The Pathological and Biochemical Characterization of Leucine-Rich Repeat Kinase 2 in Parkinson’s Disease

Parkinson\u27s disease (PD) is a debilitating and progressive neurodegenerative disorder that affects over 6 million people worldwide. Despite being the most common movement disorder in the U.S., there is still no effective treatment for halting the progression of disease. While generally considered a sporadic and idiopathic disorder, a number of mutations in genetic loci causal for PD have provided valuable insight into the etiology of disease. Mutations in the gene for leucine-rich repeat kinase 2 (LRRK2) are the single most common cause of both familial and sporadic forms of PD. LRRK2 is a large 2527‐amino acid protein with several distinct domains: leucine-rich repeats, Ras-like GTPase domain, C-terminal of ROC (COR) domain, serine/threonine kinase domain, and WD40 repeats; however the understanding of LRRK2 function or how its aberration may lead to disease is still rudimentary. In 2006, we identified and characterized 3 patients with the G2019S LRRK2 mutation; however this search was limited to a few sequenced exons. An expanded screen identified 2 new patients with LRRK2 mutation, and the clinical and neuropathological findings for all these patients are provided herein. A novel system to express and purify the full-length protein with active in-vitro kinase activity revealed that the most common disease causing alteration (G2019S) markedly increases kinase activity. This highlighted overactive kinase activity as a possible intervention point for its aberrant effects. Screening for molecular inhibitors of kinase activity identified several compounds (Gö6976, K252a, and staurosporine) that share a basic indolocarbazole structure, which act as potent inhibitors of LRRK2 at low nanomolar concentrations. Increased kinase activity in the absence of outside factors is unlikely to account for the pathogenicity of the G2019S mutation. A more careful analysis of LRRK2 kinase activity revealed that this mutation, relative to the wildtype and other pathogenic mutations, may act in a novel pathway leading to disease by disrupting LRRK2 sensitivity to manganese kinase inhibition. Furthermore, based on kinetic data, we propose a novel hypothesis that LRRK2 may act as a cellular sensor of manganese levels, and disruption of this function may contribute to disease

A novel mechanism for freshwater reef growth?

Undergraduate Research Exper.Exploration of a new habitat with distinctly reef‐like features in South Fishtail Bay of Douglas Lake, Michigan has led to the development of a series of hypotheses regarding the formation of magnetic, tubular features known as cups that are found at the site. The age of the habitat and whether or not the cups are still growing is unknown, but it is nevertheless clear from this project that the cups are distinctive microhabitats. Analysis has shown that the cups contain more organic matter than the surrounding hard substrate, but equivalent amounts of chlorophyll α. The unique chemical and structural composition of the cups could be a product of bacterial activity. Algae and groundwater may also be playing a role in creating altered local conditions at the habitat that contribute to the formation of the cups.http://deepblue.lib.umich.edu/bitstream/2027.42/64885/1/Covy_Nora_2009_REU.pd

Scarification and Cultural Practice of Four Lupine Species Native to the Great Basin

The Great Basin is North America\u27s largest desert, encompassing 135 million acres. Grazing and other anthropogenic activities in the Great Basin have put heavy demands on the landscape over the last 150 years. Heavily grazed areas lack diversity which allows the spread of exotic weed species. Cheatgrass (Bromus tectorum L [Poaceae]) has invaded and shortened fire frequency intervals from historic 30—100 years to as few as three to five years. Post-fire reseeding of native species is requisite for restoration of highly invaded ecosystems thus, preventing complete conversion to exotic weeds. Most native shrubs and grasses are available for restoration projects, but native forbs are largely unavailable or expensive. This situation led to the creation of The Great Basin Native Plant Selection and Increase Project (GBNPSIP). In 2000 this project was initiated as a joint effort between the Bureau of Land Management, Forest Service Research, and the Utah Division of Wildlife Resources in an effort to make native seed more available and less expensive for landscape scale restoration projects. To meet restoration goals the GBNPSIP project promotes cultivation of native species to increase seed supplies. This research focuses on overcoming seed dormancy issues that have hindered cultivation through scarification and evaluating germination, establishment, and seed production in a cultural setting of four lupine species: hairy big leaf lupine, (Lupinus prunophilus M.E. Jones [Fabaceae]); silky lupine, (L. sericeus Pursh); silvery lupine, (L. argenteus Pursh); and longspur lupine, (L. arbustus Dougl. ex Lind) five scarification treatments were evaluated sulphuric acid and mechanical treatments significantly improved germination on three of the four species tested. All other treatments were unpredictable and not significant. No treatments significantly improved germination of L. arbustus and three of the five treatments significantly decreased seed germination from the control. Results demonstrate that scarification method, and exposure interval, differ in effectively increasing % germination among species. Germination, establishment, and seed production were evaluated using two planting methods for each species. Broadcast plots (covered) were covered with N-Sulate fabric™ and 5 cm (2 in) of sawdust. Control plots (uncovered) were drilled and left untreated. Germination was significantly improved for all four lupine species under treatment conditions. Lupinus prunophilus and L. sericeus exhibited the greatest improvement in germination when covered. Germination of L. argenteus and L. arbustus were also significantly improved (p\u3c0.0001 and p=0.004, respectively) by the covered treatment. Higher germination in the covered treatment was mirrored in establishment for every species except L. arbustus. There is an advantage of using the covered treatment, but low yields make cultivation unprofitable

CIBERSEGURIDAD, PRODUCCIÓN INTELECTUAL Y FORMACIÓN TUTORIAL: NAVEGANDO LOS DESAFÍOS DE LA ERA DIGITAL

Cybersecurity, intellectual production and tutorial training are important aspects that are emerging in the digital age, and addressing the challenges that arise from it is crucial to protect the information and ideas that are deployed in the tutor - thesis student relationship. This article aims to expose the impact of cybersecurity for digital browsers from tutorial training, influencing the generation of knowledge. Methodologically, it is an essay based on documentary review and hermeneutics. It became evident that the security of academic and scientific information is crucial for researchers and teachers. It follows that the protection of intellectual property protects years of work, research and discoveries, avoiding plagiarism, theft or unauthorized alterations; and becomes an essential tool to support and safeguard one's own thinking, allowing the organization, classification and protection of digital documents of academic value.La ciberseguridad, la producción intelectual y la formación tutorial son aspectos importantes que se avizoran en la era digital, y abordar los desafíos que surgen desde ella es crucial para proteger la información y las ideas que se despliegan en la relación tutor – tesista. Este artículo tiene como objetivo exponer la incidencia de la ciberseguridad para los navegadores digitales desde la formación tutorial, incidiendo en la generación de conocimiento. Metodológicamente, se trata de un ensayo sustentado en la revisión documental y en la hermenéutica. Se hizo evidente que la seguridad de la información académica y científica es crucial para investigadores y docentes. Se deduce que, la protección de la propiedad intelectual resguarda años de trabajo, investigaciones y descubrimientos, evitando plagios, robos o alteraciones no autorizadas; y  se convierte en una herramienta esencial para respaldar y salvaguardar el pensamiento propio, permitiendo la organización, clasificación y protección de documentos digitales de valor académico

Screening for chemical modulators for LRRK2

After the discovery of leucine-rich repeat kinase 2 (LRRK2) as a risk factor for sporadic Parkinson's disease (PD) and mutations in LRRK2 as a cause of some forms of familial PD, there has been substantial interest in finding chemical modulators of LRRK2 function. Most of the pathogenic mutations in LRRK2 are within the enzymatic cores of the protein; therefore, many screens have focused on finding chemical modulators of this enzymatic activity. There are alternative screening approaches that could be taken to investigate compounds that modulate LRRK2 cellular functions. These screens are more often phenotypic screens. The preparation for a screen has to be rigorous and enable high-throughput accurate assessment of a compound's activity. The pipeline to beginning a drug screen and some LRRK2 inhibitor and phenotypic screens will be discussed

Is inhibition of kinase activity the only therapeutic strategy for LRRK2-associated Parkinson's disease?

Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene are a common cause of familial Parkinson's disease (PD). Variation around the LRRK2 locus also contributes to the risk of sporadic PD. The LRRK2 protein contains a central catalytic region, and pathogenic mutations cluster in the Ras of complex protein C terminus of Ras of complex protein (mutations N1437H, R1441G/C and Y1699C) and kinase (G2019S and I2020T) domains. Much attention has been focused on the kinase domain, because kinase-dead versions of mutant LRRK2 are less toxic than kinase-active versions of the same proteins. Furthermore, kinase inhibitors may be able to mimic this effect in mouse models, although the currently tested inhibitors are not completely specific. In this review, we discuss the recent progress in the development of specific LRRK2 kinase inhibitors. We also discuss non-kinase-based therapeutic strategies for LRRK2-associated PD as it is possible that different approaches may be needed for different mutations

Impaired Inflammatory Responses in Murine Lrrk2-Knockdown Brain Microglia

LRRK2, a Parkinson's disease associated gene, is highly expressed in microglia in addition to neurons; however, its function in microglia has not been evaluated. Using Lrrk2 knockdown (Lrrk2-KD) murine microglia prepared by lentiviral-mediated transfer of Lrrk2-specific small inhibitory hairpin RNA (shRNA), we found that Lrrk2 deficiency attenuated lipopolysaccharide (LPS)-induced mRNA and/or protein expression of inducible nitric oxide synthase, TNF-α, IL-1β and IL-6. LPS-induced phosphorylation of p38 mitogen-activated protein kinase and stimulation of NF-κB-responsive luciferase reporter activity was also decreased in Lrrk2-KD cells. Interestingly, the decrease in NF-κB transcriptional activity measured by luciferase assays appeared to reflect increased binding of the inhibitory NF-κB homodimer, p50/p50, to DNA. In LPS-responsive HEK293T cells, overexpression of the human LRRK2 pathologic, kinase-active mutant G2019S increased basal and LPS-induced levels of phosphorylated p38 and JNK, whereas wild-type and other pathologic (R1441C and G2385R) or artificial kinase-dead (D1994A) LRRK2 mutants either enhanced or did not change basal and LPS-induced p38 and JNK phosphorylation levels. However, wild-type LRRK2 and all LRRK2 mutant variants equally enhanced NF-κB transcriptional activity. Taken together, these results suggest that LRRK2 is a positive regulator of inflammation in murine microglia, and LRRK2 mutations may alter the microenvironment of the brain to favor neuroinflammation

Efficient Allele-Specific Targeting of LRRK2 R1441 Mutations Mediated by RNAi

Since RNA interference (RNAi) has the potential to discriminate between single nucleotide changes, there is growing interest in the use of RNAi as a promising therapeutical approach to target dominant disease-associated alleles. Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene have been linked to dominantly inherited Parkinson's disease (PD). We focused on three LRRK2 mutations (R1441G/C and the more prevalent G2109S) hoping to identify shRNAs that would both recognize and efficiently silence the mutated alleles preferentially over the wild-type alleles. Using a luciferase-based reporter system, we identified shRNAs that were able to specifically target the R1441G and R1441C alleles with 80% silencing efficiency. The same shRNAs were able to silence specifically mRNAs encoding either partial or full-length mutant LRRK2 fusion proteins, while having a minimal effect on endogenous wild-type LRRK2 expression when transfected in 293FT cells. Shifting of the mutant recognition site (MRS) from position 11 to other sites (4 and 16, within the 19-mer window of our shRNA design) reduced specificity and overall silencing efficiency. Developing an allele-specific RNAi of G2019S was problematic. Placement of the MRS at position 10 resulted in efficient silencing of reporters (75–80%), but failed to discriminate between mutant and wild-type alleles. Shifting of the MRS to positions 4, 5, 15, 16 increased the specificity of the shRNAs, but reduced the overall silencing efficiency. Consistent with previous reports, these data confirm that MRS placement influences both allele-specificity and silencing strength of shRNAs, while further modification to hairpin design or MRS position may lead to the development of effective G2019S shRNAs. In summary, the effective shRNA against LRRK2 R1441 alleles described herein suggests that RNAi-based therapy of inherited Parkinson's disease is a viable approach towards developing effective therapeutic interventions for this serious neurodegenerative disease