Constanze Riemer

Lithium induces clearance of protease resistant prion protein in prion-infected cells by induction of autophagy.

Lithium is used for several decades to treat manic‐depressive illness (bipolar affective disorder). Recently, it was found that lithium induces autophagy, thereby promoting the clearance of mutant huntingtin and α‐synucleins in experimental systems. We show here for the first time that lithium significantly reduces the amount of pathological prion protein (PrPSc) in prion‐infected neuronal and non‐neuronal cultured cells by inducing autophagy. Treatment of prion‐infected cells with 3‐methyladenine, a potent inhibitor of autophagy, counteracted the anti‐prion effect of lithium, demonstrating that induction of autophagy mediates degradation of PrPSc. Co‐treatment with lithium and rapamycin, a drug widely used to induce autophagy, had an additive effect on PrPSc clearance compared to treatment with either drug alone. In addition, we provide evidence that the ability to reduce PrPSc and to induce autophagy is common for diverse lithium compounds, not only for the drug lithium chloride, usually administered in clinical therapy. Furthermore, we show here that besides reduction of PrPSc‐aggregates, lithium‐induced autophagy also slightly reduces the levels of cellular prion protein. Limiting the substrate available for conversion of cellular prion protein into PrPSc may provide an additional mechanism for reduction of PrPSc by lithium‐induced autophagy.

Identification of Upregulated Genes in Scrapie-Infected Brain Tissue

ABSTRACT The pathogenesis of scrapie, and of neurodegenerative diseases in general, is still insufficiently understood and is therefore being intensely researched. There is abundant evidence that the activation of glial cells precedes neurodegeneration and may thus play an important role in disease development and progression. The identification of genes with altered expression patterns in the diseased brain may provide insight on the molecular level into the process which ultimately leads to neuronal loss. Differentially expressed genes in scrapie-infected brain tissue were enriched by the suppression subtractive hybridization technique, molecularly cloned, and further characterized. Northern blotting and nucleotide sequencing confirmed the identities of 19 upregulated genes, 11 of which were unknown to be affected by scrapie. A considerable number of these 19 genes, namely those encoding interferon-inducible protein 10 (IP-10), 2′,5′-oligo(A) synthetase, Mx protein, IIGP protein, major histocompatibility complex classes I and II, complement, and β2-microglobulin, were inducible by interferons (IFNs), suggesting that an IFN response is a possible mechanism of gene activation in scrapie. Among the newly found genes, that coding for 2′,5′-oligo(A) synthetase is of special interest because it could contribute to the apoptotic loss of neuronal cells via RNase L activation. In addition, upregulation of the chemokine IP-10 and B-lymphocyte chemoattractant mRNAs was seen at relatively early stages of the disease and was sustained throughout disease development.

Immunisation with a synthetic prion protein-derived peptide prolongs survival times of mice orally exposed to the scrapie agent

Several lines of evidence suggest that immunisations may be helpful in the prophylaxis and treatment of neurodegenerative amyloidoses like Alzheimers disease and prion infections. We used a synthetic prion protein-derived peptide (PrP105-125) and a recombinant PrP fragment (PrP90-230) as antigens for the active immunisation of mice, which were subsequently infected by dietary exposure to the scrapie agent. Immunisation with PrP105-125 prolonged the survival times significantly. In contrast, immunisation with PrP90-230 or adjuvants alone had no effect on the disease development. An epitope mapping of the antibodies raised against PrP90-230 revealed that reactivities against previously defined protective epitopes were either underrepresented or absent. These results point towards the possibility to prevent prion spread via the food chain by vaccinating humans or other species at risk to contract prion diseases.

Role of Interleukin-1 in Prion Disease-Associated Astrocyte Activation

Prion-induced chronic neurodegeneration has a substantial inflammatory component, and the activation of glia cells may play an important role in disease development and progression. However, the functional contribution of cytokines to the development of the gliosis in vivo was never systematically studied. We report here that the expression of interleukin-1beta (IL-1beta), IL-1beta-converting enzyme, and IL-1 receptor type 1 (IL-1RI) is up-regulated in a murine scrapie model. The scrapie-induced gliosis in IL-1RI(-/-) mice was characterized by an attenuated activation of astrocytes in the asymptomatic stage of the disease and a reduced expression of CXCR3 ligands. Furthermore, the accumulation of the misfolded isoform of the prion protein PrP(Sc) was significantly delayed in the IL-1RI(-/-) mice. These observations indicate that IL-1 is a driver of the scrapie-associated astrocytosis and possibly the accompanying amyloid deposition. In addition, scrapie-infected IL-1RI-deficient (IL-1RI(-/-)) mice showed a delayed disease onset and significantly prolonged survival times suggesting that an anti-inflammatory therapeutical approach to suppress astrocyte activation and/or glial IL-1 expression may help to delay disease onset in established prion infections of the central nervous system.

Green tea extracts interfere with the stress-protective activity of PrPC and the formation of PrPSc

A hallmark in prion diseases is the conformational transition of the cellular prion protein (PrPC) into a pathogenic conformation, designated scrapie prion protein (PrPSc), which is the essential constituent of infectious prions. Here, we show that epigallocatechin gallate (EGCG) and gallocatechin gallate, the main polyphenols in green tea, induce the transition of mature PrPC into a detergent‐insoluble conformation distinct from PrPSc. The PrP conformer induced by EGCG was rapidly internalized from the plasma membrane and degraded in lysosomal compartments. Isothermal titration calorimetry studies revealed that EGCG directly interacts with PrP leading to the destabilizing of the native conformation and the formation of random coil structures. This activity was dependent on the gallate side chain and the three hydroxyl groups of the trihydroxyphenyl side chain. In scrapie‐infected cells EGCG treatment was beneficial; formation of PrPSc ceased. However, in uninfected cells EGCG interfered with the stress‐protective activity of PrPC. As a consequence, EGCG‐treated cells showed enhanced vulnerability to stress conditions. Our study emphasizes the important role of PrPC to protect cells from stress and indicate efficient intracellular pathways to degrade non‐native conformations of PrPC.

Role of cytokines and chemokines in prion infections of the central nervous system

Prion infections of the central nervous system (CNS) are characterised by a reactive gliosis and the subsequent degeneration of neuronal tissue. The activation of glial cells, which precedes neuronal death, is likely to be initially caused by the deposition of misfolded, proteinase K‐resistant, isoforms (termed PrPres) of the prion protein (PrP) in the brain. Cytokines and chemokines released by PrPres‐activated glia cells may contribute directly or indirectly to the disease development by enhancement and generalisation of the gliosis and via cytotoxicity for neurons. However, the actual role of prion‐induced glia activation and subsequent cytokine/chemokine secretion in disease development is still far from clear. In the present work, we review our present knowledge concerning the functional biology of cytokines and chemokines in prion infections of the CNS.

Accelerated Prion Replication in, but Prolonged Survival Times of, Prion-Infected CXCR3−/− Mice

ABSTRACT Prion diseases have a significant inflammatory component. Glia activation, which is associated with increased production of cytokines and chemokines, may play an important role in disease development. Among the chemokines upregulated highly and early upregulated during scrapie infections are ligands of CXCR3. To gain more insight into the role of CXCR3 in a prion model, CXCR3-deficient (CXCR3−/−) mice were infected intracerebrally with scrapie strain 139A and characterized in comparison to similarly infected wild-type controls. CXCR3−/− mice showed significantly prolonged survival times of up to 30 days on average. Surprisingly, however, they displayed accelerated accumulation of misfolded proteinase K-resistant prion protein PrPSc and 20 times higher infectious prion titers than wild-type mice at the asymptomatic stage of the disease, indicating that these PrP isoforms may not be critical determinants of survival times. As demonstrated by immunohistochemistry, Western blotting, and gene expression analysis, CXCR3-deficient animals develop an excessive astrocytosis. However, microglia activation is reduced. Quantitative analysis of gliosis-associated gene expression alterations demonstrated reduced mRNA levels for a number of proinflammatory factors in CXCR3−/− compared to wild-type mice, indicating a weaker inflammatory response in the knockout mice. Taken together, this murine prion model identifies CXCR3 as disease-modifying host factor and indicates that inflammatory glial responses may act in concert with PrPSc in disease development. Moreover, the results indicate that targeting CXCR3 for treatment of prion infections could prolong survival times, but the results also raise the concern that impairment of microglial migration by ablation or inhibition of CXCR3 could result in increased accumulation of misfolded PrPSc.

Evaluation of drugs for treatment of prion infections of the central nervous system.

Prion diseases are fatal and at present there are neither cures nor therapies available to delay disease onset or progression in humans. Inspired in part by therapeutic approaches in the fields of Alzheimers disease and amyotrophic lateral sclerosis, we tested five different drugs, which are known to efficiently pass through the blood-brain barrier, in a murine prion model. Groups of intracerebrally prion-challenged mice were treated with the drugs curcumin, dapsone, ibuprofen, memantine and minocycline. Treatment with antibiotics dapsone and minocycline had no therapeutic benefit. Ibuprofen-treated mice showed severe adverse effects, which prevented assessment of therapeutic efficacy. Mice treated with low- but not high-dose curcumin and mice treated with memantine survived infections significantly longer than untreated controls (P<0.01). These results encourage further research efforts to improve the therapeutic effect of these drugs.

Rapid disease development in scrapie‐infected mice deficient for CD40 ligand

The inhibition of CD40–CD40L interaction‐mediated signalling was suggested as a therapeutic strategy for the treatment of Alzheimers disease. Conversely, CD40‐deficient neurons were reported to be more vulnerable to stress associated with ageing as well as nerve growth factor‐β and serum withdrawal. We studied the scrapie infection of CD40L‐deficient (CD40L−/−) mice to see whether ablation of the CD40L gene would be beneficial or detrimental in this model of a neurodegenerative amyloidosis. CD40L−/− mice died on average 40 days earlier than wild‐type control mice and exhibited a more pronounced vacuolation of the neuropil and an increased microglia activation. The experimental model indicates that a deficiency for CD40L is highly detrimental in prion diseases and reinforces the neuroprotective function of intact CD40–CD40L interactions. The stimulation of neuroprotective pathways may represent a possibility to delay therapeutically the disease onset in prion infections of the central nervous system.

Reduced Treg frequency in LFA-1-deficient mice allows enhanced T effector differentiation and pathology in EAE

The αLβ2‐integrin LFA‐1 (CD11a/CD18) is known as an important molecule for leukocyte migration. However, the precise role of LFA‐1 in the pathogenesis of EAE has so far remained unclear. We describe here the disease development in LFA‐1−/− mice compared with WT controls. Ablation of LFA‐1 resulted in more severe EAE with increased demyelination and increased numbers of myelin oligodendrocyte glycoprotein‐reactive CD4+ T cells in the CNS. However, the production of the pro‐inflammatory cytokines IL‐17 and IFN‐γ was unchanged on the level of antigen‐specific T cells. Interestingly, LFA‐1‐deficient mice showed a clearly reduced frequency of Treg in the inflamed CNS. Moreover, Treg counts in spleens and thymi of unimmunized LFA‐1−/− mice were lower in comparison to the WT controls, indicating an impairment of Treg generation. In combination, these results suggest a substantial role of LFA‐1 in Treg generation and subsequent expansion of effector T cells and highlight the importance of Treg in limiting EAE.