University of Tennessee Health Science Center
UTHSC Digital Commons (University of Tennessee Health Science Center)Not a member yet
1172 research outputs found
Sort by
Assessing the Role of Faith-Based Organizations in Health Promotion
Get PDFObjective: The objective of this analysis was to evaluate the relationship between organizational readiness (OR) and weight loss and physical activity outcomes among faith-based organizations (FBOs).
Methods: Data for this study were collected in two phases. Phase I data were based on a feasibility study and targeted African-American individuals (N = 55) who participated in an obesity prevention program. The intervention was accomplished in two stages, which included a 10-week core period followed by a 6-month maintenance period. Phase II data were based on key informant interviews that were conducted with community health leaders (CHLs) (N=6) from participating FBOs. These interviews addressed six dimensions of readiness, with each dimension receiving an independent score that ranged from 1 to 9 (no awareness to a high level of community ownership). Dimension scores were averaged and each FBO was assigned a numerical OR score. OR scores were computed from the interview data, utilizing anchored rating scales outlined in the Community Readiness Model (CRM). These scores were subsequently combined with the Phase I data and used to statistically estimate the associations of OR. Linear mixed models, using SAS/STAT® software, were used to evaluate the relationship between OR scores and weight loss and physical activity while adjusting for covariates. A qualitative analysis of the Phase II data was also performed.
Results: Approximately 12.5% of the sample had an OR score of 4, 69.6% had an OR score of 5, while 17.9% were assigned an OR score of 7. An OR score of four indicated a pre-planning stage of readiness. Those with an OR score of 5 were in the preparation stage of readiness, while those with a score of seven were in the stabilization stage.
An OR score of 5 was associated with a significant increase in weight (2.532, p=0.048) when compared with an OR score of 7. Post hoc analysis revealed significant mean differences in weight when comparing congregation 1 with congregations 4 (difference=3.452, p=0.016) and 5(difference= 4.646, p=0.0005). Congregation 2 had a significant mean difference in weight compared to both congregations 4 (difference= 5.264, p\u3c0.0006) and 5 (difference= 6.457, p\u3c0.0001).
During the maintenance period, Group(s) with an OR score of 5 gained weight compared to those with an OR score of 7 (6.093, p=0.0018). Post hoc analyses revealed significant mean differences between congregation 1 and congregations 4 (difference=7.896, p=0.001) and 5 (difference= 10.708, p=0.003).
The mean activity level of group(s) with an OR score of 4 and 5 were 166.02 minutes (p\u3c0.0001) and 177.33 minutes (p\u3c0.001) lower, respectively, than the group with an OR score of 7. Post hoc analysis revealed significant mean differences in physical activity minutes for congregation 1 compared with congregations 2 (difference= -91.698, p=0.011), and 4 (difference= -203.90, p\u3c0.0001). There were also significant mean differences between congregations 2 versus 4 (-112.20, p= 0.010) and 4 versus 5 (155.18, p\u3c.0006). There were also statistically significant differences in physical activity by OR category. Physical activity minutes among group(s) with an OR score of 4 (-1284.21, p\u3c0.0001) and 5 (-933.21, p\u3c0.0001) were lower than those with a score of 7. The post hoc analysis revealed significant mean differences between congregations 2 (-2191.82, p\u3c0.0001), and 4 (-1631.77, p\u3c0.0001) when compared with congregation 1. There were also significant mean differences in physical activity between congregations 2 versus 3 (difference= 2557.60, p=0.0009) and 5(difference=1602.11, p\u3c0.001), 3 versus 4 (difference= -1997.55, p=0.007) and 4 versus 5 (difference= 1042.06, p\u3c0.001).
Despite the enthusiasm of participating FBO, the qualitative evaluation revealed that health behavior change can be difficult to adopt and maintain. Moving into an organization that is well structured is seemingly a great formula for success; however, the strength of the organization alone is not sufficient to promote and support health behavior change. Irrespective of an organization’s position on the readiness continuum, several barriers may exist. Primary obstacles included: age of the congregation, competing activities, time frame of the initiative, recognizing the issue and appropriate problem solving, motivation and cost of healthy food options.
Conclusion: Although physical activity and weight outcomes were associated with OR scores, the post-hoc analysis revealed variations in outcomes by congregation. Congregational differences may be attributable to intra-group distinctions rather than organizational readiness levels. Therefore, health promotion coordinators must work closely with FBO to pinpoint effective recruitment, implementation, and maintenance strategies that reach the community at various sectors
Neuroanatomical Maps and Taste Reactivity to Sweet, Umami, and Bitter Taste in the PBN of C57BL/6J Mice
Get PDFHumans can distinguish at least five different taste qualities, sour, salty, bitter, sweet, and umami (the savory taste of certain amino acids). In neuroscience research, behavioral testing is used to measure the ability of rodents (including inbred mice) to discriminate between the different taste qualities. Taste reactivity and two-bottle preference are behavioral tests that are utilized to investigate different aspects of taste. These tests involve either voluntary or forced consumption of taste stimuli, respectively. Either test can be used to infer the preference and palatability of the stimulus consumed by an animal.
In order to understand the basis of taste behavior, one must understand the organization of the taste pathway. As an organism consumes a particular food or fluid, it first binds to or activates taste receptors or channels located inside taste buds found in the oral cavity. This transduction event then produces a cascade of neuronal activation via sensory nerves that innervate the taste buds –branches of three cranial nerves (VII, IX, and X). These cranial nerves then synapse centrally in the nucleus of the solitary tract (NST) where the relayed taste information is kept relatively segregated from visceral input (which arrives via cranial nerve X).
From this point, the taste information is relayed to the parabrachial nucleus (PBN) in the pons, where the taste and visceral information now overlap. The PBN has not been studied as extensively as the NST in terms of taste representation, especially in regards to umami taste. A few recent studies have indicated that taste neurons in the PBN respond to sweet and synergistic umami (i.e. a combination of glutamate and a ribonucleotide) stimuli in a similar manner, providing a rationale for further study of the representation of these taste stimuli in this area.
Sweet and umami taste share a common G-protein-coupled taste receptor subunit, T1R3, that responds in combination with either T1R1 to transduce umami stimuli or T1R2 to transduce sweet stimuli. Aside from sharing a common taste receptor, previous studies using pharmacological manipulations, electrophysiology, conditioned taste aversion (CTA), and discrimination studies have shown a strong functional link between sweet and umami taste in rodents. Compounds found to be sweet taste inhibitors either entirely or partially block the nerve response to the prototypical umami stimulus monosodium glutamate (MSG), as well as a synergistic mixture of MSG combined with the cyclic nucleotide inosine monophosphate (IMP). When the epithelial sodium channel blocker amiloride is combined with MSG, both rats and mice have difficulty determining the difference between this umami stimulus and sucrose. Overall, it appears that some umami stimuli appear to be perceived as sucrose-like in rodents, which differs dramatically from the human perception of umami stimuli. Although umami taste has not been studied as comprehensively in mice as it has been in rats, it is important to investigate due to the widespread use of a variety of genetic mouse models in taste research. Along with using behavioral models, one might gauge the uniqueness of sweet and umami stimuli using an anatomical technique, such as visualization of the immediate early gene c-fos in PBN neurons. In fact, previous research has indicated stimulation with different taste qualities produces distinctive c-fos patterns in the PBN. For this current research study, my first hypothesis was that since previous studies suggested the similarity between sweet and umami compounds in C57BL/6J (B6) mice; stimuli of both taste qualities would produce similar levels of preference, consumption, and levels of taste reactivity behaviors. Secondly, I hypothesized that taste stimulation with either sweet (sucrose) or umami (monopotassium glutamate; MPG, or the synergistic mixture of MPG+IMP) stimuli would produce a similar c-fos expression pattern in sweet and umami stimuli, and this would also be distinct from the c-fos expression patterns elicited by both the bitter stimulus, quinine hydrochloride (QHCl) and water.
Overall, the preference tests revealed that both sucrose and umami stimuli (especially MSG+IMP) were preferred and consumed at a similarly high level in B6 mice. However, the taste reactivity test did not yield any insight into whether the sweet and umami taste stimuli were perceived as similar. However, taste reactivity to the bitter stimulus, QHCl, was easily distinguishable from the other tested taste stimuli. Using c-fos immunohistochemistry to visualize neuronal activation, I then compared staining patterns of activation evoked by: water, QHCl, sucrose, saccharin, MPG, and MPG+IMP in subdivisions of the PBN in B6 mice, as well as a few other non-taste brainstem areas (locus coeruleus and mesencephalic nucleus of the trigeminal nerve). Results showed that quinine elicited significantly less c-fos positive nuclei in the entire dorsal lateral (DL) subnucleus compared to water. A few other significant effects of the tastant stimuli were found in the rostral portion of the waist, central lateral (CL), and DL PBN subnuclei, but distinct c-fos representations were not found for each stimulus tested. To determine if tastant effects might have been subtler in terms of cell density or patterning; and
therefore, could have been missed using normal cell counting methods, I decided to use a three-dimensional mapping approach to examine c-fos expression in the PBN. Results of this new mapping approach suggest its potential usage in future studies
Regulation of IRF-3-Dependent Innate Immune Signaling Pathway by the PLpro Domain of Non-Structural Protein 3 (NSP3) of Severe Acute Respiratory Syndrome (SARS) Coronavirus
Get PDFThe induction of Type I Interferons (IFNs) is a powerful and rapid innate defense mechanism against viral infection, and many viruses have developed elaborate strategies to overcome the antiviral effects of IFN, ensuring their survival and replication. Severe acute respiratory syndrome coronavirus (SARS-CoV) is a highly pathogenic virus that causes severe lung disease in humans and is associated with high mortality rates. SARS-CoV, like all other successful viruses, encode proteins that counteract the innate immune response. A number of reports have indicated the papain-like protease (PLpro) domain of SARS-CoV Non-Structural Protein 3 (NSP3) as a powerful interferon antagonist, by suppressing interferon regulatory factor 3 (IRF3) dependent innate antiviral defenses. IRF3 plays a key role in viral-induced type I IFN induction pathway. Thus, viruses are well-known to evade the establishment of an antiviral state by regulating the activation of IRF3. However, functional studies detailing the PLpro IFN antagonistic abilities, are not describe in the context of the full length nsp3 protein, in which it is contained in virus infected cells. Nsp3 is the largest replicase gene product in the coronavirus genome, which contains several functional domains that are required for coronavirus replication. Establishment of a stable and controllable CoV-nsp3 expression system will allow the physiological relevant study of the PLpro mediated function of this protein. Here, I described the development of tetracycline-inducible mammalian cell lines for stable expression of the full length nsp3 of HCoV-OC43, HCoV-NL63, MERS-CoV, and SARS-CoV, respectively. Although these cell lines exhibited stable and tight control of nsp3 expression in the presence of tetracycline, I observed a variation in CoV’s nsp3 protein expression levels. However, HeLa-Fit-SCoV-nsp3 and HeLa-Fit-SCoV-nsp3-delPLP stable cell lines expressed SARS-nsp3 and SARS-nsp3-delPLP robustly and at comparable levels. I found that expression of SARS-CoV nsp3 compromised virus-induced expression of IRF-3-dependent antiviral genes and that such ability depended on the PLpro domain. In agreement with our previous study examining the effects of the PLpro domain, the inhibitory effect was downstream of the IRF-3 kinases while upstream of IRF-3. Overall, my data demonstrates that SARS-CoV nsp3 is a bona fide interferon antagonist, which acts through PLpro-mediated suppression of IRF-3 activation
Understanding Structural And Functional Mechanisms of Emi1 Inhibition of the Anaphase Promoting Complex
Get PDFHealthy, reproducing cells create and destroy proteins in an ordered manner. Both the concentration and localization of protein pools is important to regulate the many cellular processes necessary for the life of the cell. In order to produce new proteins, cells degrade existing materials such as proteins and organelles that are dispensable or troublesome in order to recycle their raw components. Degradation is accomplished largely through two major pathways: in bulk through processes such as autophagy and phagocytosis, or in a targeted manner through the ubiquitin-proteasome pathway. Autophagy utilizes an encompassing body to encapsulate targets and surrounding materials for decomposition in a regulated but relatively non-specific manner. The ubiquitin-proteasome pathway, however, is an exquisitely precise method of degradation capable of targeting specific pools of protein substrates. So-called ubiquitination generates a signal for degradation by the 26S proteasome machinery. In order to establish the degradation signal, the cell utilizes a cascade of 3 enzymes working in concert to organize substrates and ubiquitin. By using enzymes capable of substrate specificity, the cell can regulate large pools of proteins in a specific spatio-temporal manner.
In the present study, we employ biochemical and structure-based techniques to study proteins involved in an essential ubiquitination pathway involved in maintenance of the cell cycle. A mitotic regulator called the Anaphase Promoting Complex, also called Cyclosome (APC/C), modifies myriad substrates that control cell cycle activities. The APC/C is a ubiquitin ligase complex; it is the final enzyme in a tri-enzyme cascade and catalyzes the final step of Ub transfer from an E2 enzyme directly to substrates. A protein called Emi1 is responsible for directly binding and inhibiting the APC/C throughout interphase, when APC/C substrates are stabilized. We set out to study domains of Emi1 responsible for binding and inhibiting the APC/C and its association with E2 enzymes or substrates, with the aim of charcterizing both the mechanism of the inhibitor and the essential functional requirements of the APC/C, which are not well understood. We aim to visualize the APC/C in complex with Emi1 through electron microscopy and to accurately interpret the location and orientation of Emi1 within this density.
The APC/C is a large, ~1,200 kDa multi-protein complex, and Emi1 is a single protein of only 50 kDa. Despite its small size, even a small subdomain of Emi1 potently inhibits the APC/C through a combination of not well-characterized mechanisms. Emi1 has an APC/C recognition motif called a D-box that is typically found within APC/C substrates. Emi1’s D-box is recruited to the substrate-binding sites of APC/C and serves as a pseudo-substrate inhibitor. Emi1 also inhibits through distinct mechanisms both of the E2 enzymes that coordinate with APC/C function. An essential, folded Zinc-binding Region (ZBR) and a helical “Linker” sequence cooperate to bind and block APC/C from recruiting one of two APC-specific E2 enzymes, Ubch10. Emi1 also has a conserved C-terminal motif, with charge and sequence similar to the other APC/C- specific E2 enzyme, Ube2s. Emi1 competes directly with Ube2s for APC/C recruitment, and it is through the combination of these three mechanisms that are afforded by many motifs within Emi1, that makes Emi1 a potent APC/C inhibitor. When associated with Emi1, the APC/C is inefficient at both recruiting substrates and binding E2 enzymes, allowing for stabilization of APC/C substrates, which is important for regulation of timing of cell cycle processes
Pten Signaling in Regulatory T Cells and Inflammatory Disease
Get PDFRegulatory T (Treg) cells suppress CD4+ T cell responses during homeostasis and inflammation to prevent autoimmunity and other immune disorders. Although the transcriptional and epigenetic programs impacting Treg cell function have been extensively studied, the signaling and metabolic pathways underlying Treg stability and function are not fully understood. In this study, we determined the role of the phosphatase PTEN in Treg cells. We found that specific depletion of PTEN in Treg cells results in excessive TH1 and T follicular helper cells (TFH) responses, associated with elevated germinal center (GC) B cells and spontaneous development of autoimmune and lymphoproliferative disease in vivo. Interestingly, the exaggerated TFH and GC responses and autoimmune symptoms are suppressed when IFN-γ expression is abrogated in mice containing Pten-deficient Treg cells. Thus, the uncontrolled TH1-mediated inflammation in these mice drives aberrant TFH responses and autoimmune and lymphoproliferative disease. Mechanistically, we linked PTEN to mTORC2-mediated control of transcriptional and metabolic programs that enforce Treg cell stability and function. Consistent with this notion, deletion of Rictor, the obligate component for mTORC2, restores Treg cell function and stability in the absence of Pten. Similarly, partially restoring the activity of Foxo1, a downstream transcription factor negatively regulated by mTORC2 signaling, also largely rectified the defects of PTEN-deficient Treg cells. Together, these results establish that Treg cells rely on the PTEN-mTORC2-Foxo1 axis to maintain their stability and suppressive activity in controlling TH1 and TFH cell responses
Assessment of the Zoonotic Potential of a Novel Bovine Influenza Virus
Get PDFA novel orthomyxovirus was recently identified from pigs, with subsequent work suggesting the natural reservoir being bovine populations. The virus had genome characteristics most similar to influenza C viruses (ICV) but, due to the extent of sequence divergence, was proposed as a new genus, influenza D virus (IDV). Current literature on IDV has largely focused on the agricultural significance of the virus and provided evidence for the agricultural impact via observation of widespread prevalence and pathology in laboratory infected cattle. However, only one study, which identified 1.3% seroprevalence in a small cohort, has addressed the zoonotic potential of IDV to date, despite evidence that the virus can infect multiple mammalian species. Regardless of zoonotic potential, it is clear that IDV have distinct host ranges from ICV but the molecular markers responsible are not known. In this dissertation we assessed the zoonotic potential of D/swine/Oklahoma/1334/2011 (D/OK), a representative IDV, and conducted studies to investigate receptor binding specificity, temperature sensitivity of replication kinetics, and pH of inactivation, all factors known to affect influenza A virus (IAV) host range.
In order to better address zoonotic potential of D/OK we independently verified the high seroprevalence of D/OK in cattle in the US and found evidence of D/OK circulation in this animal population since at least 2003. We also identified 1% seroprevalence in a cohort of older humans who lived in a rural community with likely exposure to cattle. This seropositivity rate was not, however, elevated compared to earlier studies in populations with low exposure to cattle suggesting that the responses measured were not specific. Further analysis of the seropositive sera indeed found that the IDV seroreactivity was most likely due to cross-reactivity of antibodies induced after prior ICV infection. Despite our inability to identify strong serologic support for zoonotic IDV infection, we did show that D/OK was able to replicate and transmit by direct contact in ferrets and that it replicated robustly in differentiated human respiratory cells, both of which are consistent with an ability to replicate in humans for IAV.
We next explored possible mechanisms for the differences in host range of IDV, which has multiple host species, and ICV, which infects primarily humans. Characterization of the HEF proteins of D/OK and a representative ICV demonstrated that D/OK exhibits altered receptor binding specificity and replicates at higher temperatures than ICV although it does bind receptors present in the human respiratory tract. Using virus-like particles with mutant hemagglutinin-esterase fusion (HEF) proteins, we found that the differences in receptor binding of D/OK were at least partially attributable to residues F143, W201, and F256 that line the putative receptor binding pocket. Surprisingly, we also found that, unlike other orthomyxoviruses, the replication of D/OK was not affected by prior incubation at low pH, raising the possibility that its replication might be pH independent.
Reassortment of orthomyxoviruses is a known mechanism of pandemic emergence of IAV and an informal proxy for genus distinction with viruses from distinct genera considered unable to successfully reassort. Contradictory to published data using conventional approaches we found that, using reverse genetics to force reassortment, D/OK genes could complement each of the corresponding genes from ICV and viable reassortants were produced. It is unclear, however, the biologic impact of these reassortments. The answer to this and other aspects of our work will require a resolution to the current US Governments pause on gain-of-function research. This observation does, however, bring into question the validity of classification of a new influenza genus despite IDV exhibiting the phylogenetic and antigenic divergence used to distinguish a novel genus.
Together the evidence described in this study show that D/OK is widespread in cattle and has characteristics consistent with a zoonotic potential, although we were unable to find convincing evidence for such transmission in a small cohort of humans. We did find that D/OK has many features such as host range, receptor usage, sensitivity to pH, and optimal replication that are distinct from ICV and, we propose, supports its classification as a new genus with the orthomyxovirus family. Continued surveillance and investigation of host species barriers is necessary to further address the public health risk presented by this novel virus
Predictors of Quality of Life for African American Women Who Assist Persons Who Receive Dialysis
Get PDFThe purpose of this predictive correlational study was to describe predictors of quality of life (QoL) for African American women who were caregivers of persons with end stage renal disease (ESRD). Five purposes, derived from a review of literature and conceptual model, guided this study. First, the study described the association between demographic factors (i.e., education, employment, age, marital status), and QoL. Second, the study explored the relationship between stress and QoL. Third, the study described the association between depressive symptoms and QoL. Fourth, the study described the association between family health/happiness and QoL. Fifth, the study described the extent to which demographic factors, stress, depressive symptoms, and family health/happiness predicted QoL. A secondary analysis of data from 75 African American women derived from a previous study of 120 caregivers of persons receiving either maintenance in-center hemodialysis or home peritoneal dialysis was conducted. Specifically data from the following instruments were included in the current analysis: The Caregiver Demographic Data Form, Quality of Life Index, Family APGAR, Burden Interview, and the Center for Epidemiological Studies Depression Scale (CES-D). Data were analyzed using descriptive statistics, Pearson’s r coefficient one -way ANOVA on ranks (Kruskal-Wallis), independent t-test, and backward elimination, forward selection, and step-type multiple regression analysis. The caregivers in this sample rated their QoL as moderate. Caregivers also reported clinically significant depressive symptom scores, little to no stress, and were satisfied with their family health/happiness. Significant negative correlations were found between hours worked, education, stress, and QoL. Significant positive relationships were found between age, educational level, recipients’ relationship to the caregiver, family health/happiness, satisfaction with support, and QoL. Four variables—education, full time employment, part time employment, marital status, and stress—explained 63% of the total variance in QoL scores (p \u3c 0.0001). Caregivers who had more education, were employed full time or part time, and reported more stress experienced the lowest QoL. In addition, caregivers who were married experienced the highest QoL. The findings of this study suggest that QoL is multifaceted and is affected by many areas of a person’s life. Social and economic factors and stress were the strongest predictors of QoL for African American women caring for persons with ESRD. The African American women in this study did not experience high levels of stress even though stress was a significant predictor of QoL. This finding may reflect a problem with the measurement of stress used in this population. Several factors, including caregiver work status, explained caregivers’ QoL. Findings from this study provide support for exploring the needs of working caregivers, as QoL was lowest in this group. Understanding better the experiences of working caregivers would inform clinical practice, health policies, and theory development that address the needs of understudied African American women who help persons manage ESRD
Antibodies to Heterogenous Nuclear Ribonucleoprotein A1 Penetrate Neurons Leading to Multiple Downstream Effects Resulting in Neurodegeneration
Get PDFMultiple sclerosis (MS) is the most common demyelinating disorder of the central nervous system. MS is believed to occur in genetically susceptible individuals due to an unknown environmental stimulus. MS patients produce autoantibodies to heterogenous nuclear ribonuclearprotein A1 (hnRNP A1), an RNA binding protein (RBP) highly expressed in neurons. hnRNP A1 functions in pre-mRNA splicing, mRNA trafficking, and translation. Furthermore, the anti-hnRNP A1 antibodies are specific to a N-terminal region termed ‘M9’ which serves as a nuclear export sequence/nuclear localization sequence (NES/NLS) responsible for nuclear/cytoplasmic transport of the protein. In this manuscript we will provide data revealing that anti-hnRNP A1 antibodies enter neuronal cells via clathrin-mediated endocytosis. Moreover, we have shown that anti-hnRNP A1 antibodies cause redistribution of endogenous hnRNP A1 protein, decrease in cellular ATP levels, and increase in apoptosis. Additionally, we present data depicting RNA binding partners of hnRNP A1 protein as well as the effect of anti-hnRNP A1 autoantibodies upon specific RNA binding partners. To further these studies we set out to determine the effect of anti-hnRNP A1 antibodies on experimental autoimmune encephalomyelitis (EAE), the murine model of MS. Specifically, we will present the effect of anti-hnRNP A1-M9 antibodies on clinical symptoms and neurodegeneration in EAE. Taken together, we present a series of experiments which will depict a journey of discovery from the initial discovery of anti-hnRNP A1 antibodies to our present understanding of trafficking, deleterious effects, and in vivo effects of anti-hnRNP A1 antibodies and their implications in the disease Multiple Sclerosis
Evaluating the Therapeutic Effect of an Hsp90 Inhibitor in Mouse Models of Alzheimer’s Disease
Get PDFThe excessive accumulation of amyloid peptides (Aβ) represents one major pathological hallmark of Alzheimer’s disease (AD), which is most notably characterized by synaptic dysfunction. Strategies targeting heat shock protein 90 (Hsp90) inhibition have been widely investigated in the treatment of cancer for over two decades. Its application in the treatment of neurodegenerative diseases however, has emerged more recently in the last decade. The role of the Hsp90 chaperone in clearing misfolded protein aggregates has been well established (in vitro only), but its function in synaptic activity remains elusive.
In our study, we utilized a widely used Hsp90 inhibitor, 17-AAG (17-allylamino-17- desmethoxygeldanamycin), to show that 17-AAG not only induces a heat shock-like response,
but also regulates (likely at the transcriptional level as modeled by qRT-PCR) select proteins enriched in the synapse, such as Post-synaptic density 95 (PSD95), synapsin I, and brain-derived neurotrophic factor (BDNF), among others. Confocal imaging demonstrated rescued dendritic spines with 17-AAG treatment after Aβ challenge in neuronal culture models. The functionality of these changes in the synapse was further confirmed by memory improvement in contextual fear conditioning tests. This work implied a potential strategy in using Hsp90 inhibitors to treat AD. Nevertheless, it must be noted that 17-AAG has limited feasibility in therapy due to its poor CNS-permeability.
In order to test the hypothesis inspired and encouraged by the initial 17-AAG experimental results, we instead used a similar compound with favorable CNS-permeability. OS47720, a radicicol derivative Hsp90 inhibitor, produced comparable effects to those of 17- AAG in inducing heat shock-like responses and promoting synaptic protein expression. Electrophysiology studies on coherence demonstrated enhancement of neuronal activity within the cortical hippocampal network. Most importantly, chronic 0S47720 administration for three months and six months in early and middle symptomatic AD mouse model (Tg2576) both rescued memory deficits without obvious systemic toxicity. OS47720, as an Hsp90 inhibitor, was the first reported compound to display therapeutic effects after long-term treatment in an AD animal model. Post-mortem studies revealed elevation in protein expression of heat shock proteins, synaptic proteins, and BDNF following treatment, without affecting Aβ load and neuroinflammation. Downregulation of heat shock factor 1 (HSF1) by either a pharmacological inhibitor or a virus-delivered shRNA abolished OS47720’s effects on memory improvement against Aβ toxicity. Overexpressing HSF1 partially improved memory. Together with the notion that HSF1-regulated BDNF mRNA, this implicated an HSF1-mediated mechanism at the transcriptional level of synaptic genes. Importantly, these observable effects bypassed several important signaling pathways, including the survival Protein kinase B (PKB or AKT) pathway which is an underlying mechanism of most neuroprotective drugs.
These findings revealed a novel function of Hsp90 inhibition in regulating synaptic gene expression and synaptic activity, further supporting the potential of using Hsp90 inhibitors in treating neurodegenerative diseases such as AD
Dissecting the Physiological Roles of ULK1/2 in the Mouse Brain
Get PDFMammalian UNC-51–like kinases 1 and 2 (ULK1 and ULK2), Caenorhabditis elegans UNC-51 and Drosophila melanogaster Atg1 are redundant serine/threonine kinases that regulate flux through the autophagy pathway in response to various types of cellular stress. C. elegans UNC-51 and D. melanogaster Atg1 also promote axonal growth and defasciculation, and disruption of these genes results in defects in axon guidance in invertebrates. Germline Ulk1/2-deficient mice die perinatally. Therefore, we used a conditional-knockout approach to investigate the roles of ULK1/2 in the brain. Mice lacking Ulk1 and Ulk2 in their central nervous systems (CNS) showed defects in axonal pathfinding and defasciculation affecting the corpus callosum (CC), anterior commissure (AC), corticothalamic axons (CTAs) and thalamocortical axons (TCAs) and mossy fibers. These defects led to impaired midline crossing of callosal axons, anterior commissure hypoplasia and disorganization of the somatosensory cortex. The axon guidance defects observed in Ulk1/2 double knockout (dko) and in CNS-specific (Nestin-Cre) Ulk1/2 conditional double knockout (cdko) mice were not recapitulated in mice lacking other autophagy genes (i.e. Atg7 or Fip200), and was associated with abnormal localization of the axon guidance molecule, transient axonal glycoprotein-1 (TAG-1) in the distal CTAs. Approximately 40% of the Ulk1/2 cdko animals died shortly after birth; the remaining animals survived up to 4 months. Although the mice showed neuronal degeneration, specifically in the hippocampal CA1 region, the neurons showed no accumulation of P62+/ubiquitin+ inclusions or abnormal membranous structures, which are observed in mice lacking other autophagy genes, such as Atg7, and Fip200. Rather, neuronal death was associated with activation of the unfolded protein response (UPR) pathway. An unbiased proteomics approach identified SEC16A as a novel ULK1/2-interacting partner. ULK-mediated phosphorylation of SEC16A regulated the assembly of endoplasmic reticulum (ER) exit sites and ER-to-Golgi trafficking of specific cargo such as, the serotonin transporter SERT, and did not require other autophagy proteins (e.g. ATG13). The defect in ER-to-Golgi trafficking activated the UPR pathway in ULK-deficient cells; both processes were reversed upon expression of SEC16A with a phosphomimetic substitution. Thus, the regulation of ER-to-Golgi trafficking by ULK1/2 is essential for cellular homeostasis. Moreover, the defect in SERT trafficking may also contribute to the disrupted formation of the barrel cortex in the Ulk1/2 cdko mice. Together, these data highlight the autophagy-independent role of ULK1 and ULK2 in maintaining cellular homeostasis and regulating axon guidance in the mammalian brain