Michael Burwinkel

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.

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.

Prion infection of mice transgenic for human APPSwe: increased accumulation of cortical formic acid extractable Aβ(1–42) and rapid scrapie disease development

Neuropathological, epidemiological and experimental data indicate a potential interrelationship between Alzheimers disease and prion diseases. Proteolytic processing of amyloid precursor protein (APP) by β‐secretase was recently suggested to be controlled by prion protein expression. Here, we characterized the prion infection of Tg2576 mice, which overexpress the human APPSwe protein. Prion infection of Tg2576‐mice led to an early death of the animals, which was preceded by a relatively short symptomatic stage. However, disease‐associated gliosis and deposition of misfolded prion protein PrPSc were identical in infected Tg2576‐mice and non‐transgenic littermate controls. To analyze the effect of prion infection on APP processing and generation of β‐amyloid we determined cortical levels of SDS‐ and formic acid (FA)‐extractable forms of β‐amyloid (1–40) and (1–42) by ELISA. Formic acid‐extractable Aβ (1–42) levels were 10‐fold higher in infected versus uninfected Tg2576 mice whereas other forms of Aβ were essentially unaffected by the prion infection. Hence, the experimental model demonstrates that a prion infection of the CNS promotes selectively formation of FA‐extractable Aβ(1–42) in Tg2576 mice.

Unchanged scrapie pathology in brain tissue of tyrosine kinase Fyn-deficient mice.

Fyn is a 59-kDa member of the Src family of tyrosine kinases synthesized on cytosolic polysomes and then targeted to the plasma membrane where it clusters in caveolae-like membrane microdomains. The cellular isoform of the prion protein (PrP) has also been identified to be a caveolar constituent and to participate in signal transduction events concerning cell survival and differentiation via recruitment of Fyn. We studied the scrapie infection of mice deficient for Fyn (Fyn–/–) to clarify the role of Fyn in an in vivo model of transmissible spongiforme encephalopathies. Fyn–/– mice died on average 9 days earlier than wild-type control mice, but no differences were seen regarding activation of astrocytes, vacuolization of the neuropil, and accumulation of misfolded prion protein. The experimental model suggests that a deficiency for Fyn is detrimental in prion diseases, although it has no major effect on the clinical course of an experimental prion infection of the CNS.

Ablation of CCAAT/Enhancer-Binding Protein Delta (C/EBPD): Increased Plaque Burden in a Murine Alzheimer’s Disease Model

Alzheimer’s disease (AD) and prion diseases carry a significant inflammatory component. The astrocytic overexpression of CCAAT/enhancer-binding protein delta (C/EBPD) in prion- and AD-affected brain tissue prompted us to study the role of this transcription factor in murine model systems of these diseases. Ablation of C/EBPD had neither in the AD model (APP/PS1double transgenic mice) nor in the prion model (scrapie-infected C57BL/6 mice) an influence on overt clinical symptoms. Moreover, the absence of C/EBPD did not affect the extent of the disease-related gliosis. However, C/EBPD-deficient APP/PS1 double transgenic mice displayed significantly increased amyloid beta (Abeta) plaque burdens while amyloid precursor protein (APP) expression and expression of genes involved in beta amyloid transport and turnover remained unchanged. Gene expression analysis in mixed glia cultures demonstrated a strong dependency of complement component C3 on the presence of C/EBPD. Accordingly, C3 mRNA levels were significantly lower in brain tissue of C/EBPD-deficient mice. Vice versa, C3 expression in U-373 MG cells increased upon transfection with a C/EBPD expression vector. Taken together, our data indicate that a C/EBPD-deficiency leads to increased Abeta plaque burden in AD model mice. Furthermore, as shown in vivo and in vitro, C/EBPD is an important driver of the expression of acute phase response genes like C3 in the amyloid-affected CNS.