Intrabody Gene Therapy Ameliorates Motor, Cognitive, and Neuropathological Symptoms in Multiple Mouse Models of Huntington's Disease

Huntington's disease (HD) is an autosomal dominant neurodegenerative disease resulting from the expansion of a glutamine repeat in the huntingtin (Htt) protein. Current therapies are directed at managing symptoms such as chorea and psychiatric disturbances. In an effort to develop a therapy directed at disease prevention we investigated the utility of highly specific, anti-Htt intracellular antibodies (intrabodies). We previously showed that V_(L)12.3, an intrabody recognizing the N terminus of Htt, and Happ1, an intrabody recognizing the proline-rich domain of Htt, both reduce mHtt-induced toxicity and aggregation in cell culture and brain slice models of HD. Due to the different mechanisms of action of these two intrabodies, we then tested both in the brains of five mouse models of HD using a chimeric adeno-associated virus 2/1 (AAV2/1) vector with a modified CMV enhancer/chicken β-actin promoter. V_(L)12.3 treatment, while beneficial in a lentiviral model of HD, has no effect on the YAC128 HD model and actually increases severity of phenotype and mortality in the R6/2 HD model. In contrast, Happ1 treatment confers significant beneficial effects in a variety of assays of motor and cognitive deficits. Happ1 also strongly ameliorates the neuropathology found in the lentiviral, R6/2, N171-82Q, YAC128, and BACHD models of HD. Moreover, Happ1 significantly prolongs the life span of N171-82Q mice. These results indicate that increasing the turnover of mHtt using AAV-Happ1 gene therapy represents a highly specific and effective treatment in diverse mouse models of HD

A Novel Role for Osteopontin in Facilitating West Nile Virus Neuroinvasion

West Nile virus (WNV) is a positive-sensed, single-stranded RNA flavivirus that can cause human neuroinvasive diseases, including encephalitis, meningitis, and flaccid paralysis. The mechanisms by which WNV enters the central nervous system and the host-factors that are involved in WNV-neuroinvasiveness are not completely understood. Osteopontin (OPN), a multifunctional glycoprotein, has been implicated as a bio-marker for a number of neuroinflammatory diseases. In particular, secreted (s)OPN has been implicated to participate in recruitment of polymorphonuclear neutrophils (PMN) to sites of its expression, while PMNs have been suggested to act as WNV reservoirs. Therefore, sOPN recruitment of PMNs may contribute to neuroinvasive WNV infection via the ‘Trojan horse’ mechanism of viral entry into the brain. Therefore, we hypothesize brain-infiltration of PMNs during neuroinvasive WNV pathogenesis is in part mediated by sOPN. Our results show that sOPN expression was significantly increased in human sera, human neuronal cells line, murine plasma, brain homogenates and primary neuronal supernatant following WNV infection, indicating a role for OPN in WNV pathogenesis. In addition, after challenge with WNV in vivo, Opn-/- mice exhibited a higher (70%) survival rate than wild-type (WT) mice (30%). Consistent with this, qPCR analysis between WT and Opn-/- mice demonstrated comparable levels of viremia; yet, reduced viral burden in the brains of Opn-/-mice compared to WT controls. Analysis of brain- infiltrating leukocytes displayed reduced PMNs and PMN-chemokine expression levels in Opn-/- mice brains. Importantly, intracerebral supplement of recombinant OPN (rOPN) into Opn-/-mice resulted in increased PMN-brain infiltration, increased viral load and reduced overall survival. Together, these data suggest OPN facilitates WNV neuroinvasion in a mouse model

Antibody therapy in Neurodegenerative Disease

Advances in medical science have led to increased life expectancy and increased median age in the population. Because the symptoms of neurodegenerative diseases generally onset in mid- to late-life, a concomitant increase in the number of persons afflicted with these devastating diseases has occurred. Developing therapies for neurodegenerative diseases is of the highest priority due to the enormous cost of medical care required, as well as for the human suffering involved. Although caused by a variety of genetic and environmental insults, such diseases share commonalities. Many of these diseases are proteinopathies-diseases caused by misfolded, aggregating proteins. Antibodies that can recognize and remove misfolded proteins are ideally suited for proteinopathy therapeutics. The numerous intriguing advances in antibody-based therapies for neurodegenerative diseases are discussed in this review

Differential Gene Expression of Mice Pellet Microbial Communities exposed to Simulated Microgravity

Microbial communities within the human gut play an important role in our overall health and wellbeing. Understanding how microgravity affects these microbial communities provides greater insight into the health of astronauts on board the ISS and what may occur during Deep Space explorations. Previous experiments have measured differential gene expression of single culture bacteria exposed to simulated microgravity, allowing us to map which genes are selected for or against in that environment. Using fecal pellets from mice exposed to simulated microgravity and space radiation, we are constructing and sequencing RNA and DNA libraries to study the metatranscriptome and changes in gut microbiota. These comparisons will allow us to gain a better understanding of how microbial communities are affected by space environments and what that may entail for the gut health of astronauts

A Directory of Threat Assessment Models

Threat assessment is a process of identifying, assessing, and managing threats of targeted violence prompted by warning behaviors. Threat assessment is an evolving field with no singular guidebook that can cover the range of settings to which it is applied. Therefore, there are many different practical models of threat assessment implementation. This directory reviews how threat assessment is practically implemented in various settings, by systematically reviewing case study literature that describes the structure and operations of existing threat assessment teams and models. The directory compiles information on 27 threat assessment models which cover a range of harms within educational settings and workplaces as well as more specific crime types such as fixated threats to public figures, violent extremism, and stalking. For each of the 27 models, the directory outlines details about their background, team details and composition, the nature and structure of their referral system, their threat assessment operations, their case management structure, and their quality assurance processes. This directory serves as the foundation for a comparative analysis of threat assessment models with a focus on learning from partner countries outside of the United States

Heat Shock Protein 40 and Immune Function in Altered Gravity

In space, astronauts are more susceptible to pathogens, viral reactivation and immunosuppression, which poses limits to their health and the mission. Interestingly, during space flight, stress-inducible heat shock proteins (HSP) are robustly induced, and the overexpression of HSPs have been implicated in immune dysregulation, therefore HSPs may be critically involved in regulating immune homeostasis. HSP40/DNAJ1 plays a major role in proper protein translation and folding. Its loss of function has been implicated in susceptibility to microbial infection, while its overexpression has been implicated in autoimmunity, collectively suggesting its complicated, but necessary, role in maintaining immunological function. To determine the role of HSP40 during stress-induced altered gravity conditions, wild-type and Hsp40 mutant Drosophila melanogaster were exposed to ground-based chronic hypergravity conditions, followed by quantitative PCR (qPCR) analysis of immune gene expression. In addition, larval hemocytes were collected to determine the functional output in response to E. coli bioparticle phagocytosis. Preliminary data indicates a required role for Hsp40 in strengthening immune function during stress-induced spaceflight in flies. In short, a critical need to evaluate the relationship between HSPs and immune suppression during space flight is necessary. Since space travel may become available to the general public in the not too distant future, and for the possibility of long-term space missions, a more comprehensive evaluation of the molecules responsible for immune dysfunction observed during space flight is required

Gravity as a Continuum: Effects of Altered Gravity on Drosophila Melanogaster Immunity

The impact of spaceflight on immune function is undoubtedly a critical focus in the area of space biology and human health research. Heat shock proteins (Hsp) are an evolutionarily conserved family of proteins that are expressed in response to cellular and physiological stressors, experienced during radiation exposure, confinement, circadian rhythm disruption, and altered gravity (hypergravity experienced at launch/landing and microgravity experienced in-flight). In particular, Hsp70 aids in the folding of proteins, facilitates the movement of proteins across the membranes during signal transductions and can stimulate innate immunity. Since Hsp70 is induced during cellular stress, and can act as a stimulator for innate immunity, we sought to address how a loss of Hsp70 affects immunity, under the stress-inducing model of acute and chronic hypergravity. Moreover, the effects of gravity as a continuum on the induction of Hsps and key immune genes were also assessed to determine if increased cellular stress, via increased gravity (g)-force, contributes to immune dysfunctions. For this, wildtype (W1118) and Hsp70 deficient (Hsp70null) Drosophila melanogaster were subjected to simulated hypergravity at increasing levels of g-force (1.2g, 3g, and 5g) for acute (1hr) and chronic (7-day) timepoints and were compared to 0g \u27non-hypergravity\u27 controls. Following simulation, whole bodies were sex-segregated, RNA was isolated and quantitative (q)PCR was performed to determine differential immune gene expression profiles. Further, functional output of hemocytes were assessed by a phagocytosis assay. Collectively, these studies evaluated the effects of Hsp70 in the context of immunity during acute and chronic hypergravity. Indeed, relevance for this work can directly translate to acute effects of launch/landing gravitational forces upon liftoff (~1.7g) and entry (~3.4g) that astronauts experience. In addition, the effects of chronic cellular stress is directly relevant to the immune health of astronauts on long duration missions, as well. Thus, as we approach the goal of returning to the Moon and landing the first humans on Mars, an evaluation of gravity as a continuum and the stress-inducing effects of altered gravity experienced during spaceflight on astronaut immunity and health are necessary

Intrabodies Binding the Proline-Rich Domains of Mutant Huntingtin Increase Its Turnover and Reduce Neurotoxicity

Although expanded polyglutamine (polyQ) repeats are inherently toxic, causing at least nine neurodegenerative diseases, the protein context determines which neurons are affected. The polyQ expansion that causes Huntington's disease (HD) is in the first exon (HDx-1) of huntingtin (Htt). However, other parts of the protein, including the 17 N-terminal amino acids and two proline (polyP) repeat domains, regulate the toxicity of mutant Htt. The role of the P-rich domain that is flanked by the polyP domains has not been explored. Using highly specific intracellular antibodies (intrabodies), we tested various epitopes for their roles in HDx-1 toxicity, aggregation, localization, and turnover. Three domains in the P-rich region (PRR) of HDx-1 are defined by intrabodies: MW7 binds the two polyP domains, and Happ1 and Happ3, two new intrabodies, bind the unique, P-rich epitope located between the two polyP epitopes. We find that the PRR-binding intrabodies, as well as VL12.3, which binds the N-terminal 17 aa, decrease the toxicity and aggregation of HDx-1, but they do so by different mechanisms. The PRR-binding intrabodies have no effect on Htt localization, but they cause a significant increase in the turnover rate of mutant Htt, which VL12.3 does not change. In contrast, expression of VL12.3 increases nuclear Htt. We propose that the PRR of mutant Htt regulates its stability, and that compromising this pathogenic epitope by intrabody binding represents a novel therapeutic strategy for treating HD. We also note that intrabody binding represents a powerful tool for determining the function of protein epitopes in living cells

Towards optimal education including self-regulated learning in technology-enhanced preschools and primary schools

At the start of preschool, four-year-old pupils differ in their development, including the capacity to self-regulate their playing and learning. In preschool and primary school, educational processes are generally adapted to the mean age of the pupils in class. The same may apply to ICT-based pupil-monitoring systems. This norm-based factor undermines the potential of ICT to support the educational differentiation needed for pupils and increases the amount of daily work for teachers. A theoretical framework is sketched in which pedagogical-didactical, organisational, and ICT guidelines enhance differentiated, self-regulated playing and learning for each pupil within preschool and primary school. To develop and check such optimal education and ICT conditions in practice, a pilot and a randomised intervention study are carried out in integrated Dutch preschools/primary schools for pupils aged 4 – 12. Pilot results support the use of a procedure to screen each child’s characteristics at the start of preschool by parents and preschool teachers, and also the immediate relevance of criterion-based and norm-based ordering (‘double diagnostics’) of playing and learning materials. Intermediate results of the longitudinal intervention study empirically support the hypothesis regarding the positive effects of optimal education on the cognitive and emotional functioning of both cognitively gifted and other pupils. The discussion emphasises the need to assist schools in realising optimal education including learners’ self-regulation and adequate ICT to enhance optimal learning. Final attention is given to further development of optimal education in a European context