202 research outputs found

    The role of attenuated astrocyte activation in infantile neuronal Ceroid Lipofuscinosis

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    Infantile neuronal ceroid lipofuscinosis (INCL) is an inherited neurodegenerative disorder affecting the CNS during infancy. INCL is caused by mutations in the CLN1 gene that leads to a deficiency in the lysosomal hydrolase, palmitoyl protein thioesterase (PPT1). A murine model of INCL, the PPT1(−/−) mouse, is an accurate phenocopy of the human disease. The first pathological change observed in the PPT1(−/−) brain is regional areas of GFAP upregulation, which predicts future areas of neurodegeneration. We hypothesized that preventing GFAP upregulation in reactive astrocytes will alter the CNS disease. To test this hypothesis, we generated mice simultaneously carrying null mutations in the GFAP, Vimentin, and PPT1 genes (GFAP(−/−)Vimentin(−/−)PPT1(−/−)). Although the clinical and pathological features of the GFAP(−/−)Vim(−/−)PPT1(−/−) mice are similar to INCL, the disease appears earlier and progresses more rapidly. One mechanism underlying this accelerated phenotype is a profound neuroinflammatory response within the CNS. Thus, our data identify a protective role for intermediate filament upregulation during astrocyte activation in INCL, a model of chronic neurodegeneration

    Bone marrow transplantation alters the tremor phenotype in the murine model of globoid-cell leukodystrophy

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    Tremor is a prominent phenotype of the twitcher mouse, an authentic genetic model of Globoid-Cell Leukodystrophy (GLD, Krabbe’s disease). In the current study, the tremor was quantified using a force-plate actometer designed to accommodate low-weight mice. The actometer records the force oscillations caused by a mouse’s movements, and the rhythmic structure of the force variations can be revealed. Results showed that twitcher mice had significantly increased power across a broad band of higher frequencies compared to wildtype mice. Bone marrow transplantation (BMT), the only available therapy for GLD, worsened the tremor in the twitcher mice and induced a measureable alteration of movement phenotype in the wildtype mice. These data highlight the damaging effects of conditioning radiation and BMT in the neonatal period. The behavioral methodology used herein provides a quantitative approach for assessing the efficacy of potential therapeutic interventions for Krabbe’s disease

    Generation of a stable packaging cell line producing high-titer PPT-deleted integration-deficient lentiviral vectors

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    The risk of insertional mutagenesis inherent to all integrating exogenous expression cassettes was the impetus for the development of various integration-defective lentiviral vector (IDLV) systems. These systems were successfully employed in a plethora of preclinical applications, underscoring their clinical potential. However, current production of IDLVs by transient plasmid transfection is not optimal for large-scale production of clinical grade vectors. Here, we describe the development of the first tetracycline-inducible stable IDLV packaging cell line comprising the D64E integrase mutant and the VSV-G envelope protein. A conditional self-inactivating (cSIN) vector and a novel polypurine tract (PPT)-deleted vector were incorporated into the newly developed stable packaging cell line by transduction and stable transfection, respectively. High-titer (~107 infectious units (IU)/ml) cSIN vectors were routinely generated. Furthermore, screening of single-cell clones stably transfected with PPT-deleted vector DNA resulted in the identification of highly efficient producer cell lines generating IDLV titers higher than 108 IU/mL, which upon concentration increased to 1010 IU/ml. IDLVs generated by stable producer lines efficiently transduce CNS tissues of rodents. Overall, the availability of high-titer IDLV lentivirus packaging cell line described here will significantly facilitate IDLV-based basic science research, as well as preclinical and clinical applications

    Preclinical studies in Krabbe disease: A model for the investigation of novel combination therapies for lysosomal storage diseases

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    Krabbe disease (KD) is a lysosomal storage disease (LSD) caused by mutations in the galc gene. There are over 50 monogenetic LSDs, which largely impede the normal development of children and often lead to premature death. At present, there are no cures for LSDs and the available treatments are generally insufficient, short acting, and not without co-morbidities or long-term side effects. The last 30 years have seen significant advances in our understanding of LSD pathology as well as treatment options. Two gene therapy-based clinical trials, NCT04693598 and NCT04771416, for KD were recently started based on those advances. This review will discuss how our knowledge of KD got to where it is today, focusing on preclinical investigations, and how what was discovered may prove beneficial for the treatment of other LSDs

    Krabbe disease successfully treated via monotherapy of intrathecal gene therapy

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    Globoid cell leukodystrophy (GLD; Krabbe disease) is a progressive, incurable neurodegenerative disease caused by deficient activity of the hydrolytic enzyme galactosylceramidase (GALC). The ensuing cytotoxic accumulation of psychosine results in diffuse central and peripheral nervous system (CNS, PNS) demyelination. Presymptomatic hematopoietic stem cell transplantation (HSCT) is the only treatment for infantile-onset GLD; however, clinical outcomes of HSCT recipients often remain poor, and procedure-related morbidity is high. There are no effective therapies for symptomatic patients. Herein, we demonstrate in the naturally occurring canine model of GLD that presymptomatic monotherapy with intrathecal AAV9 encoding canine GALC administered into the cisterna magna increased GALC enzyme activity, normalized psychosine concentration, improved myelination, and attenuated inflammation in both the CNS and PNS. Moreover, AAV-mediated therapy successfully prevented clinical neurological dysfunction, allowing treated dogs to live beyond 2.5 years of age, more than 7 times longer than untreated dogs. Furthermore, we found that a 5-fold lower dose resulted in an attenuated form of disease, indicating that sufficient dosing is critical. Finally, postsymptomatic therapy with high-dose AAV9 also significantly extended lifespan, signifying a treatment option for patients for whom HSCT is not applicable. If translatable to patients, these findings would improve the outcomes of patients treated either pre- or postsymptomatically

    School-based Agricultural Education Teachers’ Perceived Professional Development Needs Associated with Teaching, Classroom Management, and Technical Agriculture

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    Across the nation, there is a large shortage of teachers in several subject areas (Daniel, 2015; Milliard, 2015; Sutcher, Darling-Hammond, & Carver-Thomas, 2016). In agricultural education, many teachers are leaving the profession within the first five years of beginning their teaching careers (Tippens, Ricketts, Morgan, Navaro, & Flanders, 2013). Professional development opportunities for teachers can be utilized to retain teachers in the profession (Touchstone, 2015). The purpose of this study was to evaluate the professional development needs of Iowa school-based agricultural education teachers in the areas of teaching, classroom management, and technical agriculture. Motivating students to learn, teaching in a land laboratory, proper implementation of Individualized Education Program (IEP) for students with disabilities, teaching students decision-making skills, and developing performance-based assessment instruments were the teaching and classroom management items that SBAE teachers expressed the highest perceived levels of professional development needs. The items with the highest perceived training needs were teaching knowledge and skills in biotechnology, integrating current advances in agriculture technology into the curriculum, teaching knowledge and skills in agribusiness, teaching about public issues regarding agriculture, and teaching knowledge and skills in agricultural mechanic. Future research should be conducted to determine what agriculture technology and biotechnology professional development is needed by in-service teachers

    Bone Marrow Transplantation Alters the Tremor Phenotype in the Murine Model of Globoid-Cell Leukodystrophy

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    This is the publisher's version, also available electronically from "http://www.mdpi.com".Tremor is a prominent phenotype of the twitcher mouse, an authentic genetic model of Globoid-Cell Leukodystrophy (GLD, Krabbe’s disease). In the current study, the tremor was quantified using a force-plate actometer designed to accommodate low-weight mice. The actometer records the force oscillations caused by a mouse’s movements, and the rhythmic structure of the force variations can be revealed. Results showed that twitcher mice had significantly increased power across a broad band of higher frequencies compared to wildtype mice. Bone marrow transplantation (BMT), the only available therapy for GLD, worsened the tremor in the twitcher mice and induced a measureable alteration of movement phenotype in the wildtype mice. These data highlight the damaging effects of conditioning radiation and BMT in the neonatal period. The behavioral methodology used herein provides a quantitative approach for assessing the efficacy of potential therapeutic interventions for Krabbe’s disease
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