How does the age of control individuals hinder the identification of target genes for Huntington's disease?

Several studies have compared the transcriptome across various brain regions in Huntington's disease (HD) gene-positive and neurologically normal individuals to identify potential differentially expressed genes (DEGs) that could be pharmaceutical or prognostic targets for HD. Despite adhering to technical recommendations for optimal RNA-Seq analysis, none of the genes identified as upregulated in these studies have yet demonstrated success as prognostic or therapeutic targets for HD. Earlier studies included samples from neurologically normal individuals older than the HD gene-positive group. Considering the gradual transcriptional changes induced by aging in the brain, we posited that utilizing samples from older controls could result in the misidentification of DEGs. To validate our hypothesis, we reanalyzed 146 samples from this study, accessible on the SRA database, and employed Propensity Score Matching (PSM) to create a "virtual" control group with a statistically comparable age distribution to the HD gene-positive group. Our study underscores the adverse impact of using neurologically normal individuals over 75 as controls in gene differential expression analysis, resulting in false positives and negatives. We conclusively demonstrate that using such old controls leads to the misidentification of DEGs, detrimentally affecting the discovery of potential pharmaceutical and prognostic markers. This underscores the pivotal role of considering the age of control samples in RNA-Seq analysis and emphasizes its inclusion in evaluating best practices for such investigations. Although our primary focus is HD, our findings suggest that judiciously selecting age-appropriate control samples can significantly improve best practices in differential expression analysis.

Integrating Research into the Undergraduate Curriculum: 1. Early Research Experiences and Training.

Undergraduate research experiences have emerged as some of the most beneficial high-impact practices in education, providing clear benefits to students that include improved critical thinking and scientific reasoning, increased academic performance, and enhanced retention both within STEM majors and in college overall. These benefits extend to faculty members as well. Several disciplines, including neuroscience, have implemented research as part of their curriculum, yet many research opportunities target late stage undergraduates, despite evidence that early engagement can maximize the beneficial nature of such work. A 2019 Society for Neuroscience professional development workshop provided multiple examples of integrating research into an undergraduate curriculum, including early engagement (Fernandes, 2020). This article is the first in a series of three that expands upon the information presented in those workshop discussions, focusing on ways to promote early research opportunities. The benefits and challenges associated with early research engagement suggest thoughtful consideration of the best mechanisms for implementation are warranted; some options might include apprenticeship models or course-based approaches. Regardless of mechanism, early research can serve to initiate more prolonged, progressive, scaffolded experiences that span the academic undergraduate career.

Integrating Research into the Undergraduate Curriculum: 2. Scaffolding Research Skills and Transitioning toward Independent Research.

Undergraduate research experiences are widely regarded as high-impact practices that foster meaningful mentoring relationships, enhance retention and graduation, and stimulate postbaccalaureate enrollment in STEM graduate and professional programs. Through immersion in a mentored original research project, student develop and apply their skills in critical thinking, problem solving, intellectual independence, communication, collaboration, project ownership, innovation, and leadership. These skills are readily transferable to a wide array of future careers in and beyond STEM that are well-served by evidence-based approaches. The 2019 Society for Neuroscience meeting included a well-attended workshop on integrating research into the curriculum at primarily undergraduate institutions (PUIs). This article is the second of three articles that summarize, analyze, and expand the workshop discussions. In this second article, we specifically describe approaches to transitional research courses that prepare students for independent research experiences such as undergraduate research theses. Educators can intentionally scaffold research experience and skills across the curriculum, to foster participation in scientific research and enhance diversity, equity, and inclusivity in research training. This article provides an overview of important goals and considerations for intermediate undergraduate research experiences, specific examples from several institutions of transitional courses that scaffold research preparation using different structures, and a summary of lessons learned from these experiences.

Integrating Research into the Undergraduate Curriculum: 3. Research Training in the Upper-level Neuroscience Curriculum.

The benefits of undergraduate training in research are significant. Integration of such training into the undergraduate experience, however, can be challenging at institutions without extensive research programs, and may inadvertently exclude some populations of students. Therefore, inclusion of research into the academic curriculum ensures all students can access this important training. The 2019 annual meeting of the Society for Neuroscience included a workshop on integrating research into the curriculum at primarily undergraduate institutions (PUIs). In this last article of a three-part series, we describe models for integrating research into advanced stages of the undergraduate curriculum, specifically for juniors and seniors. First, we describe multiple models of faculty-mentored group-based research. Second, we detail a peer-mentored research system, in which seniors mentor groups of first through third year students. Third, we describe multiple examples of integrating research into "capstone" courses for seniors. Fourth, we describe models in which a senior thesis is a graduation requirement for all students. Lastly, we describe several models of implementing an optional honors thesis for students. Although similarities exist across these programs, their differences allow for specific secondary objectives to be met, which are often unique to institutions and/or departments. Therefore, for each of these examples, we describe the context, specific design, and required student assessments. We conclude by discussing some of the key successes and challenges of developing programs that facilitate undergraduate research by upper-level students, and suggest a number of concepts that should be considered by individuals developing and assessing new programs.

Effectiveness of a Low-Cost, Graduate Student-Led Intervention on Study Habits and Performance in Introductory Biology.

Institutions have developed diverse approaches that vary in effectiveness and cost to improve student performance in introductory science, technology, engineering, and mathematics courses. We developed a low-cost, graduate student-led, metacognition-based study skills course taught in conjunction with the introductory biology series at Miami University. Our approach aimed to improve performance for underachieving students by combining an existing framework for the process of learning (the study cycle) with concrete tools (outlines and concept maps) that have been shown to encourage deep understanding. To assess the effectiveness of our efforts, we asked 1) how effective our voluntary recruitment model was at enrolling the target cohort, 2) how the course impacted performance on lecture exams, 3) how the course impacted study habits and techniques, and 4) whether there are particular study habits or techniques that are associated with large improvements on exam scores. Voluntary recruitment attracted only 11-17% of our target cohort. While focal students improved on lecture exams relative to their peers who did not enroll, gains were relatively modest, and not all students improved. Further, although students across both semesters of our study reported improved study habits (based on pre and post surveys) and on outlines and concept maps (based on retrospectively scored assignments), gains were more dramatic in the Fall semester. Multivariate models revealed that, while changes in study habits and in the quality of outlines and concept maps were weakly associated with change in performance on lecture exams, relationships were only significant in the Fall semester and were sometimes counterintuitive. Although benefits of the course were offset somewhat by the inefficiency of voluntary recruitment, we demonstrate the effectiveness our course, which is inexpensive to implement and has advantage of providing pedagogical experience to future educators.

Remodeling of peripheral nerve ensheathment during the larval-to-adult transition in Drosophila.

Over the course of a 4-day period of metamorphosis, the Drosophila larval nervous system is remodeled to prepare for adult-specific behaviors. One example is the reorganization of peripheral nerves in the abdomen, where five pairs of abdominal nerves (A4-A8) fuse to form the terminal nerve trunk. This reorganization is associated with selective remodeling of four layers that ensheath each peripheral nerve. The neural lamella (NL), is the first to dismantle; its breakdown is initiated by 6 hours after puparium formation, and is completely removed by the end of the first day. This layer begins to re-appear on the third day of metamorphosis. Perineurial glial (PG) cells situated just underneath the NL, undergo significant proliferation on the first day of metamorphosis, and at that stage contribute to 95% of the glial cell population. Cells of the two inner layers, Sub-Perineurial Glia (SPG) and Wrapping Glia (WG) increase in number on the second half of metamorphosis. Induction of cell death in perineurial glia via the cell death gene reaper and the Diptheria toxin (DT-1) gene, results in abnormal bundling of the peripheral nerves, suggesting that perineurial glial cells play a role in the process. A significant number of animals fail to eclose in both reaper and DT-1 targeted animals, suggesting that disruption of PG also impacts eclosion behavior. The studies will help to establish the groundwork for further work on cellular and molecular processes that underlie the co-ordinated remodeling of glia and the peripheral nerves they ensheath. © 2017 Wiley Periodicals, Inc. Develop Neurobiol 77: 1144-1160, 2017.

dHb9 expressing larval motor neurons persist through metamorphosis to innervate adult-specific muscle targets and function in Drosophila eclosion.

The Drosophila larval nervous system is radically restructured during metamorphosis to produce adult specific neural circuits and behaviors. Genesis of new neurons, death of larval neurons and remodeling of those neurons that persistent collectively act to shape the adult nervous system. Here, we examine the fate of a subset of larval motor neurons during this restructuring process. We used a dHb9 reporter, in combination with the FLP/FRT system to individually identify abdominal motor neurons in the larval to adult transition using a combination of relative cell body location, axonal position, and muscle targets. We found that segment specific cell death of some dHb9 expressing motor neurons occurs throughout the metamorphosis period and continues into the post-eclosion period. Many dHb9 > GFP expressing neurons however persist in the two anterior hemisegments, A1 and A2, which have segment specific muscles required for eclosion while a smaller proportion also persist in A2-A5. Consistent with a functional requirement for these neurons, ablating them during the pupal period produces defects in adult eclosion. In adults, subsequent to the execution of eclosion behaviors, the NMJs of some of these neurons were found to be dismantled and their muscle targets degenerate. Our studies demonstrate a critical continuity of some larval motor neurons into adults and reveal that multiple aspects of motor neuron remodeling and plasticity that are essential for adult motor behaviors. © 2016 Wiley Periodicals, Inc. Develop Neurobiol 76: 1387-1416, 2016.

Glial remodeling during metamorphosis influences the stabilization of motor neuron branches in Drosophila.

Motor neurons that innervate the dorsal longitudinal (flight) muscles, DLMs, make multiple points of contact along the length of fibers. The stereotypy of the innervation lies in the number of contact points (CPs) made by each motor neuron and is established as a consequence of pruning that occurs during metamorphosis. Coincident with the onset of pruning is the arrival of glial processes that eventually ensheath persistent branches. To test a possible role for glia during pruning, the development of adult-specific glial ensheathment was disrupted using a targeted expression of dominant negative shibire. Such a manipulation resulted in fewer contact points at the DLM fibers. The development of innervation was examined during metamorphosis, specifically to test if the reduction was a consequence of increased pruning. We quantified the number of branches displaying discontinuities in their membrane, an indicator of the level of pruning. Disrupting the formation of glial ensheathment resulted in a two-fold increase in the discontinuities, indicating that pruning is enhanced. Thus glial-neuronal interactions, specifically during pruning are important for the patterning of adult innervation. Our studies also suggest that FasII plays a role in mediating this communication. At the end of the pruning phase, FasII localizes to glia, which envelops each of the stabilized contact points. When glial FasII levels are increased using the Gal4/UAS system of targeted expression, pruning of secondary branches is enhanced. Our results indicate that glia regulate pruning of secondary branches by influencing the balance between stabilization and pruning. This was confirmed by an observed rescue of the innervation phenotype of FasII hypomorphs by over expressing FasII in glia.

Founder cells regulate fiber number but not fiber formation during adult myogenesis in Drosophila.

During insect myogenesis, myoblasts are organized into a pre-pattern by specialized organizer cells. In the Drosophila embryo, these cells have been termed founder cells and play important roles in specifying muscle identity and in serving as targets for myoblast fusion. A group of adult muscles, the dorsal longitudinal (flight) muscles, DLMs, is patterned by persistent larval scaffolds; the second set, the dorso-ventral muscles, DVMs is patterned by mono-nucleate founder cells (FCs) that are much larger than the surrounding myoblasts. Both types of organizer cells express Dumbfounded, which is known to regulate fusion during embryonic myogenesis. The role of DVM founder cells as well as the DLM scaffolds was tested in genetic ablation studies using the UAS/Gal4 system of targeted transgene expression. In both cases, removal of organizer cells prior to fusion, causes formation of supernumerary fibers, suggesting that cells in the myoblast pool have the capacity to initiate fiber formation, which is normally inhibited by the organizers. In addition to the large DVM FCs, some (smaller) cells in the myoblast pool also express Dumbfounded. We propose that these cells are responsible for seeding supernumerary fibers, when DVM FCs are eliminated prior to fusion. When these cells are also eliminated, myogenesis fails to occur. In the second set of studies, targeted expression of constitutively active Ras(V12) also resulted in the appearance of supernumerary fibers. In this case, the original DVM FCs are present, suggesting alterations in cell fate. Taken together, these data suggest that DVM myoblasts are able to respond to cues other than the original founder cell, to initiate fusion and fiber formation. Thus, the role of the large DVM founder cells is to generate the correct number of fibers, but they are not required for fiber formation itself. We also present evidence that the DVM FCs may arise from the leg imaginal disc.

Substitution of critical isoleucines in the KH domains of Drosophila fragile X protein results in partial loss-of-function phenotypes.

Fragile X mental retardation proteins (FMRP) are RNA-binding proteins that interact with a subset of cellular RNAs. Several RNA-binding domains have been identified in FMRP, but the contribution of these individual domains to FMRP function in an animal model is not well understood. In this study, we have generated flies with point mutations in the KH domains of the Drosophila melanogaster fragile X gene (dfmr1) in the context of a genomic rescue fragment. The substitutions of conserved isoleucine residues within the KH domains with asparagine are thought to impair binding of RNA substrates and perhaps the ability of FMRP to assemble into mRNP complexes. The mutants were analyzed for defects in development and behavior that are associated with deletion null alleles of dfmr1. We find that these KH domain mutations result in partial loss of function or no significant loss of function for the phenotypes assayed. The phenotypes resulting from these KH domain mutants imply that the capacities of the mutant proteins to bind RNA and form functional mRNP complexes are not wholly disrupted and are consistent with biochemical models suggesting that RNA-binding domains of FMRP can function independently.

The adult abdominal neuromuscular junction of Drosophila: a model for synaptic plasticity.

During its life cycle, Drosophila makes two sets of neuromuscular junctions (NMJs), embryonic/larval and adult, which serve distinct stage-specific functions. During metamorphosis, the larval NMJs are restructured to give rise to their adult counterparts, a process that is integrated into the overall remodeling of the nervous system. The NMJs of the prothoracic muscles and the mesothoracic dorsal longitudinal (flight) muscles have been previously described. Given the diversity and complexity of adult muscle groups, we set out to examine the less complex abdominal muscles. The large bouton sizes of these NMJs are particularly advantageous for easy visualization. Specifically, we have characterized morphological attributes of the ventral abdominal NMJ and show that an embryonic motor neuron identity gene, dHb9, is expressed at these adult junctions. We quantified bouton numbers and size and examined the localization of synaptic markers. We have also examined the formation of boutons during metamorphosis and examined the localization of presynaptic markers at these stages. To test the usefulness of the ventral abdominal NMJs as a model system, we characterized the effects of altering electrical activity and the levels of the cell adhesion molecule, FasciclinII (FasII). We show that both manipulations affect NMJ formation and that the effects are specific as they can be rescued genetically. Our results indicate that both activity and FasII affect development at the adult abdominal NMJ in ways that are distinct from their larval and adult thoracic counterparts

A role for Fas II in the stabilization of motor neuron branches during pruning in Drosophila.

During insect metamorphosis, the nervous system is extensively remodeled resulting in the development of new circuits that will execute adult-specific behaviors. The peripheral remodeling seen during development of innervation to the Dorsal Longitudinal (flight) Muscle (DLM) in Drosophila involves an initial retraction of larval neuromuscular junctions followed by adult-specific branch outgrowth. Subsequently, a phase of pruning occurs during which motor neuron branches are pruned back to reveal the stereotypic pattern of multiple contact points (or arbors) along the length of each DLM fiber. In this study, we show that the cell adhesion molecule, Fasciclin II (Fas II), is important for generating the stereotypic pattern. In Fas II hypomorphs, the number of contact points is increased, and the phenotype is rescued by targeted expression of Fas II in either synaptic partner. Arbor development has three distinct phases: outgrowth and elaboration, pruning and stabilization, and expansion of stabilized arbors. Fas II is expressed during the first two phases. A subset of branches is labeled during the elaboration phase, which is likely to initiate a stabilization pathway allowing branches to survive the pruning phase. However, since not all Fas II positive branches are retained, we propose that it primes branches for stabilization. Our data suggest that Fas II functions to restrict branch length and arbor expanse.

A dominant negative form of Rac1 affects myogenesis of adult thoracic muscles in Drosophila.

Blocking Rac1 function in precursors of the indirect flight muscle of Drosophila severely disrupts muscle formation. The DLM fibers that develop using larval scaffolds are reduced in number and fiber size, while the DVMs, which develop using founder cells, are mostly absent. These adult muscle phenotypes are in part due to a reduced myoblast pool present at the third larval instar. BrDU labeling studies indicated that this is primarily due to a reduction in proliferation. In addition, DVM myoblasts display altered morphology and are unable to segregate into primordia. This defect precedes the evident block in fusion. We also show that the recently described DVM founder cells can be labeled with 22C10 and beta-3 tubulin, and that they are present under conditions of dominant negative Rac1(N17) expression. Despite the presence of founder cells, DVM fiber formation is rarely observed. Although DLM myoblasts are able to segregate around their larval scaffolds, the pace of fusion is reduced and consequently there is a delay in DLM fiber formation. Thus, in addition to its well-established role in fusion, Rac1 is also involved in the regulation of myoblast proliferation and segregation during adult myogenesis. These are two new roles for Rac1 in Drosophila.

Motoneurons regulate myoblast proliferation and patterning in Drosophila.

Motoneurons directly influence the differentiation of muscle fibers, regulating features such as muscle fiber type and receptor development. Less well understood is whether motoneurons direct earlier events, such as the patterning of the musculature. In Drosophila, the denervation of indirect flight muscles results in a diminished myoblast population and smaller or missing muscle fibers. We have examined whether the neuron-dependent control of myoblast number is due to regulation of cell division, motoneuron-dependent apoptosis, or nerve-dependent localization and migration of myoblasts. We found that denervation resulted in a reduced rate of cell division, as revealed by BrDU incorporation. There was no change in the frequency of apoptotic myoblasts following denervation. Using time lapse imaging of GFP-expressing myoblasts in vivo in pupae, we observed that despite denervation, the migration and localization of myoblasts remained unchanged. In addition to reducing myoblast proliferation, denervation also altered the segregation of myoblasts into the de novo arising dorso-ventral muscles (DVMs). To address this effect on muscle patterning, we examined the expression of the founder-cell marker Dumbfounded/Kirre (Duf) in imaginal pioneer cells. We show that there is a strong correspondence between cells that express Dumbfounded/Kirre and the number of DVM fibers, consistent with a role for these cells in establishing adult muscles. In the absence of innervation the Duf-positive cells are no longer detected, and muscle patterning is severely disrupted. Our results support a model where specialized founder cells prefigure the adult muscle fibers under the control of the nervous system.

Pruning of motor neuron branches establishes the DLM innervation pattern in Drosophila.

During the Drosophila life-cycle two sets of neuromuscular junctions are generated: the embryonic/larval NMJs develop during the first half, followed by the period of metamorphosis during which the adult counterpart is generated. Development of the adult innervation pattern is preceded by a withdrawal of larval NMJs, which occurs at the onset of metamorphosis, and is followed by adult-specific motor neuron outgrowth to innervate the newly developing adult fibers. Establishment of the adult innervation pattern occurs in the context of a broader restructuring of the nervous system, which results in the development of neural circuits that are necessary to carry out behaviors specific to the adult. In this article, we follow development of the dorsal longitudinal muscle (DLM) innervation pattern through metamorphosis. We find that the initial period of motor neuron elaboration is followed by a phase of extensive pruning resulting in a threefold reduction of neuromuscular contacts. This event establishes the adult pattern of second order branching. Subsequent higher order branching from the second order "contact" points generates the characteristic multiterminal innervation pattern of the DLMs. Boutons begin to appear after the pruning phase, and are much smaller than their larval counterparts. Additionally, we demonstrate that the DLM innervation is altered in the hyperexcitable double mutant, ether a go-go Shaker, and that the phenotype is suppressed by the hypoexcitable mutant, nap(ts1). Our results demonstrate that electrical activity regulates the patterning of DLM innervation during metamorphosis.

Evaluation of landscape coverings to reduce soil lead hazards in urban residential yards: The Safer Yards Project.

This study was designed primarily to evaluate the effectiveness of landscape coverings to reduce the potential for exposure to lead-contaminated soil in an urban neighborhood. Residential properties were randomized in to three groups: application of ground coverings/barriers plus placement of a raised garden bed (RB), application of ground coverings/barriers only (no raised bed, NRB), and control. Outcomes evaluated soil lead concentration (employing a weighting method to assess acute hazard soil lead [areas not fully covered] and potential hazard soil lead [all soil surfaces regardless of covering status]), density of landscape coverings (6 = heavy, > 90% covered; 1 = bare, < 10% covered), lead tracked onto carpeted entryway floor mats, and entryway floor dust lead loadings. Over 1 year, the intervention groups had significantly reduced acute hazard soil lead concentration (median change: RB, -478 ppm; NRB, -698 ppm; control, +52 ppm; Kruskal-Wallis, P = 0.02), enhanced landscape coverings (mean change in score: RB, +0.6; NRB, +1.5; control, -0.6; ANOVA, P < 0.001), and a 50% decrease in lead tracked onto the floor mats. The potential hazard soil lead concentration and the entryway floor dust lead loading did not change significantly. Techniques evaluated by this study are feasible for use by property owners but will require continued maintenance. The long-term sustainability of the method needs further examination.