Effects of Elk-PrPC expression levels on CWD strain properties

Chronic Wasting Disease (CWD) is a contagious prion disease affecting various species of free-ranging and/or captive cervids on three continents. Species-specific prion protein (PrPC) polymorphisms influence prion conversion into PrPCWD. PrPC amino acid variation can also regulate disease susceptibility to particular prion strains and has been implicated in the diversification of prion strain conformers [1, 2, 3]. Elk and deer PrPC differ at residue E226Q and this amino acid difference has been implicated in the selection of CWD1 and CWD2 prion strains [4]. As PrPC expression has been suggested to affect prion strain evolution [5], we hypothesized that elk PrPC levels affect CWD strain generation. To test this hypothesis, transgenic (tg) FVB mice over-expressing elk PrPC [6] were crossed with prnp knock-out FVB mice to generate tg-elk with different PrPC expression levels. Both tg-elk+/+ and tg-elk± were exposed to white-tailed deer CWD strains (Wisc-1 and H95+) [2, 3]. The H95+ strain was a mixture generated on passage of Wisc-1 in deer heterozygous for H95G96 and Q95S96 [2]. Tg-elk+/+ mice succumbed to Wisc-1 with a mean incubation period of 116 ± 7 days post infection (dpi) compared to 164 ± 11 dpi for the H95+ strain mixture. Consistent with the reduced PrPC expression, the same deer prion strains resulted in longer incubation periods (157 ± 21 dpi and >180 dpi, respectively) when passaged in tg-elk± mice. After first passage, transmission of Wisc-1 and H95+ in tg-elk+/+ mice resulted in a single neuropathological profile that differed from the profile produced by passage of elk prions (described as the CWD2 strain [1]). Our results show that, upon first passage, the E226Q polymorphism did not affect the strain properties of deer prions and indicates a single strain (Wisc-1) was selected by the tg-elk+/+ mice. The comparative analysis of neuropathological profiles between high and low expression tg-elk on first and second passage will be presented

Chronic wasting disease prions in mule deer interdigital glands

Chronic wasting disease (CWD) is a geographically expanding, fatal neurodegenerative disease in cervids. The disease can be transmitted directly (animal-animal) or indirectly via infectious prions shed into the environment. The precise mechanisms of indirect CWD transmission are unclear but known sources of the infectious prions that contaminate the environment include saliva, urine and feces. We have previously identified PrPC expression in deer interdigital glands, sac-like exocrine structures located between the digits of the hooves. In this study, we assayed for CWD prions within the interdigital glands of CWD infected deer to determine if they could serve as a source of prion shedding and potentially contribute to CWD transmission. Immunohistochemical analysis of interdigital glands from a CWD-infected female mule deer identified disease-associated PrPCWD within clusters of infiltrating leukocytes adjacent to sudoriferous and sebaceous glands, and within the acrosyringeal epidermis of a sudoriferous gland tubule. Proteinase K-resistant PrPCWD material was amplified by serial protein misfolding cyclic amplification (sPMCA) from soil retrieved from between the hoof digits of a clinically affected mule deer. Blinded testing of interdigital glands from 11 mule deer by real-time quake-induced conversion (RT-QuIC) accurately identified CWD-infected animals. The data described suggests that interdigital glands may play a role in the dissemination of CWD prions into the environment, warranting future investigation

In vitro and in vivo evidence towards fibronectin’s protective effects against prion infection

A distinctive signature of the prion diseases is the accumulation of the pathogenic isoform of the prion protein, PrPSc, in the central nervous system of prion-affected humans and animals. PrPSc is also found in peripheral tissues, raising concerns about the potential transmission of pathogenic prions through human food supplies and posing a significant risk to public health. Although muscle tissues are considered to contain levels of low prion infectivity, it has been shown that myotubes in culture efficiently propagate PrPSc. Given the high consumption of muscle tissue, it is important to understand what factors could influence the establishment of a prion infection in muscle tissue. Here we used in vitro myotube cultures, differentiated from the C2C12 myoblast cell line (dC2C12), to identify factors affecting prion replication. A range of experimental conditions revealed that PrPSc is tightly associated with proteins found in the systemic extracellular matrix, mostly fibronectin (FN). The interaction of PrPSc with FN decreased prion infectivity, as determined by standard scrapie cell assay. Interestingly, the prion-resistant reserve cells in dC2C12 cultures displayed a FN-rich extracellular matrix while the prion-susceptible myotubes expressed FN at a low level. In agreement with the in vitro results, immunohistopathological analyses of tissues from sheep infected with natural scrapie demonstrated a prion susceptibility phenotype linked to an extracellular matrix with undetectable levels of FN. Conversely, PrPSc deposits were not observed in tissues expressing FN. These data indicate that extracellular FN may act as a natural barrier against prion replication and that the extracellular matrix composition may be a crucial feature determining prion tropism in different tissues

Optical Drug Monitoring: Photoacoustic Imaging of Nanosensors to Monitor Therapeutic Lithium in Vivo

Personalized medicine could revolutionize how primary care physicians treat chronic disease and how researchers study fundamental biological questions. To realize this goal, we need to develop more robust, modular tools and imaging approaches for in vivo monitoring of analytes. In this report, we demonstrate that synthetic nanosensors can measure physiologic parameters with photoacoustic contrast, and we apply that platform to continuously track lithium levels in vivo. Photoacoustic imaging achieves imaging depths that are unattainable with fluorescence or multiphoton microscopy. We validated the photoacoustic results that illustrate the superior imaging depth and quality of photoacoustic imaging with optical measurements. This powerful combination of techniques will unlock the ability to measure analyte changes in deep tissue and will open up photoacoustic imaging as a diagnostic tool for continuous physiological tracking of a wide range of analytes

Remdesivir does not affect mitochondrial DNA copy number or deletion mutation frequency in aged male rats: A short report.

Remdesivir is a leading therapy in patients with moderate to severe coronavirus 2 (SARS-CoV-2) infection; the majority of whom are older individuals. Remdesivir is a nucleoside analog that incorporates into nascent viral RNA, inhibiting RNA-directed RNA polymerases, including that of SARS-CoV-2. Less is known about remdesivir's effects on mitochondria, particularly in older adults where mitochondria are known to be dysfunctional. Furthermore, its effect on age-induced mitochondrial mutations and copy number has not been previously studied. We hypothesized that remdesivir adversely affects mtDNA copy number and deletion mutation frequency in aged rodents. To test this hypothesis, 30-month-old male F333BNF1 rats were treated with remdesivir for three months. To determine if remdesivir adversely affects mtDNA, we measured copy number and mtDNA deletion frequency in rat hearts, kidneys, and skeletal muscles using digital PCR. We found no effects from three months of remdesivir treatment on mtDNA copy number or deletion mutation frequency in 33-month-old rats. These data support the notion that remdesivir does not compromise mtDNA quality or quantity at old age in mammals. Future work should focus on examining additional tissues such as brain and liver, and extend testing to human clinical samples

Age- and time-dependent mitochondrial genotoxic and myopathic effects of beta-guanidinopropionic acid, a creatine analog, on rodent skeletal muscles

Beta-guanidinopropionic acid (GPA) is a creatine analog suggested as a treatment for hypertension, diabetes, and obesity, which manifest primarily in older adults. A notable side effect of GPA is the induction of mitochondrial DNA deletion mutations. We hypothesized that mtDNA deletions contribute to muscle aging and used the mutation promoting effect of GPA to examine the impact of mtDNA deletions on muscles with differential vulnerability to aging. Rats were treated with GPA for up to 4 months starting at 14 or 30 months of age. We examined quadriceps and adductor longus muscles as the quadriceps exhibits profound age-induced deterioration, while adductor longus is maintained. GPA decreased body and muscle mass and mtDNA copy number while increasing mtDNA deletion frequency. The interactions between age and GPA treatment observed in the quadriceps were not observed in the adductor longus. GPA had negative mitochondrial effects in as little as 4 weeks. GPA treatment exacerbated mtDNA deletions and muscle aging phenotypes in the quadriceps, an age-sensitive muscle, while the adductor longus was spared. GPA has been proposed for use in age-associated diseases, yet the pharmacodynamics of GPA differ with age and include the detrimental induction of mtDNA deletions, a mitochondrial genotoxic stress that is pronounced in muscles that are most vulnerable to aging. Further research is needed to determine if the proposed benefits of GPA on hypertension, diabetes, and obesity outweigh the detrimental mitochondrial and myopathic side effects

PrP^CWD immunolabeling adjacent to sudoriferous and sebaceous glands of a female mule deer hind interdigital gland.

(A) Immune cell infiltrates between sebaceous and sudoriferous glands with PrPCWD immunolabeling (red). (B) Immune cell infiltrates near the epidermis with PrPCWD immunolabeling (arrows) adjacent to a sebaceous glandular element. Inset shows PrPCWD with increased magnification. PrPCWD was immunolabeled with anti-PrP BAR224 (1:2,000). (C-D). Adjacent section hematoxylin and eosin staining. Abbreviations: APM, arrector pili muscle; E, epidermis; Se, sebaceous gland; Su, sudoriferous gland.</p

PrP^CWD immunolabeling between sudoriferous glands of a female mule deer hind interdigital gland.

(A-B) Adjacent sections of immune cell infiltrates between sudoriferous glands with PrPCWD immunolabeling (red). PrPCWD was immunolabeled with anti-PrP BAR224 (1:2,000). (C) Negative control section without primary antibody. (D) Hematoxylin and eosin staining.</p

Conceptual diagram of sample origin and use.

Conceptual diagram of sample origin and use.</p

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