Interventional strategies against prion diseases

September 6, 2001 Nature Reviews Neuroscience 2, 745-749 by Adriano 
Aguzzi, et al.
Only a few years ago, the idea that transmissible spongiform encephalopathies could be treated pharmacologically would have met with considerable scepticism. Even now, there is no way to cure a patient or animal suffering from a manifest prion disease. But recent, exciting developments seem to indicate that immunological and pharmacological interventions could have some potential for the pre-exposure and post-exposure prophylaxis of prion diseases. Although it is unlikely that we will be able to cure the clinically overt stages of prion diseases in the foreseeable future, palliative and even life-prolonging interventions might no longer be confined to the realm of science fiction.

Prion diseases are inevitably fatal neurodegenerative conditions that affect humans and a wide variety of animals. diseases are also called transmissible spongiform encephalopathies (TSEs), underlining their infectious nature. Although fewer than 1% of reported cases of Creutzfeldt-Jakob disease (CJD) can be traced to a defined infectious source, the bovine spongiform encephalopathy (BSE) epizootic has highlighted prion-contaminated meat-and-bone meal as an efficient vector for bovine prion diseases. The , the most widely accepted hypothesis on the nature of the infectious agent that causes TSEs (the prion), predicates that it consists essentially of , an abnormally folded, protease-resistant, -sheet-rich isoform of a normal cellular protein termed (Ref. ; see glossary).

Interventional approaches to prion diseases can be classified as curative, palliative and prophylactic (either pre-exposure or post-exposure). Although none of these approaches has been shown to work in the clinical setting, several exciting developments have recently opened up new perspectives on potential therapeutic approaches to TSEs (). Table 1 | Rate-limiting steps and cellular targets in prion therapeutics and prophylaxis

Curative approaches

Prion diseases are invariably fatal: there is no evidence that any patients or experimental animals suffering from a clinically manifest TSE have ever been cured. Plausible reasons for this apparent irreversibility of prion-induced pathology become clear on the histopathological examination of the brains of patients suffering from the early stages of CJD. Such observations typically arise from biopsies taken by neurosurgeons for diagnostic purposes. In addition, one of us (A.A.) has carried out a post-mortem analysis of the brain of a patient with incipient CJD, who died early in the course of the disease from another cause. From these observations, it is clear that the brain suffers severe damage during the course of TSEs, including widespread cortical and subcortical neuronal loss, brisk astrogliosis and excessive microglial activation (). It would be unrealistic to expect that the extensive damage that is already visible in the early clinical phase of the disease could be easily reversed by pharmacological treatment.

Figure 1 | Neuropathological features of transmissible spongiform encephalopathies.

Histological and immunohistochemical analysis of frontal cortex samples from the brain of a patient who died of non-cerebral causes (upper row) and of a patient suffering from Creutzfeldt-Jakob disease (CJD; lower row). Brain sections were stained with haematoxylin-eosin (H-E, left panels), with antibodies against glial fibrillary acidic protein (GFAP, middle panels) and with antibodies against the prion protein (PrP, right panels). Neuronal loss and prominent spongiosis are visible in the H-E stain. Strong proliferation of reactive astrocytes (gliosis) and perivacuolar prion protein deposits are detectable in the GFAP and PrP immunostains of the CJD brain samples. Reproduced with permission from Ref. © 2001 Macmillan Magazines Ltd.

Replacement of damaged portions of the brain, through regeneration or transplantation, might be the only way to cure clinically overt prion disease. Although there is much optimism at present about the potential of stem cells to repair neuronal damage, it is difficult to conceive that stem cells could be used to replace a significant proportion of cortical neurons. One could speculate that there might be a phase during the course of prion infection that is characterized by reversible impairment of neural function - perhaps as a result of synaptic damage to neurons that are still able to re-sprout on the cessation of injury. Transgenic experiments involving conditional expression of a mutated huntingtin gene imply that, at least in the case of polyglutamine expansion diseases, this might be the case. However, similar experiments using switchable prion protein (Prnp) transgenes have not proved to be as clear.

Palliative interventions

A more realistic goal than curative therapy might be deceleration of the pace of the disease, which could prolong survival and improve quality of life, without eradicating the disease. The need for such an approach is particularly evident since the appearance of new-variant CJD (vCJD), which has affected more than 100 patients in Great Britain, France and Ireland, and which appears to result from the direct transmission of BSE to humans.

Most attempts at prion therapy have focused on causal intervention: inhibition of accumulation of the disease-associated prion protein, PrPSc. This assumes that the production and deposition of PrPSc is the crucial pathogenetic event in prion diseases - a suggestion for which there is now considerable evidence.

Much public debate has surrounded the recent publication that phenothiazine and acridine derivatives appear to suppress, to varying degrees, prion replication in cultured cells. Korth et al. found that chlorpromazine and quinacrine are effective in clearing PrPSc and prion infectivity from scrapie-infected neuroblastoma N2A cells. This agrees with previous findings that chlorpromazine marginally delays the incubation time of scrapie and that quinacrine can interfere with prion replication in neuroblastoma cells.

Clinical trials have now begun in the United States and the United Kingdom, in which quinacrine is administered to patients suffering from sporadic CJD or vCJD. Quinacrine is a well-known drug, for which a large body of clinical, pharmacokinetic and toxicological information is available. Given that CJD is invariably lethal, the 'compassionate treatment' of individual cases is certainly justifiable.

This might be true not only for patients with incipient sporadic CJD or vCJD, but also, importantly, for families suffering from hereditary prion diseases. The latter are autosomal-dominant diseases with typically high penetrance, invariably accompanied by mutations in the Prnp reading frame. Therefore, genetic diagnosis is possible at an early, presymptomatic stage. It would be exciting to discover that some of the proposed antiprion drugs could have therapeutic potential for individuals carrying these mutations, who at present - like those carrying pathogenic mutations in the amyloid precursor protein (APP) or TAU genes - have no option but to bear the Damocles' sword of late-onset neurodegeneration.

There is, however, another side to this argument: supporting evidence that quinacrine or chlorpromazine might be effective in vivo is almost non-existent. Many substances have proved to be effective in quenching the build-up of PrPSc and/or prion infectivity in in vitro systems: these include Congo red, amphotericin B, anthracycline derivatives, sulphated polyanions, pentosan polysulphate, soluble lymphotoxin- (LT) receptors (LTRs), porphyrins, branched polyamines and -sheet breaker peptides. Some of these substances interact directly with PrPSc aggregates and might disturb their geometry. It will be of interest to determine whether a similar mechanism applies to acridine and phenothiazine derivatives. The last word might not have been spoken on the practical usefulness of any of these compounds, but none has, so far, proved to be particularly effective for the therapy of overtly sick animals - let alone human patients. One problem is that even the more promising substances work only when administered concomitantly with the infectious agent, or very shortly thereafter, if at all. Some of these limitations could well be due to bioavailability and pharmacokinetics issues (including the ability to cross the blood-brain barrier), and it is hoped that derivatives and further developments might eventually overcome these problems. However, these facts should lead us to proceed cautiously when experimenting with prionostatic compounds in CJD patients before attempting to provide evidence that these compounds have any effect in experimental animals.

Post-exposure prophylaxis

Prion diseases typically have a very long latency period between infection and clinical manifestation; this is why these diseases were originally thought to be caused by 'slow viruses'. From the viewpoint of interventional approaches, this peculiarity might open a window of intervention after infection has occurred, but before brain damage begins. Prions spend much of this latency time in achieving neuroinvasion - the process of reaching the central nervous system after entering the body from peripheral sites, . During this process, little or no damage occurs to brain, and its interruption might therefore prevent neurodegeneration.

Some of the components of the neuroinvasion machinery have been identified: for example, specialized cells within the intestinal epithelium - M cells - mediate prion entry from the gastrointestinal lumen into the body. However, it has not been possible to manipulate the maturation or functional characteristics of M cells in the context of prion neuroinvasion, mainly because no genetic markers of M cells are known. We anticipate that this lack of tools could be obviated in the near future. Signalling through the tumour necrosis factor (TNF) receptor I (TNFR1) and the LTR is required for the development and maintenance of mature (FDCs), which are thought to be essential for prion replication and for the accumulation of disease-associated PrPSc within . Inhibition of the LT signalling pathway with a soluble receptor that depletes FDCs abolishes prion replication in the spleen and prolongs the latency of scrapie after intraperitoneal challenge, . B-cell-deficient MT mice, which bear a deletion of the transmembrane exon, are resistant to intraperitoneal inoculation with prions, perhaps because of impaired FDC maturation, . Neuroinvasion generally seems to depend on the presence of B lymphocytes, with one possible exception. TNF and lymphotoxin signal through TNF receptors, whereas signalling of membrane-bound lymphotoxin-/LT heterotrimers through LTRs is necessary for the development and maintenance of secondary lymphoid organs. The lymphotoxin and TNF signalling pathways are not equivalent: the former appears to be essential for prion colonization of lymph nodes, whereas the latter is required for the invasion of spleen (M.P., F.M. and A.A., unpublished observations). by components is also likely to be relevant, as mice that are genetically engineered to lack complement factors, or mice depleted of the C3 complement component by the administration of cobra venom, are resistant to peripheral prion inoculation.

All of the above indicates that the lymphoreticular phase of prion replication is a rate-limiting step for neuroinvasion. Because prions seem to have to go through a lymphoreticular bottleneck before reaching the brain, the cells and molecules involved in 'lymphoinvasion' represent attractive targets for post-exposure prophylaxis.

The sympathetic nervous system seems to be important in the transmission of infectivity from the lymphoreticular to the central nervous compartment. The peripheral injection of 6-hydroxydopamine (6-OHDA) or antibodies directed against nerve growth factor causes efficient and protracted sympathectomy in mice. We found that permanent or transient sympathectomy significantly delayed, or even prevented, the development of terminal scrapie after intraperitoneal inoculation with prions.

In further investigations, we administered prions to transgenic mice that overexpressed nerve growth factor, which show massive sympathetic hyperinnervation of lymphoid organs. The incubation time of scrapie was significantly reduced in these mice. Significantly, hyperinnervated spleens contain >100-fold more infectivity than control spleens, indicating that splenic nerve endings might act as prion reservoirs. Although sympathectomy is far too invasive to represent a viable option for post-exposure prophylaxis, these results indicate that the sympathetic nervous system might be worth exploring as a target for measures aimed at preventing or halting neuroinvasion.

Pre-exposure prophylaxis

For many conventional viral agents, vaccination is the most effective method of infection control. But is it possible to induce protective immunity in vivo against prions? After all, prions are extremely sturdy agents, with a resistance against sterilization procedures that is proverbial. Wouldn't one expect that 'soft' biological reagents such as antibodies would be too blunt as weapons for quenching prions, a goal that is failed even by autoclaving at 121 °C for prolonged periods of time? In vitro pre-incubation with anti-PrP antisera has been reported to reduce the prion titre of infectious hamster brain homogenates by up to 2 log units, and an anti-PrP antibody can inhibit the formation of PrPSc in a cell-free system. Furthermore, two recent papers have shown that antibodies and F(ab) fragments against certain domains of PrP can suppress prion replication in cultured cells. However, the blocking of prions in vivo and prevention of scrapie by specific anti-PrP antibodies was not reported: so, no evidence has been forthcoming that effective vaccines can be developed for the prevention of prion diseases.

One problem is that it is difficult to induce humoral immune responses against PrPC and PrPSc. This is probably due to of the mammalian immune system to PrPC, which is a ubiquitous, endogenous protein. Ablation of the Prnp gene, which encodes PrPC, renders mice highly susceptible to immunization with prions, and many of the best available monoclonal antibodies to the prion protein have been generated in Prnp-/- mice. However, Prnp-/- mice are unsuitable for testing vaccines, because they do not support prion pathogenesis.

In a recent set of experiments, we asked whether it might be possible to engineer a neutralizing immune response to prions in vivo. We reasoned that genes encoding high-affinity anti-PrP antibodies generated in Prnp-/- mice could be used to reprogramme the B-cell responses of prion-susceptible mice that express PrPC. This approach proved to be effective: the introduction of the -interacting region of the heavy chain of an anti-PrP monoclonal antibody (derived from hybridoma 6H4; Ref. ) into the germ line of mice produced high-titre anti-PrPC immunity. At the age of 150 days, the 6H4 heavy chain transgene induced similar anti-PrPC titres in Prnp-/-, Prnp-/+ and Prnp+/+ mice, indicating that the deletion of autoreactive B cells in prion-expressing mice does not prevent anti-PrP immunity.

Having established that transgenic mice expressing an anti-PrPC heavy chain were viable and had a spontaneous anti-PrPC titre, the most exciting experiment was to inoculate these mice with prions. Expression of the 6H4 heavy chain protected the mice against scrapie on the intraperitoneal inoculation of the prion agent. PrPC is a normal protein that is expressed by most tissues of the body. Therefore, an anti-PrP immune response could conceivably induce an autoimmune disease, and defeat any realistic prospect for prion vaccination. Interestingly, we did not observe any blatant autoimmune disease as a consequence of anti-prion immunization, unless PrPC was artificially, transgenically expressed at extremely high, non-physiological levels.

The strategy outlined above delivers the proof of principle that a protective humoral response against prions can be mounted by the mammalian immune system, and indicates that B cells are not intrinsically tolerant to PrPC. If the latter is generally true, lack of immunity to prions could be due to T-helper tolerance. The latter problem is not trivial, but could perhaps be overcome by presenting PrPC to the immune system along with highly active . These findings encourage a reassessment of the possible value of active and passive immunization, and perhaps of reprogramming B-cell repertoires by -chain transfer in the prophylaxis or therapy of prion diseases.

A perspective for the near future

Despite some tantalizing advances, therapy of manifest CJD continues to be an unattainable target. Therefore, it might be most sensible to concentrate on working towards post-exposure and pre-exposure prophylaxis. Many promising approaches to these goals are being developed: they involve small therapeutic molecules and cytokine antagonists (post-exposure), as well as specific anti-PrPC immunity (pre-exposure). Of course, pharmacological prophylaxis makes sense only if one can define the population at risk: this is why progress in this field must go hand in hand with progress in prion diagnostics. The latter is proceeding at a very rapid pace, especially as an enormous commercial interest in prion assays has arisen in both veterinary and human medicine. Therapy, however, is unlikely to be profitable, as the number of CJD patients is very small and will, we hope, not increase too much. Therefore, charities will continue to be the main promoters of studies aimed at prion therapy. It is to be hoped that national and international funding agencies will agree to consider this area of funding with priority, so that the most interesting of the approaches outlined above will be developed further, until their usefulness can be proved in clinical settings.

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Acknowledgements Supported by grants of the Bundesamt für Bildung und Wissenschaft, the National Centre of Competence in Research (NCCR-Neuro) and the Swiss National Foundation. We acknowledge technical help from P. Schwarz. F.L.H. is a Human Frontier Science Program and Stammbach Foundation fellow, and M.P. is a Deutsche Forschungsgemeinschaft fellow.

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