NF-κB/NLRP3 Translational Inhibition by Nanoligomer Therapy Mitigates Ethanol and Advanced Age-Related Neuroinflammation.

Binge alcohol use is increasing among aged adults (>65 years). Alcohol-related toxicity in aged adults is associated with neurodegeneration, yet the molecular underpinnings of age-related sensitivity to alcohol are not well described. Studies utilizing rodent models of neurodegenerative disease reveal heightened activation of Nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and Nod like receptor 3 (NLRP3) mediate microglia activation and associated neuronal injury. Our group, and others, have implicated hippocampal-resident microglia as key producers of inflammatory mediators, yet the link between inflammation and neurodegeneration has not been established in models of binge ethanol exposure and advanced age. Here, we report binge ethanol increased the proportion of NLRP3+ microglia in the hippocampus of aged (18-20 months) female C57BL/6N mice compared to young (3-4 months). In primary microglia, ethanol-induced expression of reactivity markers and NLRP3 inflammasome activation were more pronounced in microglia from aged mice compared to young. Making use of an NLRP3-specific inhibitor (OLT1177) and a novel brain-penetrant Nanoligomer that inhibits NF-κB and NLRP3 translation (SB_NI_112), we find ethanol-induced microglial reactivity can be attenuated by OLT1177 and SB_NI_112 in microglia from aged mice. In a model of intermittent binge ethanol exposure, SB_NI_112 prevented ethanol-mediated microglia reactivity, IL-1ß production, and tau hyperphosphorylation in the hippocampus of aged mice. These data suggest early indicators of neurodegeneration occurring with advanced age and binge ethanol exposure are NF-κB- and NLRP3-dependent. Further investigation is warranted to explore the use of targeted immunosuppression via Nanoligomers to attenuate neuroinflammation after alcohol consumption in the aged.

Targeting Neuroinflammation by Pharmacologic Downregulation of Inflammatory Pathways Is Neuroprotective in Protein Misfolding Disorders.

Neuroinflammation plays a crucial role in the development of neurodegenerative protein misfolding disorders. This category of progressive diseases includes, but is not limited to, Alzheimer's disease, Parkinson's disease, and prion diseases. Shared pathogenesis involves the accumulation of misfolded proteins, chronic neuroinflammation, and synaptic dysfunction, ultimately leading to irreversible neuronal loss, measurable cognitive deficits, and death. Presently, there are few to no effective treatments to halt the advancement of neurodegenerative diseases. We hypothesized that directly targeting neuroinflammation by downregulating the transcription factor, NF-κB, and the inflammasome protein, NLRP3, would be neuroprotective. To achieve this, we used a cocktail of RNA targeting therapeutics (SB_NI_112) shown to be brain-penetrant, nontoxic, and effective inhibitors of both NF-κB and NLRP3. We utilized a mouse-adapted prion strain as a model for neurodegenerative diseases to assess the aggregation of misfolded proteins, glial inflammation, neuronal loss, cognitive deficits, and lifespan. Prion-diseased mice were treated either intraperitoneally or intranasally with SB_NI_112. Behavioral and cognitive deficits were significantly protected by this combination of NF-κB and NLRP3 downregulators. Treatment reduced glial inflammation, protected against neuronal loss, prevented spongiotic change, rescued cognitive deficits, and significantly lengthened the lifespan of prion-diseased mice. We have identified a nontoxic, systemic pharmacologic that downregulates NF-κB and NLRP3, prevents neuronal death, and slows the progression of neurodegenerative diseases. Though mouse models do not always predict human patient success and the study was limited due to sample size and number of dosing methods utilized, these findings serve as a proof of principle for continued translation of the therapeutic SB_NI_112 for prion disease and other neurodegenerative diseases. Based on the success in a murine prion model, we will continue testing SB_NI_112 in a variety of neurodegenerative disease models, including Alzheimer's disease and Parkinson's disease.

Targeted-Neuroinflammation Mitigation Using Inflammasome-Inhibiting Nanoligomers is Therapeutic in an Experimental Autoimmune Encephalomyelitis Mouse Model.

Multiple sclerosis (MS) is a debilitating autoimmune disease that impacts millions of patients worldwide, disproportionately impacting women (4:1), and often presenting at highly productive stages of life. This disease affects the spinal cord and brain and is characterized by severe neuroinflammation, demyelination, and subsequent neuronal damage, resulting in symptoms like loss of mobility. While untargeted and pan-immunosuppressive therapies have proven to be disease-modifying and manage (or prolong the time between) symptoms in many patients, a significant fraction are unable to achieve remission. Recent work has suggested that targeted neuroinflammation mitigation through selective inflammasome inhibition can offer relief to patients while preserving key components of immune function. Here, we show a screening of potential therapeutic targets using inflammasome-inhibiting Nanoligomers (NF-κB1, TNFR1, TNF-α, IL-6) that meet or far-exceed commercially available small-molecule counterparts like ruxolitinib, MCC950, and deucravacitinib. Using the human brain organoid model, top Nanoligomer combinations (NF-κB1 + TNFR1: NI111, and NF-κB1 + NLRP3: NI112) were shown to significantly reduce neuroinflammation without any observable negative impact on organoid function. Further testing of these top Nanoligomer combinations in an aggressive experimental autoimmune encephalomyelitis (EAE) mouse model for MS using intraperitoneal (IP) injections showed that NF-κB1 and NLRP3 targeting Nanoligomer combination NI112 rescues mice without observable loss of mobility or disability, minimal inflammation in brain and spinal cord histology, and minimal to no immune cell infiltration of the spinal cord and no demyelination, similar to or at par with mice that received no EAE injections (negative control). Mice receiving NI111 (NF-κB1 + TNFR1) also showed reduced neuroinflammation compared to saline (sham)-treated EAE mice and at par/similar to other inflammasome-inhibiting small molecule treatments, although it was significantly higher than NI112 leading to subsequent worsening clinical outcomes. Furthermore, treatment with an oral formulation of NI112 at lower doses showed a significant reduction in EAE severity, albeit with higher variance owing to administration and formulation/fill-and-finish variability. Overall, these results point to the potential of further development and testing of these inflammasome-targeting Nanoliogmers as an effective neuroinflammation treatment for multiple neurodegenerative diseases and potentially benefit several patients suffering from such debilitating autoimmune diseases like MS.

Nanoligomers targeting NF-κB and NLRP3 reduce neuroinflammation and improve cognitive function with aging and tauopathy.

Neuroinflammation contributes to impaired cognitive function in brain aging and neurodegenerative disorders like Alzheimer's disease, which is characterized by the aggregation of pathological tau. One major driver of both age- and tau-associated neuroinflammation is the NF-κB and NLRP3 signaling axis. However, current treatments targeting NF-κB or NLRP3 may have adverse/systemic effects, and most have not been clinically translatable. Here, we tested the efficacy of a novel, nucleic acid therapeutic (Nanoligomer) cocktail specifically targeting both NF-κB and NLRP3 in the brain for reducing neuroinflammation and improving cognitive function in old wildtype mice, and in a mouse model of tauopathy. We found that 4 weeks of NF-κB/NLRP3-targeting Nanoligomer treatment strongly reduced neuro-inflammatory cytokine profiles in the brain and improved cognitive-behavioral function in both old and tauopathy mice. These effects of NF-κB/NLRP3-targeting Nanoligomer treatment were associated with reduced glial cell activation in old wildtype mice, less pathology in tauopathy mice, favorable changes in transcriptome signatures of inflammation (reduced) and neuronal health (increased) in both mouse models, and positive systemic effects. Collectively, our results provide a basis for future translational studies targeting NF-κB/NLRP3 in the brain, perhaps using Nanoligomers, to inhibit neuroinflammation and improve cognitive function with aging and neurodegenerative disease.

Adipose-Derived Mesenchymal Stromal Cells Co-Cultured with Primary Mixed Glia to Reduce Prion-Induced Inflammation.

Mesenchymal stromal cells (MSCs) are potent regulators of inflammation through the production of anti-inflammatory cytokines, chemokines, and growth factors. These cells show an ability to regulate neuroinflammation in the context of neurodegenerative diseases such as prion disease and other protein misfolding disorders. Prion diseases can be sporadic, acquired, or genetic; they can result from the misfolding and aggregation of the prion protein in the brain. These diseases are invariably fatal, with no available treatments. One of the earliest signs of disease is the activation of astrocytes and microglia and associated inflammation, which occurs prior to detectable prion aggregation and neuronal loss; thus, the anti-inflammatory and regulatory properties of MSCs can be harvested to treat astrogliosis in prion disease. Recently, we showed that adipose-derived MSCs (AdMSCs) co-cultured with BV2 cells or primary mixed glia reduce prion-induced inflammation through paracrine signaling. This paper describes a reliable treatment using stimulated AdMSCs to decrease prion-induced inflammation. A heterozygous population of AdMSCs can easily be isolated from murine adipose tissue and expanded in culture. Stimulating these cells with inflammatory cytokines enhances their ability to both migrate toward prion-infected brain homogenate and produce anti-inflammatory modulators in response. Together, these techniques can be used to investigate the therapeutic potential of MSCs on prion infection and can be adapted for other protein misfolding and neuroinflammatory diseases.

Biological agents and the aging brain: glial inflammation and neurotoxic signaling.

Neuroinflammation is a universal characteristic of brain aging and neurological disorders, irrespective of the disease state. Glial inflammation mediates this signaling, through astrocyte and microglial polarization from neuroprotective to neurotoxic phenotypes. Glial reactivity results in the loss of homeostasis, as these cells no longer provide support to neurons, in addition to the production of chronically toxic pro-inflammatory mediators. These glial changes initiate an inflammatory brain state that injures the central nervous system (CNS) over time. As the brain ages, glia are altered, including increased glial cell numbers, morphological changes, and either a pre-disposition or inability to become reactive. These alterations induce age-related neuropathologies, ultimately leading to neuronal degradation and irreversible damage associated with disorders of the aged brain, including Alzheimer's Disease (AD) and other related diseases. While the complex interactions of these glial cells and the brain are well studied, the role additional stressors, such as infectious agents, play on age-related neuropathology has not been fully elucidated. Both biological agents in the periphery, such as bacterial infections, or in the CNS, including viral infections like SARS-CoV-2, push glia into neuroinflammatory phenotypes that can exacerbate pathology within the aging brain. These biological agents release pattern associated molecular patterns (PAMPs) that bind to pattern recognition receptors (PRRs) on glial cells, beginning an inflammatory cascade. In this review, we will summarize the evidence that biological agents induce reactive glia, which worsens age-related neuropathology.

Gliosis, misfolded protein aggregation, and neuronal loss in a guinea pig model of pulmonary tuberculosis.

Tuberculosis, caused by Mycobacterium tuberculosis infection, is an ongoing epidemic with an estimated ten million active cases of the disease worldwide. Pulmonary tuberculosis is associated with cognitive and memory deficits, and patients with this disease are at an increased risk for Parkinson's disease and dementia. Although epidemiological data correlates neurological effects with peripheral disease, the pathology in the central nervous system is unknown. In an established guinea pig model of low-dose, aerosolized Mycobacterium tuberculosis infection, we see behavior changes and memory loss in infected animals. We correlate these findings with pathological changes within brain regions related to motor, cognition, and sensation across disease progression. This includes microglial and astrocytic proliferation and reactivity. These cellular changes are followed by the aggregation of neurotoxic amyloid ß and phosphorylated tau and, ultimately, neuronal degeneration in the hippocampus. Through these data, we have obtained a greater understanding of the neuropathological effects of a peripheral disease that affects millions of persons worldwide.

Activated gliosis, accumulation of amyloid ß, and hyperphosphorylation of tau in aging canines with and without cognitive decline.

Canine cognitive dysfunction (CCD) syndrome is a well-recognized naturally occurring disease in aged dogs, with a remarkably similar disease course, both in its clinical presentation and neuropathological changes, as humans with Alzheimer's disease (AD). Similar to human AD patients this naturally occurring disease is found in the aging canine population however, there is little understanding of how the canine brain ages pathologically. It is well known that in neurodegenerative diseases, there is an increase in inflamed glial cells as well as an accumulation of hyperphosphorylation of tau (P-tau) and amyloid beta (Aß1-42). These pathologies increase neurotoxic signaling and eventual neuronal loss. We assessed these brain pathologies in aged canines and found an increase in the number of glial cells, both astrocytes and microglia, and the activation of astrocytes indicative of neuroinflammation. A rise in the aggregated protein Aß1-42 and hyperphosphorylated tau, at Threonine 181 and 217, in the cortical brain regions of aging canines. We then asked if any of these aged canines had CCD utilizing the only current diagnostic, owner questionnaires, verifying positive or severe CCD had pathologies of gliosis and accumulation of Aß1-42 like their aged, matched controls. However uniquely the CCD dogs had P-tau at T217. Therefore, this phosphorylation site of tau at threonine 217 may be a predictor for CCD.

Intranasally delivered mesenchymal stromal cells decrease glial inflammation early in prion disease.

Mesenchymal stromal cells (MSCs) are an intriguing avenue for the treatment of neurological disorders due to their ability to migrate to sites of neuroinflammation and respond to paracrine signaling in those sites by secreting cytokines, growth factors, and other neuromodulators. We potentiated this ability by stimulating MSCs with inflammatory molecules, improving their migratory and secretory properties. We investigated the use of intranasally delivered adipose-derived MSCs (AdMSCs) in combating prion disease in a mouse model. Prion disease is a rare, lethal neurodegenerative disease that results from the misfolding and aggregation of the prion protein. Early signs of this disease include neuroinflammation, activation of microglia, and development of reactive astrocytes. Later stages of disease include development of vacuoles, neuronal loss, abundant aggregated prions, and astrogliosis. We demonstrate the ability of AdMSCs to upregulate anti-inflammatory genes and growth factors when stimulated with tumor necrosis factor alpha (TNFα) or prion-infected brain homogenates. We stimulated AdMSCs with TNFα and performed biweekly intranasal deliveries of AdMSCs on mice that had been intracranially inoculated with mouse-adapted prions. At early stages in disease, animals treated with AdMSCs showed decreased vacuolization throughout the brain. Expression of genes associated with Nuclear Factor-kappa B (NF-κB) and Nod-Like Receptor family pyrin domain containing 3 (NLRP3) inflammasome signaling were decreased in the hippocampus. AdMSC treatment promoted a quiescent state in hippocampal microglia by inducing changes in both number and morphology. Animals that received AdMSCs showed a decrease in both overall and reactive astrocyte number, and morphological changes indicative of homeostatic astrocytes. Although this treatment did not prolong survival or rescue neurons, it demonstrates the benefits of MSCs in combatting neuroinflammation and astrogliosis.

Effects of Mycobacterium vaccae NCTC 11659 and Lipopolysaccharide Challenge on Polarization of Murine BV-2 Microglial Cells.

Previous studies have shown that the in vivo administration of soil-derived bacteria with anti-inflammatory and immunoregulatory properties, such as Mycobacterium vaccae NCTC 11659, can prevent a stress-induced shift toward an inflammatory M1 microglial immunophenotype and microglial priming in the central nervous system (CNS). It remains unclear whether M. vaccae NCTC 11659 can act directly on microglia to mediate these effects. This study was designed to determine the effects of M. vaccae NCTC 11659 on the polarization of naïve BV-2 cells, a murine microglial cell line, and BV-2 cells subsequently challenged with lipopolysaccharide (LPS). Briefly, murine BV-2 cells were exposed to 100 µg/mL whole-cell, heat-killed M. vaccae NCTC 11659 or sterile borate-buffered saline (BBS) vehicle, followed, 24 h later, by exposure to 0.250 µg/mL LPS (Escherichia coli 0111: B4; n = 3) in cell culture media vehicle (CMV) or a CMV control condition. Twenty-four hours after the LPS or CMV challenge, cells were harvested to isolate total RNA. An analysis using the NanoString platform revealed that, by itself, M. vaccae NCTC 11659 had an "adjuvant-like" effect, while exposure to LPS increased the expression of mRNAs encoding proinflammatory cytokines, chemokine ligands, the C3 component of complement, and components of inflammasome signaling such as Nlrp3. Among LPS-challenged cells, M. vaccae NCTC 11659 had limited effects on differential gene expression using a threshold of 1.5-fold change. A subset of genes was assessed using real-time reverse transcription polymerase chain reaction (real-time RT-PCR), including Arg1, Ccl2, Il1b, Il6, Nlrp3, and Tnf. Based on the analysis using real-time RT-PCR, M. vaccae NCTC 11659 by itself again induced "adjuvant-like" effects, increasing the expression of Il1b, Il6, and Tnf while decreasing the expression of Arg1. LPS by itself increased the expression of Ccl2, Il1b, Il6, Nlrp3, and Tnf while decreasing the expression of Arg1. Among LPS-challenged cells, M. vaccae NCTC 11659 enhanced LPS-induced increases in the expression of Nlrp3 and Tnf, consistent with microglial priming. In contrast, among LPS-challenged cells, although M. vaccae NCTC 11659 did not fully prevent the effects of LPS relative to vehicle-treated control conditions, it increased Arg1 mRNA expression, suggesting that M. vaccae NCTC 11659 induces an atypical microglial phenotype. Thus, M. vaccae NCTC 11659 acutely (within 48 h) induced immune-activating and microglial-priming effects when applied directly to murine BV-2 microglial cells, in contrast to its long-term anti-inflammatory and immunoregulatory effects observed on the CNS when whole-cell, heat-killed preparations of M. vaccae NCTC 11659 were given peripherally in vivo.

Adipose-derived mesenchymal stromal cells decrease prion-induced glial inflammation in vitro.

Prion diseases are characterized by the cellular prion protein, PrPC, misfolding and aggregating into the infectious prion protein, PrPSc, which leads to neurodegeneration and death. An early sign of disease is inflammation in the brain and the shift of resting glial cells to reactive astrocytes and activated microglia. Few therapeutics target this stage of disease. Mesenchymal stromal cells produce anti-inflammatory molecules when exposed to inflammatory signals and damaged tissue. Here, we show that adipose-derived mesenchymal stromal cells (AdMSCs) migrate toward prion-infected brain homogenate and produce the anti-inflammatory molecules transforming growth factor ß (TGFß) and tumor necrosis factor-stimulated gene 6 (TSG-6). In an in vitro model of prion exposure of both primary mixed glia and BV2 microglial cell line, co-culturing with AdMSCs led to a significant decrease in inflammatory cytokine mRNA and markers of reactive astrocytes and activated microglia. This protection against in vitro prion-associated inflammatory responses is independent of PrPSc replication. These data support a role for AdMSCs as a beneficial therapeutic for decreasing the early onset of glial inflammation and reprogramming glial cells to a protective phenotype.

Assessment of risk factors in dogs with presumptive advanced canine cognitive dysfunction.

Objectives: The aim of this study was to investigate the potential risk factors involved in the development of presumptive advanced canine cognitive dysfunction (pACCD). Materials and methods: A questionnaire was developed to identify dogs with presumptive canine cognitive dysfunction (CCD) based on an adapted Canine Dementia Scale and to evaluate for potential risk factors among the presumptive advanced cognitive dysfunction group. The questionnaire was distributed to 7,574 owners of dogs (≥8 years of age) who presented to the CSU VTH between 2017 and 2020. Dogs were classified into four groups based on the Canine Dementia Scale score (normal, mild, moderate, and severe cognitive impairment) and two subgroups for the cognitively impaired groups based on the presence or absence of underlying medical conditions. Comparisons between normal and presumptive advanced cognitively impaired groups, with and without underlying medical conditions, were made against various risk factors. Chi-square tests and logistic regression analysis were used to determine associations between categorical variables and a p-value of <0.05 was considered indicative of evidence of association. Results: The completed response rate for the questionnaire was 14.2% (1,079/7,574). Among those, 231 dogs were classified as having presumptive advanced cognitive dysfunction. The prevalence of presumptive advanced cognitive dysfunction in the included age groups was 8.1% in ages 8 to <11 years, 18.8% in ages 11 to <13 years, 45.3% in ages 13 to <15 years, 67.3% in ages 15 to <17 years, and 80% in ages >17 years. Dogs with a thin body condition score had the largest contribution to the chi-square statistic. Based on the logistic regression model, both age (p < 0.001) and BCS (p = 0.0057) are associated with presumptive ACCD. Conclusion and relevance: The chi-square test and logistic regression analysis both suggested an association between a thin body condition and an increased chance of cognitive decline. However, it is difficult to determine if the thin BCS in this group could be secondary to another confounding factor. The prevalence of cognitive dysfunction rapidly increased with age in this study. These findings warrant continued studies including veterinary evaluations to explore risk factors of canine dementia.

Survey of risk factors and frequency of clinical signs observed with feline cognitive dysfunction syndrome.

OBJECTIVES: The aims of this study were to distribute a survey to cat owners to identify common clinical signs of feline cognitive dysfunction (FCD) and to evaluate for potential risk factors. METHODS: A questionnaire was developed and adapted based on previously validated canine cognitive dysfunction questionnaires. This questionnaire was distributed to 4342 cat owners who had presented to Colorado State University Veterinary Teaching Hospital between 2015 and 2020. Cats aged ⩾8 years with signs of cognitive dysfunction and no underlying medical conditions were classified as the FCD-positive group. Cats aged ⩾8 years with no signs of cognitive dysfunction were classified as the FCD-negative control group. Chi-square or Fisher's exact tests were used to determine associations between categorical variables and a P value <0.05 was considered indicative of evidence of association. RESULTS: A total of 615 completed survey responses were recorded, which was a response rate of 14.2%. Among those, 80 (13%) cats were identified as the FCD-positive group and 114 (18.5%) were identified as the FCD-negative control group. The most common clinical sign in the FCD-positive group was inappropriate vocalization (32/80, 40.0%). The only variable determined to have an association with the FCD group (positive or negative), with a P value of 0.033, was the environmental setting. Cats living in a rural environment (FCD-positive or -negative) had the largest contribution to the χ2 statistic. CONCLUSION AND RELEVANCE: The observed number of FCD-positive cats living in a rural community was less than the expected value based on the χ2 tests. This is suggestive of an association between living in a rural environment and a reduced chance of cognitive dysfunction. There are many factors such as air pollution, social interactions and environmental enrichment that need to be studied further to determine how they relate to FCD as this could not be concluded from this study.

Nontransgenic Guinea Pig Strains Exhibit Hallmarks of Human Brain Aging and Alzheimer's Disease.

Older age is the primary risk factor for most chronic diseases, including Alzheimer's disease (AD). Current preclinical models to study brain aging and AD are mainly transgenic and harbor mutations intended to mirror brain pathologies associated with human brain aging/AD (eg, by increasing production of the amyloid precursor protein, amyloid beta [Aß], and/or phosphorylated tau, all of which are key pathological mediators of AD). Although these models may provide insight on pathophysiological processes in AD, none completely recapitulate the disease and its strong age-dependence, and there has been limited success in translating preclinical results and treatments to humans. Here, we describe 2 nontransgenic guinea pig (GP) models, a standard PigmEnTed (PET) strain, and lesser-studied Dunkin-Hartley (DH) strain, that may naturally mimic key features of brain aging and AD in humans. We show that brain aging in PET GP is transcriptomically similar to human brain aging, whereas older DH brains are transcriptomically more similar to human AD. Both strains/models also exhibit increased neurofilament light chain (NFL, a marker of neuronal damage) with aging, and DH animals display greater S100 calcium-binding protein B (S100ß), ionized calcium-binding adapter molecule 1 (Iba1), and Aß and phosphorylated tau-which are all important markers of neuroinflammation-associated AD. Collectively, our results suggest that both the PET and DH GP may be useful, nontransgenic models to study brain aging and AD, respectively.

Tissue-specific biochemical differences between chronic wasting disease prions isolated from free-ranging white-tailed deer (Odocoileus virginianus).

Chronic wasting disease (CWD) is an invariably fatal prion disease affecting cervid species worldwide. Prions can manifest as distinct strains that can influence disease pathology and transmission. CWD is profoundly lymphotropic, and most infected cervids likely shed peripheral prions replicated in lymphoid organs. However, CWD is a neurodegenerative disease, and most research on prion strains has focused on neurogenic prions. Thus, a knowledge gap exists comparing neurogenic prions to lymphogenic prions. In this study, we compared prions from the obex and lymph nodes of naturally exposed white-tailed deer to identify potential biochemical strain differences. Here, we report biochemical evidence of strain differences between the brain and lymph node from these animals. Conformational stability assays, glycoform ratio analyses, and immunoreactivity scanning across the structured domain of the prion protein that refolds into the amyloid aggregate of the infectious prion reveal significantly more structural and glycoform variation in lymphogenic prions than neurogenic prions. Surprisingly, we observed greater biochemical differences among neurogenic prions than lymphogenic prions across individuals. We propose that the lymphoreticular system propagates a diverse array of prions from which the brain selects a more restricted pool of prions that may be quite different than those from another individual of the same species. Future work should examine the biological and zoonotic impact of these biochemical differences and examine more cervids from multiple locations to determine if these differences are conserved across species and locations.

Adaptive selection of a prion strain conformer corresponding to established North American CWD during propagation of novel emergent Norwegian strains in mice expressing elk or deer prion protein.

Prions are infectious proteins causing fatal, transmissible neurodegenerative diseases of animals and humans. Replication involves template-directed refolding of host encoded prion protein, PrPC, by its infectious conformation, PrPSc. Following its discovery in captive Colorado deer in 1967, uncontrollable contagious transmission of chronic wasting disease (CWD) led to an expanded geographic range in increasing numbers of free-ranging and captive North American (NA) cervids. Some five decades later, detection of PrPSc in free-ranging Norwegian (NO) reindeer and moose marked the first indication of CWD in Europe. To assess the properties of these emergent NO prions and compare them with NA CWD we used transgenic (Tg) and gene targeted (Gt) mice expressing PrP with glutamine (Q) or glutamate (E) at residue 226, a variation in wild type cervid PrP which influences prion strain selection in NA deer and elk. Transmissions of NO moose and reindeer prions to Tg and Gt mice recapitulated the characteristic features of CWD in natural hosts, revealing novel prion strains with disease kinetics, neuropathological profiles, and capacities to infect lymphoid tissues and cultured cells that were distinct from those causing NA CWD. In support of strain variation, PrPSc conformers comprising emergent NO moose and reindeer CWD were subject to selective effects imposed by variation at residue 226 that were different from those controlling established NA CWD. Transmission of particular NO moose CWD prions in mice expressing E at 226 resulted in selection of a kinetically optimized conformer, subsequent transmission of which revealed properties consistent with NA CWD. These findings illustrate the potential for adaptive selection of strain conformers with improved fitness during propagation of unstable NO prions. Their potential for contagious transmission has implications for risk analyses and management of emergent European CWD. Finally, we found that Gt mice expressing physiologically controlled PrP levels recapitulated the lymphotropic properties of naturally occurring CWD strains resulting in improved susceptibilities to emergent NO reindeer prions compared with over-expressing Tg counterparts. These findings underscore the refined advantages of Gt models for exploring the mechanisms and impacts of strain selection in peripheral compartments during natural prion transmission.

Network Simulations Reveal Molecular Signatures of Vulnerability to Age-Dependent Stress and Tau Accumulation.

Alzheimer's disease (AD) is the leading cause of dementia and one of the most common causes of death worldwide. As an age-dependent multifactorial disease, the causative triggers of AD are rooted in spontaneous declines in cellular function and metabolic capacity with increases in protein stressors such as the tau protein. This multitude of age-related processes that cause neurons to change from healthy states to ones vulnerable to the damage seen in AD are difficult to simultaneously investigate and even more difficult to quantify. Here we aimed to diminish these gaps in our understanding of neuronal vulnerability in AD development by using simulation methods to theoretically quantify an array of cellular stress responses and signaling molecules. This temporally-descriptive molecular signature was produced using a novel multimethod simulation approach pioneered by our laboratory for biological research; this methodology combines hierarchical agent-based processes and continuous equation-based modeling in the same interface, all while maintaining intrinsic distributions that emulate natural biological stochasticity. The molecular signature was validated for a normal organismal aging trajectory using experimental longitudinal data from Caenorhabditis elegans and rodent studies. In addition, we have further predicted this aging molecular signature for cells impacted by the pathogenic tau protein, giving rise to distinct stress response conditions needed for cytoprotective aging. Interestingly, our simulation experiments showed that oxidative stress signaling (via daf-16 and skn-1 activities) does not substantially protect cells from all the early stressors of aging, but that it is essential in preventing a late-life degenerative cellular phenotype. Together, our simulation experiments aid in elucidating neurodegenerative triggers in the onset of AD for different genetic conditions. The long-term goal of this work is to provide more detailed diagnostic and prognostic tools for AD development and progression, and to provide more comprehensive preventative measures for this disease.

The cellular prion protein promotes neuronal regeneration after acute nasotoxic injury.

Adult neurogenesis, analogous to early development, is comprised of several, often concomitant, processes including proliferation, differentiation, and formation of synaptic connections. However, due to continual, asynchronous turn-over, newly-born adult olfactory sensory neurons (OSNs) must integrate into existing circuitry. Additionally, OSNs express high levels of cellular prion protein (PrPC), particularly in the axon, which implies a role in this cell type. The cellular prion has been shown to be important for proper adult OSN neurogenesis primarily by stabilizing mature olfactory neurons within this circuitry. However, the role of PrPC on each specific adult neurogenic processes remains to be investigated in detail. To tease out the subtle effects of prion protein expression level, a large population of regenerating neurons must be investigated. The thyroid drug methimazole (MTZ) causes nearly complete OSN loss in rodents and is used as a model of acute olfactory injury, providing a mechanism to induce synchronized OSN regeneration. This study investigated the effect of PrPC on adult neurogenesis after acute nasotoxic injury. Altered PrPC levels affected olfactory sensory epithelial (OSE) regeneration, cell proliferation, and differentiation. Attempts to investigate the role of PrPC level on axon regeneration did not support previous studies, and glomerular targeting did not recover to vehicle-treated levels, even by 20 weeks. Together, these studies demonstrate that the cellular prion protein is critical for regeneration of neurons, whereby increased PrPC levels promote early neurogenesis, and that lack of PrPC delays the regeneration of this tissue after acute injury.

Primary structural differences at residue 226 of deer and elk PrP dictate selection of distinct CWD prion strains in gene-targeted mice.

Although the unifying hallmark of prion diseases is CNS neurodegeneration caused by conformational corruption of host prion protein (PrP) to its infective counterpart, contagious transmission of chronic wasting disease (CWD) results from shedding of prions produced at high titers in the periphery of diseased cervids. While deer and elk PrP primary structures are equivalent except at residue 226, which is glutamate in elk and glutamine in deer, the effect of this difference on CWD pathogenesis is largely unknown. Using a gene-targeting approach where the mouse PrP coding sequence was replaced with elk or deer PrP, we show that the resulting GtE226 and GtQ226 mice had distinct kinetics of disease onset, prion conformations, and distributions of prions in the brains of diseased mice following intracerebral CWD challenge. These findings indicate that amino acid differences at PrP residue 226 dictate the selection and propagation of divergent strains in deer and elk with CWD. Because prion strain properties largely dictate host-range potential, our findings suggest that prion strains from elk and deer pose distinct risks to sympatric species or humans exposed to CWD. GtE226 and GtQ226 mice were also highly susceptible to CWD prions following intraperitoneal and oral exposures, a characteristic that stood in stark contrast to previously produced transgenic models. Remarkably, disease transmission was effective when infected mice were cohoused with naïve cagemates. Our findings indicate that gene-targeted mice provide unprecedented opportunities to accurately investigate CWD peripheral pathogenesis, CWD strains, and mechanisms of horizontal CWD transmission.

Art Therapy, Community Building, Activism, and Outcomes.

This article is a descriptive study of two groups who came together through service-learning: The first group is graduate art therapy students enrolled in a research class, who partnered with six community agencies to help them prepare assignments for undergraduate service-learning students in a subsequent semester. The art therapy research students also assisted the agencies with program evaluation. The second group is the six directors of the community agencies who were preparing for service-learning students enrolled in an art history class titled Art as A Social Practice. Service-learning is an experiential pedagogy where community service is integrated into an academic course, and where the services performed meet genuine community needs. The hyphen in service-learning represents the ideal that both the students and community agencies experience benefits from the relationship, although in reality, it is often the experiences of the students rather than the agencies that receive greater attention in the scholarly research literature. The present article places focus on the community agencies that, in the process of planning for service-learners, made two unexpected requests: First they requested that the service-learners stay longer than one semester, and secondly, they requested assistance with evaluating the effectiveness of their programs. This article is about the efforts to respond to these requests through the assistance of art therapy research students. With growing trends in community-based art therapy practice, greater attention to the community agencies where art therapists work is necessary and valuable to art therapy preparation. The present article describes six distinctive communities, illustrating new frontiers of practice. The research students' experiences and the experiences of the community partners were assessed using qualitative methods that included pre and post-questionnaires, written reflections of students, interviews of agency directors and agency, student, and researcher focus group transcripts. This study will inform other art therapy programs who may want to use a service-learning approach to teaching research. A discussion of the promising practices of service-learning and research, as well as the challenges leads to recommendations for art therapy education.