Sabine Gilch

Chronic wasting disease (CWD) is an expanding prion disease of cervids. CWD prions persist in the environment, are shed in excreta, and accumulate in tissues of infected cervids, raising concerns about its zoonotic potential. Using cynomolgus macaques, we explored the zoonotic potential of CWD. Although most inoculated macaques remained asymptomatic, sensitive in vitro prion amplification assays revealed low levels of prions in macaque tissues. Inoculation of transgenic mice and bank voles with macaque tissues induced prion disease, achieving 100% transmission rates upon serial passage. One interpretation of these findings is that CWD prions retain infectivity across species and that primate infection may manifest atypically while still enabling transmission. Our results challenge earlier conclusions that minimize the zoonotic risk of CWD and underscore the need for continued surveillance.

Prion diseases are fatal neurodegenerative disorders that affect humans and animals, caused by the conformational conversion of the normal cellular prion protein (PrP^C) into its misfolded, infectious isoform PrP^Sc. Recently, camel prion disease (CPrD) was identified in dromedary camels (Camelus dromedarius) in Algeria. Due to the potential implications for animal and human health, as well as the possible socio-economic impact in Mediterranean regions where camels play a pivotal role as a source of food, in-depth characterization of camel prions is important to increase our understanding of camel prion disease. We developed a neuronal cell line model for studying the molecular features of camel prion infection. We genetically edited mouse neuronal CAD5 cells to generate CAD5 PrP knockout (KO) cells. We then used lentiviral transduction to generate CAD5 cells expressing camel PrP (CAD5-camel-PrP). Following infection of these cells with a CPrD-positive camel brain homogenate, we observed PrP^Sc signals at various passages, as indicated by immunoblotting analysis. RT-QuIC (Real-Time Quaking-Induced Conversion) assays further supported these findings, demonstrating transient prion conversion activity in the CPrD-infected CAD5-camel-PrP cells. Taken together, our data describe the first neuronal cell line permissive to camel prion infection, a novel in vitro tool for mechanistic studies of camel prion disease.

Chronic wasting disease (CWD) is a prion disease of cervids marked by growing strain diversity and variation in host susceptibility. Central to this complexity are prion protein gene (Prnp) polymorphisms, which can modulate pathogenesis by altering the ability of cellular prion protein (PrP^C) to misfold into infectious prions (PrP^Sc), or by promoting the emergence of novel strains. Studies in cervids and transgenic rodent models demonstrate that individual polymorphisms influence PrP stability, conversion efficiency, and the selection of PrP^Sc conformers, with most variants conferring partial resistance but none offering complete protection. These host–strain interactions define transmission barriers and disease phenotype. Understanding how Prnp genotypes shape CWD strain diversity is essential for predicting transmission dynamics, refining surveillance, and assessing zoonotic potential as the disease continues to expand geographically and genetically.

Prion diseases are fatal, infectious, and incurable neurodegenerative conditions affecting humans and animals, caused by the misfolding of the cellular prion protein (PrP ^C ) into its pathogenic isoform, PrP ^Sc . In humans, sporadic Creutzfeldt-Jakob disease (sCJD) is the most prevalent prion disease. Recently, we demonstrated that treatment with the FDA-approved anti-HIV drug efavirenz (EFV) significantly reduced PrP ^Sc and extended survival of scrapie prion–infected mice. Among other effects, EFV activates the brain-specific cholesterol-metabolizing enzyme, CYP46A1, which converts cholesterol into 24S-hydroxycholesterol (24S-HC). However, drugs effective against scrapie prions often fail in human prion diseases, and a relation of the antiprion effects of EFV to CYP46A1 activation is not established. Thus, we evaluated EFV treatment in mice overexpressing human PrP ^C infected with human sCJD prions. Oral, low-dose EFV treatment starting at 30 or 130 days postinfection significantly slowed disease progression and extended their survival. At early clinical stage, we observed reduced PrP ^Sc accumulation, decreased cholesterol and lipid droplet content, and elevated CYP46A1 and 24S-HC levels in EFV-treated mice. Overexpression of CYP46A1 in prion-infected neuronal cells reduced PrP ^Sc levels and increased 24S-HC, indicating that antiprion effects of EFV correlate with CYP46A1 activation. These findings highlight EFV as a safe and efficacious therapeutic candidate for human prion diseases.

Prions can exist as different strains that consist of conformational variants of the misfolded, pathogenic prion protein isoform PrP^Sc. Defined by stably transmissible biological and biochemical properties, strains have been identified in a spectrum of prion diseases, including chronic wasting disease (CWD) of wild and farmed cervids. CWD is highly contagious and spreads via direct and indirect transmission involving extraneural sites of infection, peripheral replication and neuroinvasion of prions. Here, we investigated the impact of infection route on CWD prion conformational selection and propagation. We used gene-targeted mouse models expressing deer PrP for intracerebral or intraperitoneal inoculation with fractionated or unfractionated brain homogenates from white-tailed deer, harboring CWD strains Wisc-1 or 116AG. Upon intracerebral inoculation, Wisc-1 and 116AG-inoculated mice differed in conformational stability of PrP^Sc. In brains of mice infected intraperitoneally with either inoculum, PrP^Sc propagated with identical conformational stability and fewer PrP^Sc deposits in most brain regions than intracerebrally inoculated animals. For either inoculum, PrP^Sc conformational stability in brain and spinal cord was similar upon intracerebral infection but significantly higher in spinal cords of intraperitoneally infected animals. Inoculation with fractionated brain homogenates resulted in lower variance of survival times upon intraperitoneal compared to intracerebral infection. In summary, we demonstrate that extraneural infection mitigates the impact of PrP^Sc quaternary structure on infection and reduces conformational variability of PrP^Sc propagated in the brain. These findings provide new insights into the evolution of stable CWD strains in natural, extraneural transmissions.

Chronic wasting disease (CWD) is a prion disease affecting deer, elk and moose in North America and reindeer, moose and red deer in Northern Europe. Pathogenesis is driven by the accumulation of PrP^Sc, a pathological form of the host’s cellular prion protein (PrP^C), in the brain. CWD is contagious among North American cervids and Norwegian reindeer, with prions commonly found in lymphatic tissue. In Nordic moose and red deer CWD appears exclusively in older animals, and prions are confined to the CNS and undetectable in lymphatic tissues, indicating a sporadic origin. We aimed to determine transmissibility, neuroinvasion and lymphotropism of Nordic CWD isolates using gene-targeted mice expressing either wild-type (138SS/226QQ) or S138N (138NN/226QQ) deer PrP. When challenged with North American CWD strains, mice expressing S138N PrP did not develop clinical disease but harbored prion seeding activity in brain and spleen. Here, we infected these models intracerebrally or intraperitoneally with Norwegian moose, red deer and reindeer CWD isolates. The moose isolate was the first CWD type to cause full-blown disease in the 138NN/226QQ model in the first passage, with 100% attack rate and shortened survival times upon second passage. Furthermore, we detected prion seeding activity or PrP^Sc in brains and spinal cords, but not spleens, of 138NN/226QQ mice inoculated intraperitoneally with the moose isolate, providing evidence of prion neuroinvasion. We also demonstrate, for the first time, that transmissibility of the red deer CWD isolate was restricted to transgenic mice overexpressing elk PrP^C (138SS/226EE), identical to the PrP primary structure of the inoculum. Our findings highlight that susceptibility to clinical disease is determined by the conformational compatibility between prion inoculum and host PrP primary structure. Our study indicates that neuroinvasion of Norwegian moose prions can occur without, or only very limited, replication in the spleen, an unprecedented finding for CWD.

Infectious prions consist of PrP^Sc, a misfolded, aggregation-prone isoform of the host’s prion protein. PrP^Sc assemblies encode distinct biochemical and biological properties. They harbor a specific profile of PrP^Sc species, from small oligomers to fibrils in different ratios, where the highest infectivity aligns with oligomeric particles. To investigate the impact of PrP^Sc aggregate complexity on prion propagation, biochemical properties, and disease pathogenesis, we fractionated elk prions by sedimentation velocity centrifugation, followed by sub-passages of individual fractions in cervidized mice. Upon first passage, different fractions generated PrP^Sc with distinct biochemical, biophysical, and neuropathological profiles. Notably, low or high molecular weight PrP^Sc aggregates caused different clinical signs of hyperexcitability or lethargy, respectively, which were retained over passage, whereas other properties converged. Our findings suggest that PrP^Sc quaternary structure determines an initial selection of a specific replication environment, resulting in transmissible features that are independent of PrP^Sc biochemical and biophysical properties.

During aging, the cellular response to unfolded proteins is believed to decline, resulting in diminished proteostasis. In model organisms, such as Caenorhabditis elegans, proteostatic decline with age has been linked to proteome solubility shifts and the onset of protein aggregation. However, this correlation has not been extensively characterized in aging mammals. To uncover age-dependent changes in the insoluble portion of a mammalian proteome, we analyzed the detergent-insoluble fraction of mouse brain tissue by mass spectrometry. We identified a group of 171 proteins, including the small heat shock protein α-crystallin, that become enriched in the detergent-insoluble fraction obtained from old mice. To enhance our ability to detect features associated with proteins in that fraction, we complemented our data with a meta-analysis of studies reporting the detergent-insoluble proteins in various mouse models of aging and neurodegeneration. Strikingly, insoluble proteins from young and old mice are distinct in several features in our study and across the collected literature data. In younger mice, proteins are more likely to be disordered, part of membraneless organelles, and involved in RNA binding. These traits become less prominent with age, as an increased number of structured proteins enter the pellet fraction. This analysis suggests that age-related changes to proteome organization lead a group of proteins with specific features to become detergent-insoluble. Importantly, these features are not consistent with those associated with proteins driving membraneless organelle formation. We see no evidence in our system of a general increase of condensate proteins in the detergent-insoluble fraction with age.

Alzheimer’s disease (AD) is an incurable, progressive and devastating neurodegenerative disease. Pathogenesis of AD is associated with the aggregation and accumulation of amyloid beta (Aβ), a major neurotoxic mediator that triggers neuroinflammation and memory impairment. Recently, we found that cellulose ether compounds (CEs) have beneficial effects against prion diseases by inhibiting protein misfolding and replication of prions, which share their replication mechanism with Aβ. CEs are FDA-approved safe additives in foods and pharmaceuticals. Herein, for the first time we determined the therapeutic effects of the representative CE (TC-5RW) in AD using in vitro and in vivo models. Our in vitro studies showed that TC-5RW inhibits Aβ aggregation, as well as neurotoxicity and immunoreactivity in Aβ-exposed human and murine neuroblastoma cells. In in vivo studies, for the first time we observed that single and weekly TC-5RW administration, respectively, improved memory functions of transgenic 5XFAD mouse model of AD. We further demonstrate that TC-5RW treatment of 5XFAD mice significantly inhibited Aβ oligomer and plaque burden and its associated neuroinflammation via regulating astrogliosis, microgliosis and proinflammatory mediator glial maturation factor beta (GMFβ). Additionally, we determined that TC-5RW reduced lipopolysaccharide-induced activated gliosis and GMFβ in vitro. In conclusion, our results demonstrate that CEs have therapeutic effects against Aβ pathologies and cognitive impairments, and direct, potent anti-inflammatory activity to rescue neuroinflammation. Therefore, these FDA-approved compounds are effective candidates for developing therapeutics for AD and related neurodegenerative diseases associated with protein misfolding.

Currently, no effective therapeutics exist for the treatment of incurable neurodegenerative diseases such as Alzheimer’s disease (AD). The cellular prion protein (PrP^C) acts as a high-affinity receptor for amyloid beta oligomers (AβO), a main neurotoxic species mediating AD pathology. The interaction of AβO with PrP^C subsequently activates Fyn tyrosine kinase and neuroinflammation. Herein, we used our previously developed peptide aptamer 8 (PA8) binding to PrP^C as a therapeutic to target the AβO–PrP–Fyn axis and prevent its associated pathologies. Our in vitro results indicated that PA8 prevents the binding of AβO with PrP^C and reduces AβO-induced neurotoxicity in mouse neuroblastoma N2a cells and primary hippocampal neurons. Next, we performed in vivo experiments using the transgenic 5XFAD mouse model of AD. The 5XFAD mice were treated with PA8 and its scaffold protein thioredoxin A (Trx) at a 14.4 µg/day dosage for 12 weeks by intraventricular infusion through Alzet^® osmotic pumps. We observed that treatment with PA8 improves learning and memory functions of 5XFAD mice as compared to Trx-treated 5XFAD mice. We found that PA8 treatment significantly reduces AβO levels and Aβ plaques in the brain tissue of 5XFAD mice. Interestingly, PA8 significantly reduces AβO–PrP interaction and its downstream signaling such as phosphorylation of Fyn kinase, reactive gliosis as well as apoptotic neurodegeneration in the 5XFAD mice compared to Trx-treated 5XFAD mice. Collectively, our results demonstrate that treatment with PA8 targeting the AβO–PrP–Fyn axis is a promising and novel approach to prevent and treat AD.