Dystonin loss-of-function leads to impaired autophagy-endolysosomal pathway dynamics

The neuronal dystonin protein (DST-a) is a large cytoskeletal linker important for integrating the various components of the cytoskeleton. Recessive Dst mutations lead to a sensory neuropathy in mice known as dystonia musculorum (DstThe accepted manuscript in pdf format is listed with the files at the bottom of this page. The presentation of the authors' names and (or) special characters in the title of the manuscript may differ slightly between what is listed on this page and what is listed in the pdf file of the accepted manuscript; that in the pdf file of the accepted manuscript is what was submitted by the author

An Online Pediatric Palliative Care Education and Mentoring (Project ECHO) in Nepal: A Program Implementation Case Study and Assessment of Changes in Healthcare Providers’ Knowledge, Confidence, and Attitudes

OBJECTIVES The goal of this implementation study was to describe the implementation and evaluation of the impact of an online pediatric palliative care training program in Nepal, using the Project ECHO model. METHODS The study used mixed methods, including a program case study describing the online learning program and before-and-after surveys of program participants, assessing learning through changes in knowledge, comfort, and attitudes. An end-of-program survey was used to evaluate participants’ overall experiences with the learning program and use of the learning resources. RESULTS A literature review, stakeholder surveys, and expert input informed the design of the intervention. The course used the Project ECHO model of online education, with modifications based on the leadership team's previous ECHO experiences and local stakeholder input. The intervention occurred over 9 months, with 22 online teaching sessions. Each session consisted of a didactic lecture, case presentation, and interactive discussion with expert clinical teachers. Fifty-five clinicians in Nepal participated, including physicians (47%), nurses (44%), and psychotherapists (5%). Clinicians reported improvements in knowledge, skills, and attitudes after program participation. Program acceptability scores were high, with 93% of participants reporting that the course provided effective learning. CONCLUSIONS Project ECHO can be successfully implemented to deliver continuing professional development in Nepal. Delivering palliative care education online using the Project ECHO model, leads to improved knowledge, skills, and attitudes for clinicians. Project ECHO suggests an innovative solution which can provide training and support to clinicians in settings where educational opportunities in palliative care are limited

sj-docx-2-mde-10.1177_23821205241234541 - Supplemental material for An Online Pediatric Palliative Care Education and Mentoring (Project ECHO) in Nepal: A Program Implementation Case Study and Assessment of Changes in Healthcare Providers’ Knowledge, Confidence, and Attitudes

Supplemental material, sj-docx-2-mde-10.1177_23821205241234541 for An Online Pediatric Palliative Care Education and Mentoring (Project ECHO) in Nepal: A Program Implementation Case Study and Assessment of Changes in Healthcare Providers’ Knowledge, Confidence, and Attitudes by Anisha Lynch-Godrei, Sudhir Sapkota, Jennifer Rowe, Bishnu Dutta Paudel, Garima Aryal and Megan Doherty in Journal of Medical Education and Curricular Development</p

sj-docx-3-mde-10.1177_23821205241234541 - Supplemental material for An Online Pediatric Palliative Care Education and Mentoring (Project ECHO) in Nepal: A Program Implementation Case Study and Assessment of Changes in Healthcare Providers’ Knowledge, Confidence, and Attitudes

Supplemental material, sj-docx-3-mde-10.1177_23821205241234541 for An Online Pediatric Palliative Care Education and Mentoring (Project ECHO) in Nepal: A Program Implementation Case Study and Assessment of Changes in Healthcare Providers’ Knowledge, Confidence, and Attitudes by Anisha Lynch-Godrei, Sudhir Sapkota, Jennifer Rowe, Bishnu Dutta Paudel, Garima Aryal and Megan Doherty in Journal of Medical Education and Curricular Development</p

sj-docx-1-mde-10.1177_23821205241234541 - Supplemental material for An Online Pediatric Palliative Care Education and Mentoring (Project ECHO) in Nepal: A Program Implementation Case Study and Assessment of Changes in Healthcare Providers’ Knowledge, Confidence, and Attitudes

Supplemental material, sj-docx-1-mde-10.1177_23821205241234541 for An Online Pediatric Palliative Care Education and Mentoring (Project ECHO) in Nepal: A Program Implementation Case Study and Assessment of Changes in Healthcare Providers’ Knowledge, Confidence, and Attitudes by Anisha Lynch-Godrei, Sudhir Sapkota, Jennifer Rowe, Bishnu Dutta Paudel, Garima Aryal and Megan Doherty in Journal of Medical Education and Curricular Development</p

Cytoskeletal Linker Protein Dystonin Is Not Critical to Terminal Oligodendrocyte Differentiation or CNS Myelination

<div><p>Oligodendrocyte differentiation and central nervous system myelination require massive reorganization of the oligodendrocyte cytoskeleton. Loss of specific actin- and tubulin-organizing factors can lead to impaired morphological and/or molecular differentiation of oligodendrocytes, resulting in a subsequent loss of myelination. Dystonin is a cytoskeletal linker protein with both actin- and tubulin-binding domains. Loss of function of this protein results in a sensory neuropathy called Hereditary Sensory Autonomic Neuropathy VI in humans and <i>dystonia musculorum</i> in mice. This disease presents with severe ataxia, dystonic muscle and is ultimately fatal early in life. While loss of the neuronal isoforms of dystonin primarily leads to sensory neuron degeneration, it has also been shown that peripheral myelination is compromised due to intrinsic Schwann cell differentiation abnormalities. The role of this cytoskeletal linker in oligodendrocytes, however, remains unclear. We sought to determine the effects of the loss of neuronal dystonin on oligodendrocyte differentiation and central myelination. To address this, primary oligodendrocytes were isolated from a severe model of <i>dystonia musculorum</i>, <i>Dst</i>^{<i>dt-27J</i>}, and assessed for morphological and molecular differentiation capacity. No defects could be discerned in the differentiation of <i>Dst</i>^{<i>dt-27J</i>} oligodendrocytes relative to oligodendrocytes from wild-type littermates. Survival was also compared between <i>Dst</i>^{<i>dt-27J</i>} and wild-type oligodendrocytes, revealing no significant difference. Using a recently developed migration assay, we further analysed the ability of primary oligodendrocyte progenitor cell motility, and found that <i>Dst</i>^{<i>dt-27J</i>} oligodendrocyte progenitor cells were able to migrate normally. Finally, <i>in vivo</i> analysis of oligodendrocyte myelination was done in phenotype-stage optic nerve, cerebral cortex and spinal cord. The density of myelinated axons and g-ratios of <i>Dst</i>^{<i>dt-27J</i>} optic nerves was normal, as was myelin basic protein expression in both cerebral cortex and spinal cord. Together these data suggest that, unlike Schwann cells, oligodendrocytes do not have an intrinsic requirement for neuronal dystonin for differentiation and myelination.</p></div

<i>Dst</i>^{<i>dt-27J</i>} OLs exhibit normal molecular differentiation.

<p>A. Immunofluorescence micrographs of WT and <i>Dst</i>^{<i>dt-27J</i>} showing maturation marker expression at DD3. B. Quantification of the proportion of NG2⁺, MAG⁺/MBP^-, and MAG⁺/MBP⁺ OLs at DD3. C. Immunofluorescence micrographs of WT and <i>Dst</i>^{<i>dt-27J</i>} showing maturation marker expression at DD6. D. Quantification of the proportion of NG2⁺, MAG⁺/MBP^-, and MAG⁺/MBP⁺ OLs at DD6. B, D: n = 3; all comparisons non-significant by two-tailed Student’s t-test. Data represent mean ± SEM. Arrowheads: yellow = NG2⁺, orange = MAG⁺/MBP^-, grey = MAG⁺/MBP⁺, white = contaminating cell. Scale bars = 50 μm.</p

Proliferating OPCs and differentiating OLs express neuronal <i>Dst</i> transcripts.

<p>Top. Representative RT-PCR products from <i>Dst</i>-<i>A1</i>, <i>-A2</i> and <i>-A3</i> with <i>actb</i> loading control in primary proliferating OPCs (left lanes) and differentiating OLs (right lanes). Bottom. qRT-PCR analysis of <i>Dst</i>-<i>A1</i>, <i>-A2</i> and <i>-A3</i> expression in primary proliferating OPCs and differentiating OLs. n = 4–6; ΔΔCt, <i>Dst</i> normalized to <i>actb</i>. * p<0.05, n.s. = p≥0.05; two-tailed Student’s t-test. Data represent mean ± SEM.</p

Migration is normal in <i>Dst</i>^{<i>dt-27J</i>} OPCs.

<p>A. Immunofluorescence micrographs of WT and <i>Dst</i>^{<i>dt-27J</i>} of OPC aggregates 4 hours post-seeding. B. Quantification of the proportion of NG2⁺ OPCs migrated at 4 hours within rings set at 50 μm increments from the aggregate. C. Immunofluorescence micrographs of WT and <i>Dst</i>^{<i>dt-27J</i>} of OPC aggregate 24 hours post-seeding. D. Quantification of the proportion of NG2⁺ OPCs migrated at 24 hours within rings set at 100 μm increments from the center of the aggregate. B, D: n = 3; all comparisons non-significant by two-tailed Student’s t-test. Data represent mean ± SEM. Scale bars = 50 μm.</p

Apoptosis is not increased in <i>Dst</i>^{<i>dt-27J</i>} OLs.

<p>A. Immunofluorescence micrographs of WT and <i>Dst</i>^{<i>dt-27J</i>} showing colocalization of nuclear CC3 and Olig2 in apoptotic OLs at DD3. B. Quantification of the proportion of CC3⁺/Olig2⁺ relative to total Olig2⁺ OLs at DD3. C. Immunofluorescence micrographs of WT and <i>Dst</i>^{<i>dt-27J</i>} showing colocalization of nuclear CC3 and Olig2 in apoptotic OLs at DD6. D. Quantification of the proportion of CC3⁺/Olig2⁺ relative to total Olig2⁺ OLs at DD6. A, C: Arrowheads represent CC3⁺/Olig2⁺ OLs. B, D: n = 3; all comparisons non-significant by two-tailed Student’s t-test. Data represent mean ± SEM. Scale bars = 50 μm.</p