The Ubiquitin-Proteasome Reporter GFPu Does Not Accumulate in Neurons of the R6/2 Transgenic Mouse Model of Huntington's Disease

Impairment of the ubiquitin-proteasome system (UPS) has long been considered an attractive hypothesis to explain the selective dysfunction and death of neurons in polyglutamine disorders such as Huntington's disease (HD). The fact that inclusion bodies in HD mouse models and patient brains are rich in ubiquitin and proteasome components suggests that the UPS may be hindered directly or indirectly by inclusion bodies or their misfolded monomeric or oligomeric precursors. However, studies into UPS function in various polyglutamine disease models have yielded conflicting results, suggesting mutant polyglutamine tracts may exert different effects on the UPS depending on protein context, expression level, subcellular localisation and cell-type. To investigate UPS function in a well-characterised mouse model of HD, we have crossed R6/2 HD mice with transgenic UPS reporter mice expressing the GFPu construct. The GFPu construct comprises GFP fused to a constitutive degradation signal (CL-1) that promotes its rapid degradation under conditions of a healthy UPS. Using a combination of immunoblot analysis, fluorescence and immunofluorescence microscopy studies, we found that steady-state GFPu levels were not detectably different between R6/2 and non-R6/2 brain. We observed no correlation between inclusion body formation and GFPu accumulation, suggesting no direct relationship between protein aggregation and global UPS inhibition in R6/2 mice. These findings suggest that while certain branches of the UPS can be impaired by mutant polyglutamine proteins, such proteins do not necessarily cause total blockade of UPS-dependent degradation. It is therefore likely that the relationship between mutant polyglutamine proteins and the UPS is more complex than originally anticipated

A Landscape and Climate Data Logistic Model of Tsetse Distribution in Kenya

, biologically transmitted by the tsetse fly in Africa, are a major cause of illness resulting in both high morbidity and mortality among humans, cattle, wild ungulates, and other species. However, tsetse fly distributions change rapidly due to environmental changes, and fine-scale distribution maps are few. Due to data scarcity, most presence/absence estimates in Kenya prior to 2000 are a combination of local reports, entomological knowledge, and topographic information. The availability of tsetse fly abundance data are limited, or at least have not been collected into aggregate, publicly available national datasets. Despite this limitation, other avenues exist for estimating tsetse distributions including remotely sensed data, climate information, and statistical tools.Here we present a logistic regression model of tsetse abundance. The goal of this model is to estimate the distribution of tsetse fly in Kenya in the year 2000, and to provide a method by which to anticipate their future distribution. Multiple predictor variables were tested for significance and for predictive power; ultimately, a parsimonious subset of variables was identified and used to construct the regression model with the 1973 tsetse map. These data were validated against year 2000 Food and Agriculture Organization (FAO) estimates. Mapcurves Goodness-Of-Fit scores were used to evaluate the modeled fly distribution against FAO estimates and against 1973 presence/absence data, each driven by appropriate climate data.Logistic regression can be effectively used to produce a model that projects fly abundance under elevated greenhouse gas scenarios. This model identifies potential areas for tsetse abandonment and expansion

An mRNA decapping mutant deficient in P body assembly limits mRNA stabilization in response to osmotic stress

Yeast is exposed to changing environmental conditions and must adapt its genetic program to provide a homeostatic intracellular environment. An important stress for yeast in the wild is high osmolarity. A key response to this stress is increased mRNA stability primarily by the inhibition of deadenylation. We previously demonstrated that mutations in decapping activators (edc3∆ lsm4∆C), which result in defects in P body assembly, can destabilize mRNA under unstressed conditions. We wished to examine whether mRNA would be destabilized in the edc3∆ lsm4∆C mutant as compared to the wild-type in response to osmotic stress, when P bodies are intense and numerous. Our results show that the edc3∆ lsm4∆C mutant limits the mRNA stability in response to osmotic stress, while the magnitude of stabilization was similar as compared to the wild-type. The reduced mRNA stability in the edc3∆ lsm4∆C mutant was correlated with a shorter PGK1 poly(A) tail. Similarly, the MFA2 mRNA was more rapidly deadenylated as well as significantly stabilized in the ccr4∆ deadenylation mutant in the edc3∆ lsm4∆C background. These results suggest a role for these decapping factors in stabilizing mRNA and may implicate P bodies as sites of reduced mRNA degradation

Bifunctional Anti-Huntingtin Proteasome-Directed Intrabodies Mediate Efficient Degradation of Mutant Huntingtin Exon 1 Protein Fragments

Huntington's disease (HD) is a fatal autosomal dominant neurodegenerative disorder caused by a trinucleotide (CAG)n repeat expansion in the coding sequence of the huntingtin gene, and an expanded polyglutamine (>37Q) tract in the protein. This results in misfolding and accumulation of huntingtin protein (htt), formation of neuronal intranuclear and cytoplasmic inclusions, and neuronal dysfunction/degeneration. Single-chain Fv antibodies (scFvs), expressed as intrabodies that bind htt and prevent aggregation, show promise as immunotherapeutics for HD. Intrastriatal delivery of anti-N-terminal htt scFv-C4 using an adeno-associated virus vector (AAV2/1) significantly reduces the size and number of aggregates in HDR6/1 transgenic mice; however, this protective effect diminishes with age and time after injection. We therefore explored enhancing intrabody efficacy via fusions to heterologous functional domains. Proteins containing a PEST motif are often targeted for proteasomal degradation and generally have a short half life. In ST14A cells, fusion of the C-terminal PEST region of mouse ornithine decarboxylase (mODC) to scFv-C4 reduces htt exon 1 protein fragments with 72 glutamine repeats (httex1-72Q) by ∼80–90% when compared to scFv-C4 alone. Proteasomal targeting was verified by either scrambling the mODC-PEST motif, or via proteasomal inhibition with epoxomicin. For these constructs, the proteasomal degradation of the scFv intrabody proteins themselves was reduced<25% by the addition of the mODC-PEST motif, with or without antigens. The remaining intrabody levels were amply sufficient to target N-terminal httex1-72Q protein fragment turnover. Critically, scFv-C4-PEST prevents aggregation and toxicity of httex1-72Q fragments at significantly lower doses than scFv-C4. Fusion of the mODC-PEST motif to intrabodies is a valuable general approach to specifically target toxic antigens to the proteasome for degradation

Social factors affecting seasonal variation in bovine trypanosomiasis on the Jos Plateau, Nigeria

BACKGROUND: African Animal Trypanosomiasis (AAT) is a widespread disease of livestock in Nigeria and presents a major constraint to rural economic development. The Jos Plateau was considered free from tsetse flies and the trypanosomes they transmit due to its high altitude and this trypanosomiasis free status attracted large numbers of cattle-keeping pastoralists to the area. The Jos Plateau now plays a major role in the national cattle industry in Nigeria, accommodating approximately 7% of the national herd, supporting 300,000 pastoralists and over one million cattle. During the past two decades tsetse flies have invaded the Jos Plateau and animal trypanosomiasis has become a significant problem for livestock keepers. Here we investigate the epidemiology of trypanosomiasis as a re-emerging disease on the Plateau, examining the social factors that influence prevalence and seasonal variation of bovine trypanosomiasis. METHODS: In 2008 a longitudinal two-stage cluster survey was undertaken on the Jos Plateau. Cattle were sampled in the dry, early wet and late wet seasons. Parasite identification was undertaken using species-specific polymerase chain reactions to determine the prevalence and distribution of bovine trypanosomiasis. Participatory rural appraisal was also conducted to determine knowledge, attitudes and practices concerning animal husbandry and disease control. RESULTS: Significant seasonal variation between the dry season and late wet season was recorded across the Jos Plateau, consistent with expected variation in tsetse populations. However, marked seasonal variations were also observed at village level to create 3 distinct groups: Group 1 in which 50% of villages followed the general pattern of low prevalence in the dry season and high prevalence in the wet season; Group 2 in which 16.7% of villages showed no seasonal variation and Group 3 in which 33.3% of villages showed greater disease prevalence in the dry season than in the wet season. CONCLUSIONS: There was high seasonal variation at the village level determined by management as well as climatic factors. The growing influence of management factors on the epidemiology of trypanosomiasis highlights the impact of recent changes in land use and natural resource competition on animal husbandry decisions in the extensive pastoral production system

Proteasome Activator Enhances Survival of Huntington's Disease Neuronal Model Cells

In patients with Huntington's disease (HD), the proteolytic activity of the ubiquitin proteasome system (UPS) is reduced in the brain and other tissues. The pathological hallmark of HD is the intraneuronal nuclear protein aggregates of mutant huntingtin. We determined how to enhance UPS function and influence catalytic protein degradation and cell survival in HD. Proteasome activators involved in either the ubiquitinated or the non-ubiquitinated proteolysis were overexpressed in HD patients' skin fibroblasts or mutant huntingtin-expressing striatal neurons. Following compromise of the UPS, overexpression of the proteasome activator subunit PA28γ, but not subunit S5a, recovered proteasome function in the HD cells. PA28γ also improved cell viability in mutant huntingtin-expressing striatal neurons exposed to pathological stressors, such as the excitotoxin quinolinic acid and the reversible proteasome inhibitor MG132. These results demonstrate the specific functional enhancements of the UPS that can provide neuroprotection in HD cells

Triptans attenuate capsaicin-induced CREB phosphorylation within the trigeminal nucleus caudalis: a mechanism to prevent central sensitization?

The c-AMP-responsive element binding protein (CREB) and its phosphorylated product (P-CREB) are nuclear proteins expressed after stimulation of pain-producing areas of the spinal cord. There is evidence indicating that central sensitization within dorsal horn neurons is dependent on P-CREB transcriptional regulation. The objectives of the study were to investigate the expression of P-CREB in cells in rat trigeminal nucleus caudalis after noxious stimulation and to determine whether pre-treatment with specific anti-migraine agents modulate this expression. CREB and P-CREB labelling was investigated within the trigeminal caudalis by immunohistochemistry after capsaicin stimulation. Subsequently, the effect of i.v. pre-treatment with either sumatriptan (n = 5), or naratriptan (n = 7) on P-CREB expression was studied. Five animals pre-treated with i.v. normal saline were served as controls. CREB and P-CREB labelling was robust in all animal groups within Sp5C. Both naratriptan and sumatriptan decreased P-CREB expression (p = 0.0003 and 0.0013) within the Sp5C. Triptans attenuate activation of CREB within the central parts of the trigeminal system, thereby leading to potential inhibition of central sensitization. P-CREB may serve as a new marker for post-synaptic neuronal activation within Sp5C in animal models relevant to migraine

High prevalence of lack of knowledge of symptoms of acute myocardial infarction inPakistan and its contribution to delayed presentationto the hospital

<p>Abstract</p> <p>Background</p> <p>We conducted an observational study to determine the delay in presentation to hospital, and its associates among patients experiencing first Acute Myocardial Infarction (AMI) in Karachi, Pakistan.</p> <p>Methods</p> <p>A hospital based cross-sectional study was conducted at National Institute of Cardiovascular Disease (NICVD) in Karachi. A structured questionnaire was used to collect data. The primary outcome was delay in presentation, defined as a time interval of six or more hours from the onset of symptoms to presentation to hospital. Logistic regression analysis was performed to determine the factors associated with prehospital delay.</p> <p>Results</p> <p>A total of 720 subjects were interviewed; 22% were females. The mean age (SD) of the subjects was 54 (± 12) years. The mean (SE) and median (IQR) time to presentation was 12.3 (1.7) hours and 3.04 (6.0) hours respectively. About 34% of the subjects presented late. Lack of knowledge of any of the symptoms of heart attack (odds ratio (95% CI)) (1.82 (1.10, 2.99)), and mild chest pain (10.05 (6.50, 15.54)) were independently associated with prehospital delay.</p> <p>Conclusion</p> <p>Over one-third of patients with AMI in Pakistan present late to the hospital. Lack of knowledge of symptoms of heart attack, and low severity of chest pain were the main predictors of prehospital delay. Strategies to reduce delayed presentation in this population must focus on education about symptoms of heart attack.</p