupture of the cell membrane. For a screen of the transcriptional changes taking place during the transformation from a CD95L-resistant to a CD95L-sensitive cell, we employed a small custom-spotted oligonucleotide microarray. The significantly regulated mRNA species were then further analyzed over a 24 h period by quantitative PCR. We observed a complex pattern of transcriptional regulations showing changes of pro-apoptotic genes (cd95, caspase-8, bid, bak, caspase-11), as well as anti-apoptotic genes (c-flip, iap-1, iap-2/3, bcl-2). Since inflammatory astrocyte sensitization increased linearly with the time of cytokine-treatment the anti-apoptotic genes never seemed to be able to take over a dominating role in this model. Finally, the response of activated astrocytes to CD95 stimulation was compared with several other death-inducing stimuli. Cells became also more sensitive towards the classical apoptosis inducer staurosporine, but not towards necrotic stimuli such as H2O2 and N-Methyl-N-nitro-N-nitrosoguanidine

Specific Modulation of Astrocyte Inflammation by Inhibition of Mixed Lineage Kinases with CEP-1347

Inflammatory conversion of murine astrocytes correlates with the activation of various MAPK, and inhibition of terminal MAPKs like JNK or p38 dampens the inflammatory reaction. Mixed lineage kinases (MLKs), a family of MAPK kinase kinases, may therefore be involved in astrocyte inflammation. In this study, we explored the effect of the MLK inhibitors CEP-1347 and CEP-11004 on the activation of murine astrocytes by either TNF plus IL-1 or by a complete cytokine mix containing additional IFN-{gamma}. The compounds blocked NO-, PG-, and IL-6 release with a median inhibitory concentration of ~100 nM. This activity correlated with a block of the JNK and the p38 pathways activated in complete cytokine mix-treated astrocytes. Although CEP-1347 did not affect the activation of NF-{kappa}B, it blocked the expression of cyclooxygenase-2 and inducible NO synthase at the transcriptional level. Quantitative transcript profiling of 17 inflammation-linked genes revealed a specific modulation pattern of astrocyte activation by MLK inhibition, for instance, characterized by up-regulation of the anti-stress factors inhibitor of apoptosis protein-2 and activated transcription factor 4, no effect on manganese superoxide dismutase and caspase-11, and down-regulation of major inflammatory players like TNF, GM-CSF, urokinase-type plasminogen activator, and IL-6. In conclusion, MLK inhibitors like CEP-1347 are highly potent astrocyte immune modulators with a novel spectrum of activity

Molecular basis for detection of invading pathogens in the brain

Classical immunology textbooks have described the central nervous system as an immune-privileged site, i.e., as devoid of inflammatory and host-vs.-graft immunoreactions. This view has been refined, since we now know that hematopoietic cells infiltrate the CNS under certain circumstances and that CNS-resident cells are capable of launching an innate immune response. Microglia cells express an extensive repertoire of pattern-recognition receptors and act as sentinels surveilling the CNS for possible damage or infection. Astrocytes are the most abundant cell type in the brain, and they are capable of launching a strong supportive innate immune response. Novel findings show that both astrocytes and, surprisingly, even neurons express pattern-recognition receptors. Activation of these receptors leads to a functional response, indicating that cells other than microglia are capable of initiating a primary innate immune response against CNS-invading pathogens. Here, we put these findings into context with what has been learned from recent in vitro and in vivo experiments about the initiation of an innate immune response in the brain

The inflammatory transcriptome of reactive murine astrocytes and implications for their innate immune function

Upon injury, astrocytes assume an activated state associated with the release of inflammatory mediators. To model this, we stimulated murine primary astrocytes with a complete inflammatory cytokine mix consisting of TNF-α, IL-1β and IFN-γ. We analysed the transcriptional response of 480 genes at 4 and 16 h after stimulation on a chip designed to give a representative overview over the inflammation-relevant part of the transcriptome of macrophage-like cells. The list of the 182 genes found to be significantly regulated in astrocytes revealed an intriguing co-ordinate regulation of genes linked to the biological processes of antiviral/antimicrobial defence, antigen presentation and facilitation of leucocyte invasion. The latter group was characterized by very high up-regulations of chemokine genes. We also identified regulations of a thymidylate kinase and an interferon-regulated protein with a tetratricopeptide motive, both up to now only known from macrophages. The transcriptional regulations were confirmed on the protein level by a proteomic analysis. These findings taken together suggest that activated astrocytes in brain behave similarly in many respects to inflamed macrophages in the periphery