Of the many distinctive features that separate prion diseases from viral, bacterial, fungal, and parasitic disorders, the most remarkable is that PrP prion diseases are not only acquired but also can manifest as inherited and sporadic disorders. Yet, in all three etiologies, infectious prions are generated in the brain and are composed of PrP^Sc molecules with the amino acid sequence encoded by the Prnp gene of the affected host. When PrP prions are transmitted to a different host species, there is typically a “transmission barrier,” in part related to differences in PrP sequences, resulting in inefficient transmission.¹⁰,¹¹,¹²
If interspecies transmission does occur, the prions generated in the brain of the host carry the amino acid sequence encoded by the Prnp gene of the host species and not the PrP sequence found in the original inoculum. In other words, in interspecies infection, such as from sheep to cattle or from cattle to humans, the prions that replicate in the host brain are not the same as those that initiate replication. In contrast, serial transmission in the same host, in which the inoculum and host PrP sequences match, is generally more rapid and efficient. This scenario is profoundly different from what happens during a viral infection.

Phenotypically distinct strains of prions were first identified following transmission of sheep scrapie to goats.¹³ Although this was long used as an argument for the prion containing a nucleic acid, none has ever been found.¹⁴ It was later understood that PrP prion strains represent structurally different conformations of PrP^Sc.¹⁵,¹⁶ Yeast prions, which also display this phenomenon, have been important in demonstrating the structural basis of prion strains.¹⁷,¹⁸ The strain phenomenon has also been observed for Aβ, tau, and α-synuclein prions,¹⁹,²⁰,²¹ which may help explain clinical variability and raises important questions about potential therapeutic specificity.

Determining prion inactivation requires quantifiable assays that are ideally rapid and easy to perform. Due to the lack of nucleic acid in prions, quantifying inactivation has been challenging to achieve, with the gold standard still being animal bioassays in wild-type or Tg rodents.

Experimental transmission of sheep scrapie was first demonstrated in the 1930s.²² Subsequently, both kuru, an acquired human prion disease, and CJD were transmitted to chimpanzees.²³,²⁴ However, the transmission of prions to mice²⁵ and hamsters²⁶ greatly accelerated research by providing models with incubation times measured in weeks rather than years. The development of Tg mice expressing PrP from other species, particularly in combination with ablation of endogenous mouse PrP, has provided a range of tools to interrogate prion biology.²⁷ However, even with these accelerated models, survival time after inoculation is the central metric, which leads to time-consuming and expensive experiments.

A limited number of mouse cell lines have been identified that propagate PrP prions, including N2a,²⁸ GT1,²⁹ and CAD5 cells.³⁰ Interestingly, most of these lines only propagate a subset of mouse-passaged PrP prion strains, the reasons for which are poorly understood. Some success has been achieved by overexpressing heterologous PrP in rabbit RK13 cells,³¹ and recent studies have shown infection of some human prions in stem cell-derived astrocytes.³² However, a scalable cell line for the propagation of the most common strains of human PrP prions is still lacking.

Cell-free replication of PrP prions was first demonstrated by the incorporation of PrP^C into a proteaseresistant conformation following incubation with partially denatured PrP^Sc.³³ Subsequent studies using serial sonication, believed to break up PrP prion aggregates, ultimately gave rise to the protein misfolding cyclic amplification (PMCA) assay.³⁴ Although this method was demonstrated to replicate PrP prion infectivity,³⁵ the difficulties in generating reproducible results hampered its adoption by many labs. In parallel, assays based on shaking a recombinant PrP substrate led to the development of the real-time quaking-induced conversion (RT-QuIC) assay.³⁶ Although this is a promising technique for measuring low PrP prion levels, seeding ability in RT-QuIC does not necessarily translate into the replication of PrP prion infectivity, requiring animal bioassays for confirmatory testing.

Experimental models have also been developed for the quantification of Aβ, tau, and α-synuclein prions. Long-term Aβ transmission studies were performed in primates,³⁷ but the demonstrated transmission of Aβ pathology to Tg mice³⁸,³⁹ provided experimentally tractable tools. Consistent with the prion hypothesis, distinct Aβ strains are serially passaged with high fidelity in Tg mice.⁴⁰,⁴¹,⁴² However, novel cell lines propagating Aβ prions have only recently been reported.⁴³ Conversely, although the inoculation models available for studying the tauopathies are less robust than those for Aβ,⁴⁴,⁴⁵ highly sensitive cell models have been used to identify different tau prion strains from various tauopathy patient samples.²⁰,⁴⁶

In contrast to the PrP inoculation model, in which uninoculated animals typically do not have any spontaneous disease, many of the Aβ and tau inoculation models use Tg mice overexpressing the respective human protein, typically with disease-associated mutations. As a result, these mouse lines often exhibit spontaneous disease in older animals. In such cases, there is a “window” between inoculation and spontaneous prion formation that facilitates measuring induced prion propagation. Moreover, these assays typically rely on a neuropathologic readout rather than onset of disease, which can be time-consuming and more difficult to quantify across research groups.

In the five decades since the initial transmission studies of human PrP prions,²³,²⁴ the first new human neurodegenerative disease model to produce a lethal phenotype in an animal model came from a serendipitous discovery. A Tg mouse line, termed TgM83, expresses human α-synuclein with the familial PD-associated mutation A53T. Homozygous TgM83^+/+ mice develop spontaneous disease beginning at approximately 8 months old, but hemizygous TgM83^+/+ mice show no signs of disease for at least 20 months.⁴⁷ Inoculation of brain homogenate from spontaneously ill TgM83^+/+ mice, or fibrils of synthetic α-synuclein,
into 2- to 4-month-old TgM83^+/+ mice induced disease onset 3 to 4 months later.⁴⁸,⁴⁹ In an attempt to transmit α-synucleinopathy from PD, we inoculated TgM83^+/+ mice with brain homogenate from PD patients and from patients who had died from a different α-synucleinopathy, multiple system atrophy (MSA), as a control. To our surprise, whereas the PD samples did not transmit disease to the mice, the MSA samples induced a lethal phenotype approximately 4 months after inoculation.⁵⁰ These findings were subsequently confirmed using a much larger cohort of MSA samples from three continents.⁵¹ The MSA strain differs from the spontaneous TgM83^+/+ strain, exhibiting a shorter incubation period with serial passaging.⁵¹ In contrast to the limitation of cell models for PrP prion diseases, we developed a rapid cell assay for MSA,⁵² which correlates well with disease onset in the animal bioassay.⁵¹ This 4-day assay uses human embryonic kidney (HEK) cells that express human α-synuclein with the A53T mutation, conjugated to the yellow fluorescent protein (YFP). Application of exogenous α-synuclein prions induces aggregation of α-synuclein-YFP, which can be automatically identified as puncta by high-content fluorescence microscopy. Importantly, MSA prions propagated in the cell assay also transmitted a lethal disease to TgM83^+/+ mice following inoculation,⁵³ which had previously only been demonstrated with CWD and mouse-adapted scrapie strains.

The unusual resistance of PrP prions to inactivation was first identified when formalin-treated sheep’s brain and spinal cord, used to immunize animals against the louping-ill virus, resulted in the transmission of scrapie. This was ultimately attributed to the inclusion of tissue from an asymptomatic scrapie-infected sheep in a specific batch of inoculum.⁵⁴ Subsequent work showed resistance of PrP prions to heat and chemical denaturants⁵⁵,⁵⁶ and to ultraviolet irradiation.⁵⁷

Resistance to formalin fixation has also been observed for other human prions. MSA brain tissue fixed in formalin for up to 20 years still showed robust α-synuclein prion infectivity in TgM83^+/+ mice.⁵⁸ A similar phenomenon was observed using a Tg mouse model expressing the A30P mutation in human α-synuclein; formalin-fixed tissue from aged animals was able to induce an early onset of neurologic disease when inoculated into young, asymptomatic mice.⁵⁹ Likewise, formalin-fixed tissue from AD patients induced robust Aβ-amyloidosis in reporter mice, and, experimentally, formalin fixation only slightly reduced Aβ prion infectivity compared with brain homogenate from frozen tissue.⁶⁰ Using an alternative approach, fixed AD patient samples also transmitted tau prions to HEK cells expressing a tau-YFP reporter protein.⁶¹