Pharmacological PINK1 activation ameliorates Pathology in Parkinson's Disease models.

PINK1 loss-of-function mutations and exposure to mitochondrial toxins are causative for Parkinson's disease (PD) and Parkinsonism, respectively. We demonstrate that pathological α-synuclein deposition, the hallmark pathology of idiopathic PD, induces mitochondrial dysfunction, and impairs mitophagy as evidenced by the accumulation of the PINK1 substrate pS65-Ubiquitin (pUb). We discovered MTK458, a brain penetrant small molecule that binds to PINK1 and stabilizes its active complex, resulting in increased rates of mitophagy. Treatment with MTK458 mediates clearance of accumulated pUb and α-synuclein pathology in α-synuclein pathology models in vitro and in vivo. Our findings from preclinical PD models suggest that pharmacological activation of PINK1 warrants further clinical evaluation as a therapeutic strategy for disease modification in PD.

Pharmacological PINK1 activation ameliorates Pathology in Parkinson's Disease models.

PINK1 loss-of-function mutations and exposure to mitochondrial toxins are causative for Parkinson's disease (PD) and Parkinsonism, respectively. We demonstrate that pathological α-synuclein deposition, the hallmark pathology of idiopathic PD, induces mitochondrial dysfunction and impairs mitophagy, driving accumulation of the PINK1 substrate pS65-Ubiquitin (pUb) in primary neurons and in vivo. We synthesized MTK458, a brain penetrant small molecule that binds to PINK1 and stabilizes an active heterocomplex, thereby increasing mitophagy. MTK458 mediates clearance of α-synuclein pathology in PFF seeding models in vitro and in vivo and reduces pUb. We developed an ultrasensitive assay to quantify pUb levels in plasma and observed an increase in pUb in PD subjects that correlates with disease progression, paralleling our observations in PD models. Our combined findings from preclinical PD models and patient biofluids suggest that pharmacological activation of PINK1 is worthy of further study as a therapeutic strategy for disease modification in PD. Highlights: Discovery of a plasma Parkinson's Disease biomarker candidate, pS65-Ubiquitin (pUb)Plasma pUb levels correlate with disease status and progression in PD patients.Identification of a potent, brain penetrant PINK1 activator, MTK458MTK458 selectively activates PINK1 by stimulating dimerization and stabilization of the PINK1/TOM complexMTK458 drives clearance of α-synuclein pathology and normalizes pUb in in vivo Parkinson's models.

Immunotherapy targeting the C-terminal domain of TDP-43 decreases neuropathology and confers neuroprotection in mouse models of ALS/FTD.

Effective therapies are urgently needed to safely target TDP-43 pathology as it is closely associated with the onset and development of devastating diseases such as frontotemporal lobar degeneration with TDP-43 pathology (FTLD-TDP) and amyotrophic lateral sclerosis (ALS). In addition, TDP-43 pathology is present as a co-pathology in other neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease. Our approach is to develop a TDP-43-specific immunotherapy that exploits Fc gamma-mediated removal mechanisms to limit neuronal damage while maintaining physiological TDP-43 function. Thus, using both in vitro mechanistic studies in conjunction with the rNLS8 and CamKIIa inoculation mouse models of TDP-43 proteinopathy, we identified the key targeting domain in TDP-43 to accomplish these therapeutic objectives. Targeting the C-terminal domain of TDP-43 but not the RNA recognition motifs (RRM) reduces TDP-43 pathology and avoids neuronal loss in vivo. We demonstrate that this rescue is dependent on Fc receptor-mediated immune complex uptake by microglia. Furthermore, monoclonal antibody (mAb) treatment enhances phagocytic capacity of ALS patient-derived microglia, providing a mechanism to restore the compromised phagocytic function in ALS and FTD patients. Importantly, these beneficial effects are achieved while preserving physiological TDP-43 activity. Our findings demonstrate that a mAb targeting the C-terminal domain of TDP-43 limits pathology and neurotoxicity, enabling clearance of misfolded TDP-43 through microglia engagement, and supporting the clinical strategy to target TDP-43 by immunotherapy. SIGNIFICANCE STATEMENT: TDP-43 pathology is associated with various devastating neurodegenerative disorders with high unmet medical needs such as frontotemporal dementia (FTD), amyotrophic lateral sclerosis (ALS) and Alzheimer's disease. Thus, safely and effectively targeting pathological TDP-43 represents a key paradigm for biotechnical research as currently there is little in clinical development. After years of research, we have determined that targeting the C-terminal domain of TDP-43 rescues multiple patho-mechanisms involved in disease progression in two animal models of FTD/ALS. In parallel, importantly, our studies establish that this approach does not alter the physiological functions of this ubiquitously expressed and indispensable protein. Together, our findings substantially contribute to the understanding of TDP-43 pathobiology and support the prioritization for clinical testing of immunotherapy approaches targeting TDP-43.

Aß and tau prion-like activities decline with longevity in the Alzheimer's disease human brain.

The hallmarks of Alzheimer's disease (AD) are the accumulation of Aß plaques and neurofibrillary tangles composed of hyperphosphorylated tau. We developed sensitive cellular assays using human embryonic kidney-293T cells to quantify intracellular self-propagating conformers of Aß in brain samples from patients with AD or other neurodegenerative diseases. Postmortem brain tissue from patients with AD had measurable amounts of pathological Aß conformers. Individuals over 80 years of age had the lowest amounts of prion-like Aß and phosphorylated tau. Unexpectedly, the longevity-dependent decrease in self-propagating tau conformers occurred in spite of increasing amounts of total insoluble tau. When corrected for the abundance of insoluble tau, the ability of postmortem AD brain homogenates to induce misfolded tau in the cellular assays showed an exponential decrease with longevity, with a half-life of about one decade over the age range of 37 to 99 years. Thus, our findings demonstrate an inverse correlation between longevity in patients with AD and the abundance of pathological tau conformers. Our cellular assays can be applied to patient selection for clinical studies and the development of new drugs and diagnostics for AD.

A long-lived Aß oligomer resistant to fibrillization.

The hydrophobic Aß peptide is highly aggregation prone; it first forms soluble oligomers, which then convert into the amyloid fibrils found in the cerebral plaques of Alzheimer's disease. It is generally understood that as the peptide concentration of Aß increases, the fibrillization process is accelerated, but we examine the limits on this phenomenon. We found that once a threshold concentration of Aß is exceeded, a stable oligomer is formed at the expense of fibril formation. The suppression of fibril formation was observed by amyloid-binding dye Thioflavin T and solution nuclear magnetic resonance (NMR). Small-angle X-ray scattering, size exclusion chromatography, and analytical ultracentrifugation demonstrated that Aß peptides form a range of compact species, with a dimer being an early highly populated oligomer. Solution NMR allowed us to define the secondary structure of this Aß dimer, which shows interlocking contacts between C-terminal peptide strands. Thus, we present a novel Aß oligomer that resists conversion to fibrils and remains stable for more than one year.

Structural heterogeneity and intersubject variability of Aß in familial and sporadic Alzheimer's disease.

Point mutations in the amyloid-ß (Aß) coding region produce a combination of mutant and WT Aß isoforms that yield unique clinicopathologies in familial Alzheimer's disease (fAD) and cerebral amyloid angiopathy (fCAA) patients. Here, we report a method to investigate the structural variability of amyloid deposits found in fAD, fCAA, and sporadic AD (sAD). Using this approach, we demonstrate that mutant Aß determines WT Aß conformation through prion template-directed misfolding. Using principal component analysis of multiple structure-sensitive fluorescent amyloid-binding dyes, we assessed the conformational variability of Aß deposits in fAD, fCAA, and sAD patients. Comparing many deposits from a given patient with the overall population, we found that intrapatient variability is much lower than interpatient variability for both disease types. In a given brain, we observed one or two structurally distinct forms. When two forms coexist, they segregate between the parenchyma and cerebrovasculature, particularly in fAD patients. Compared with sAD samples, deposits from fAD patients show less intersubject variability, and little overlap exists between fAD and sAD deposits. Finally, we examined whether E22G (Arctic) or E22Q (Dutch) mutants direct the misfolding of WT Aß, leading to fAD-like plaques in vivo. Intracerebrally injecting mutant Aß40 fibrils into transgenic mice expressing only WT Aß induced the deposition of plaques with many biochemical hallmarks of fAD. Thus, mutant Aß40 prions induce a conformation of WT Aß similar to that found in fAD deposits. These findings indicate that diverse AD phenotypes likely arise from one or more initial Aß prion conformations, which kinetically dominate the spread of prions in the brain.

A 31-residue peptide induces aggregation of tau's microtubule-binding region in cells.

The self-propagation of misfolded conformations of tau underlies neurodegenerative diseases, including Alzheimer's. There is considerable interest in discovering the minimal sequence and active conformational nucleus that defines this self-propagating event. The microtubule-binding region, spanning residues 244-372, reproduces much of the aggregation behaviour of tau in cells and animal models. Further dissection of the amyloid-forming region to a hexapeptide from the third microtubule-binding repeat resulted in a peptide that rapidly forms fibrils in vitro. We show that this peptide lacks the ability to seed aggregation of tau244-372 in cells. However, as the hexapeptide is gradually extended to 31 residues, the peptides aggregate more slowly and gain potent activity to induce aggregation of tau244-372 in cells. X-ray fibre diffraction, hydrogen-deuterium exchange and solid-state NMR studies map the beta-forming region to a 25-residue sequence. Thus, the nucleus for self-propagating aggregation of tau244-372 in cells is packaged in a remarkably small peptide.

Zinc-binding structure of a catalytic amyloid from solid-state NMR.

Throughout biology, amyloids are key structures in both functional proteins and the end product of pathologic protein misfolding. Amyloids might also represent an early precursor in the evolution of life because of their small molecular size and their ability to self-purify and catalyze chemical reactions. They also provide attractive backbones for advanced materials. When ß-strands of an amyloid are arranged parallel and in register, side chains from the same position of each chain align, facilitating metal chelation when the residues are good ligands such as histidine. High-resolution structures of metalloamyloids are needed to understand the molecular bases of metal-amyloid interactions. Here we combine solid-state NMR and structural bioinformatics to determine the structure of a zinc-bound metalloamyloid that catalyzes ester hydrolysis. The peptide forms amphiphilic parallel ß-sheets that assemble into stacked bilayers with alternating hydrophobic and polar interfaces. The hydrophobic interface is stabilized by apolar side chains from adjacent sheets, whereas the hydrated polar interface houses the Zn2+-binding histidines with binding geometries unusual in proteins. Each Zn2+ has two bis-coordinated histidine ligands, which bridge adjacent strands to form an infinite metal-ligand chain along the fibril axis. A third histidine completes the protein ligand environment, leaving a free site on the Zn2+ for water activation. This structure defines a class of materials, which we call metal-peptide frameworks. The structure reveals a delicate interplay through which metal ions stabilize the amyloid structure, which in turn shapes the ligand geometry and catalytic reactivity of Zn2.

Structural Biology of PrP Prions.

Prion diseases are characterized by the deposition of amyloids, misfolded conformers of the prion protein. The misfolded conformation is self-replicating, by a mechanism solely enciphered in the conformation of the protein. Because of low solubility and heterogeneous aggregate sizes, the detailed atomic structure of the infectious isoform is still unknown. Progress has, however, been made, and has allowed insights into the structural and disease-related mechanisms of prions. Many structural models have been proposed, and a number of them support a consensus trimeric ß-helical model, significantly more complex than simple amyloid models. There is evidence that such complexity may be a necessary property of prion structure. Knowledge of the structure of prions will provide a greater understanding of the protein isoform conversion mechanism, and could eventually lead to rationally designed intervention strategies.

Structural Polymorphism of Alzheimer's ß-Amyloid Fibrils as Controlled by an E22 Switch: A Solid-State NMR Study.

The amyloid-ß (Aß) peptide of Alzheimer's disease (AD) forms polymorphic fibrils on the micrometer and molecular scales. Various fibril growth conditions have been identified to cause polymorphism, but the intrinsic amino acid sequence basis for this polymorphism has been unclear. Several single-site mutations in the center of the Aß sequence cause different disease phenotypes and fibrillization properties. The E22G (Arctic) mutant is found in familial AD and forms protofibrils more rapidly than wild-type Aß. Here, we use solid-state NMR spectroscopy to investigate the structure, dynamics, hydration and morphology of Arctic E22G Aß40 fibrils. (13)C, (15)N-labeled synthetic E22G Aß40 peptides are studied and compared with wild-type and Osaka E22Δ Aß40 fibrils. Under the same fibrillization conditions, Arctic Aß40 exhibits a high degree of polymorphism, showing at least four sets of NMR chemical shifts for various residues, while the Osaka and wild-type Aß40 fibrils show a single or a predominant set of chemical shifts. Thus, structural polymorphism is intrinsic to the Arctic E22G Aß40 sequence. Chemical shifts and inter-residue contacts obtained from 2D correlation spectra indicate that one of the major Arctic conformers has surprisingly high structural similarity with wild-type Aß42. (13)C-(1)H dipolar order parameters, (1)H rotating-frame spin-lattice relaxation times and water-to-protein spin diffusion experiments reveal substantial differences in the dynamics and hydration of Arctic, Osaka and wild-type Aß40 fibrils. Together, these results strongly suggest that electrostatic interactions in the center of the Aß peptide sequence play a crucial role in the three-dimensional fold of the fibrils, and by inference, fibril-induced neuronal toxicity and AD pathogenesis.

Truncated forms of the prion protein PrP demonstrate the need for complexity in prion structure.

Self-propagation of aberrant protein folds is the defining characteristic of prions. Knowing the structural basis of self-propagation is essential to understanding prions and their related diseases. Prion rods are amyloid fibrils, but not all amyloids are prions. Prions have been remarkably intractable to structural studies, so many investigators have preferred to work with peptide fragments, particularly in the case of the mammalian prion protein PrP. We compared the structures of a number of fragments of PrP by X-ray fiber diffraction, and found that although all of the peptides adopted amyloid conformations, only the larger fragments adopted conformations that modeled the complexity of self-propagating prions, and even these fragments did not always adopt the PrP structure. It appears that the relatively complex structure of the prion form of PrP is not accessible to short model peptides, and that self-propagation may be tied to a level of structural complexity unobtainable in simple model systems. The larger fragments of PrP, however, are useful to illustrate the phenomenon of deformed templating (heterogeneous seeding), which has important biological consequences.

Propagation of prions causing synucleinopathies in cultured cells.

Increasingly, evidence argues that many neurodegenerative diseases, including progressive supranuclear palsy (PSP), are caused by prions, which are alternatively folded proteins undergoing self-propagation. In earlier studies, PSP prions were detected by infecting human embryonic kidney (HEK) cells expressing a tau fragment [TauRD(LM)] fused to yellow fluorescent protein (YFP). Here, we report on an improved bioassay using selective precipitation of tau prions from human PSP brain homogenates before infection of the HEK cells. Tau prions were measured by counting the number of cells with TauRD(LM)-YFP aggregates using confocal fluorescence microscopy. In parallel studies, we fused α-synuclein to YFP to bioassay α-synuclein prions in the brains of patients who died of multiple system atrophy (MSA). Previously, MSA prion detection required ∼120 d for transmission into transgenic mice, whereas our cultured cell assay needed only 4 d. Variation in MSA prion levels in four different brain regions from three patients provided evidence for three different MSA prion strains. Attempts to demonstrate α-synuclein prions in brain homogenates from Parkinson's disease patients were unsuccessful, identifying an important biological difference between the two synucleinopathies. Partial purification of tau and α-synuclein prions facilitated measuring the levels of these protein pathogens in human brains. Our studies should facilitate investigations of the pathogenesis of both tau and α-synuclein prion disorders as well as help decipher the basic biology of those prions that attack the CNS.

Prion Protein-Antibody Complexes Characterized by Chromatography-Coupled Small-Angle X-Ray Scattering.

Aberrant self-assembly, induced by structural misfolding of the prion proteins, leads to a number of neurodegenerative disorders. In particular, misfolding of the mostly α-helical cellular prion protein (PrP(C)) into a ß-sheet-rich disease-causing isoform (PrP(Sc)) is the key molecular event in the formation of PrP(Sc) aggregates. The molecular mechanisms underlying the PrP(C)-to-PrP(Sc) conversion and subsequent aggregation remain to be elucidated. However, in persistently prion-infected cell-culture models, it was shown that treatment with monoclonal antibodies against defined regions of the prion protein (PrP) led to the clearing of PrP(Sc) in cultured cells. To gain more insight into this process, we characterized PrP-antibody complexes in solution using a fast protein liquid chromatography coupled with small-angle x-ray scattering (FPLC-SAXS) procedure. High-quality SAXS data were collected for full-length recombinant mouse PrP [denoted recPrP(23-230)] and N-terminally truncated recPrP(89-230), as well as their complexes with each of two Fab fragments (HuM-P and HuM-R1), which recognize N- and C-terminal epitopes of PrP, respectively. In-line measurements by fast protein liquid chromatography coupled with SAXS minimized data artifacts caused by a non-monodispersed sample, allowing structural analysis of PrP alone and in complex with Fab antibodies. The resulting structural models suggest two mechanisms for how these Fabs may prevent the conversion of PrP(C) into PrP(Sc).

Structural studies of truncated forms of the prion protein PrP.

Prions are proteins that adopt self-propagating aberrant folds. The self-propagating properties of prions are a direct consequence of their distinct structures, making the understanding of these structures and their biophysical interactions fundamental to understanding prions and their related diseases. The insolubility and inherent disorder of prions have made their structures difficult to study, particularly in the case of the infectious form of the mammalian prion protein PrP. Many investigators have therefore preferred to work with peptide fragments of PrP, suggesting that these peptides might serve as structural and functional models for biologically active prions. We have used x-ray fiber diffraction to compare a series of different-sized fragments of PrP, to determine the structural commonalities among the fragments and the biologically active, self-propagating prions. Although all of the peptides studied adopted amyloid conformations, only the larger fragments demonstrated a degree of structural complexity approaching that of PrP. Even these larger fragments did not adopt the prion structure itself with detailed fidelity, and in some cases their structures were radically different from that of pathogenic PrP(Sc).

Mechanism of scrapie prion precipitation with phosphotungstate anions.

The phosphotungstate anion (PTA) is widely used to facilitate the precipitation of disease-causing prion protein (PrP(Sc)) from infected tissue for applications in structural studies and diagnostic approaches. However, the mechanism of this precipitation is not understood. In order to elucidate the nature of the PTA interaction with PrP(Sc) under physiological conditions, solutions of PTA were characterized by NMR spectroscopy at varying pH. At neutral pH, the parent [PW12O40](3-) ion decomposes to give a lacunary [PW11O39](7-) (PW11) complex and a single orthotungstate anion [WO4](2-) (WO4). To measure the efficacy of each component of PTA, increasing concentrations of PW11, WO4, and mixtures thereof were used to precipitate PrP(Sc) from brain homogenates of scrapie prion-infected mice. The amount of PrP(Sc) isolated, quantified by ELISA and immunoblotting, revealed that both PW11 and WO4 contribute to PrP(Sc) precipitation. Incubation with sarkosyl, PTA, or individual components of PTA resulted in separation of higher-density PrP aggregates from the neuronal lipid monosialotetrahexosylganglioside (GM1), as observed by sucrose gradient centrifugation. These experiments revealed that yield and purity of PrP(Sc) were greater with polyoxometalates (POMs), which substantially supported the separation of lipids from PrP(Sc) in the samples. Interaction of POMs and sarkosyl with brain homogenates promoted the formation of fibrillar PrP(Sc) aggregates prior to centrifugation, likely through the separation of lipids like GM1 from PrP(Sc). We propose that this separation of lipids from PrP is a major factor governing the facile precipitation of PrP(Sc) by PTA from tissue and might be optimized further for the detection of prions.

Distinct synthetic Aß prion strains producing different amyloid deposits in bigenic mice.

An increasing number of studies continue to show that the amyloid ß (Aß) peptide adopts an alternative conformation and acquires transmissibility; hence, it becomes a prion. Here, we report on the attributes of two strains of Aß prions formed from synthetic Aß peptides composed of either 40 or 42 residues. Modifying the conditions for Aß polymerization increased both the protease resistance and prion infectivity compared with an earlier study. Approximately 150 d after intracerebral inoculation, both synthetic Aß40 and Aß42 prions produced a sustained rise in the bioluminescence imaging signal in the brains of bigenic Tg(APP23:Gfap-luc) mice, indicative of astrocytic gliosis. Pathological investigations showed that synthetic Aß40 prions produced amyloid plaques containing both Aß40 and Aß42 in the brains of inoculated bigenic mice, whereas synthetic Aß42 prions stimulated the formation of smaller, more numerous plaques composed predominantly of Aß42. Synthetic Aß40 preparations consisted of long straight fibrils; in contrast, the Aß42 fibrils were much shorter. Addition of 3.47 mM (0.1%) SDS to the polymerization reaction produced Aß42 fibrils that were indistinguishable from Aß40 fibrils produced in the absence or presence of SDS. Moreover, the Aß amyloid plaques in the brains of bigenic mice inoculated with Aß42 prions prepared in the presence of SDS were similar to those found in mice that received Aß40 prions. From these results, we conclude that the composition of Aß plaques depends on the conformation of the inoculated Aß polymers, and thus, these inocula represent distinct synthetic Aß prion strains.

Serial propagation of distinct strains of Aß prions from Alzheimer's disease patients.

An increasing number of studies argues that self-propagating protein conformations (i.e., prions) feature in the pathogenesis of several common neurodegenerative diseases. Mounting evidence contends that aggregates of the amyloid-ß (Aß) peptide become self-propagating in Alzheimer's disease (AD) patients. An important characteristic of prions is their ability to replicate distinct strains, the biological information for which is enciphered within different conformations of protein aggregates. To investigate whether distinct strains of Aß prions can be discerned in AD patients, we performed transmission studies in susceptible transgenic mice using brain homogenates from sporadic or heritable (Arctic and Swedish) AD cases. Mice inoculated with the Arctic AD sample exhibited a pathology that could be distinguished from mice inoculated with the Swedish or sporadic AD samples, which was judged by differential accumulation of Aß isoforms and the morphology of cerebrovascular Aß deposition. Unlike Swedish AD- or sporadic AD-inoculated animals, Arctic AD-inoculated mice, like Arctic AD patients, displayed a prominent Aß38-containing cerebral amyloid angiopathy. The divergent transmission behavior of the Arctic AD sample compared with the Swedish and sporadic AD samples was maintained during second passage in mice, showing that Aß strains are serially transmissible. We conclude that at least two distinct strains of Aß prions can be discerned in the brains of AD patients and that strain fidelity was preserved on serial passage in mice. Our results provide a potential explanation for the clinical and pathological heterogeneity observed in AD patients.

Short peptides self-assemble to produce catalytic amyloids.

Enzymes fold into unique three-dimensional structures, which underlie their remarkable catalytic properties. The requirement to adopt a stable, folded conformation is likely to contribute to their relatively large size (>10,000 Da). However, much shorter peptides can achieve well-defined conformations through the formation of amyloid fibrils. To test whether short amyloid-forming peptides might in fact be capable of enzyme-like catalysis, we designed a series of seven-residue peptides that act as Zn(2+)-dependent esterases. Zn(2+) helps stabilize the fibril formation, while also acting as a cofactor to catalyse acyl ester hydrolysis. These results indicate that prion-like fibrils are able to not only catalyse their own formation, but they can also catalyse chemical reactions. Thus, they might have served as intermediates in the evolution of modern-day enzymes. These results also have implications for the design of self-assembling nanostructured catalysts including ones containing a variety of biological and non-biological metal ions.

Seeded fibrillation as molecular basis of the species barrier in human prion diseases.

Prion diseases are transmissible spongiform encephalopathies in humans and animals, including scrapie in sheep, bovine spongiform encephalopathy (BSE) in cattle, chronic wasting disease (CWD) in deer, and Creutzfeldt-Jakob disease (CJD) in humans. The hallmark of prion diseases is the conversion of the host-encoded prion protein (PrP(C)) to its pathological isoform PrP(Sc), which is accompanied by PrP fibrillation. Transmission is not restricted within one species, but can also occur between species. In some cases a species barrier can be observed that results in limited or unsuccessful transmission. The mechanism behind interspecies transmissibility or species barriers is not completely understood. To analyse this process at a molecular level, we previously established an in vitro fibrillation assay, in which recombinant PrP (recPrP) as substrate can be specifically seeded by PrP(Sc) as seed. Seeding with purified components, with no additional cellular components, is a direct consequence of the "prion-protein-only" hypothesis. We therefore hypothesise, that the species barrier is based on the interaction of PrP(C) and PrP(Sc). Whereas in our earlier studies, the interspecies transmission in animal systems was analysed, the focus of this study lies on the transmission from animals to humans. We therefore combined seeds from species cattle, sheep and deer (BSE, scrapie, CWD) with human recPrP. Homologous seeding served as a control. Our results are consistent with epidemiology, other in vitro aggregation studies, and bioassays investigating the transmission between humans, cattle, sheep, and deer. In contrast to CJD and BSE seeds, which show a seeding activity we can demonstrate a species barrier for seeds from scrapie and CWD in vitro. We could show that the seeding activity and therewith the molecular interaction of PrP as substrate and PrP(Sc) as seed is sufficient to explain the phenomenon of species barriers. Therefore our data supports the hypothesis that CWD is not transmissible to humans.

Prion protein aggregation and fibrillogenesis in vitro.

This chapter focuses on the structural conversion of natural and recombinant prion proteins in vitro. They key event in prion diseases is the conversion of the cellular prion protein (PrP(C)) into its disease causing isoform PrP(Sc). This conversion is represented by a conformational change from an ß-helical dominated isoform into the mostly ß-sheeted PrP(Sc). Represented is an overview of in vitro conversion systems that result in ß-structured recombinant prion proteins including the current achievements in the generation of synthetic mammalian prions as proof of the protein-only hypothesis. In addition to the conversion of recombinant PrP the chapter features a summary of the protein misfolding cyclic amplification (PMCA) technique which has gained enormous popularity in prion research. Given is a general overview about the technique itself and the broad spectrum of utilization as detection method for prions. The spontaneous generation of prions by the protein misfolding amplification (PMCA) are also discussed.