Cell-Autonomous Death of Cerebellar Purkinje Neurons with Autophagy in Niemann-Pick Type C Disease

Niemann-Pick type C is a neurodegenerative lysosomal storage disorder caused by mutations in either of two genes, npc1 and npc2. Cells lacking Npc1, which is a transmembrane protein related to the Hedgehog receptor Patched, or Npc2, which is a secreted cholesterol-binding protein, have aberrant organelle trafficking and accumulate large quantities of cholesterol and other lipids. Though the Npc proteins are produced by all cells, cerebellar Purkinje neurons are especially sensitive to loss of Npc function. Since Niemann-Pick type C disease involves circulating molecules such as sterols and steroids and a robust inflammatory response within the brain parenchyma, it is crucial to determine whether external factors affect the survival of Purkinje cells (PCs). We investigated the basis of neurodegeneration in chimeric mice that have functional npc1 in only some cells. Death of mutant npc1 cells was not prevented by neighboring wild-type cells, and wild-type PCs were not poisoned by surrounding mutant npc1 cells. PCs undergoing cell-autonomous degeneration have features consistent with autophagic cell death. Chimeric mice exhibited a remarkable delay and reduction of wasting and ataxia despite their substantial amount of mutant tissue and dying cells, revealing a robust mechanism that partially compensates for massive PC death

Towards the Development of a Mobile Application in Movement Competency Training Grounded on the User-Centered Design Model: The Case of a State University in the Philippines

The corona-virus disease (COVID)-19 pandemic has caused extreme disruption in the delivery of instruction in many educational institutions all over the world. However, this circumstance may not only be a challenge but also an opportunity to foster learning through exploration of various technologies that can be developed, more specifically in a course characterized with a strong skills-based orientation such as physical education (PE). Therefore, the aim set in this study was to develop a mobile application for Movement Competency Training (MCT) grounded on the User-Centered Design Model. This descriptive mixed-method research started with a preliminary needs assessment analysis which was participated by 121 Filipino students enrolled in the MCT course and 10 PE teachers. Results revealed the skills that require a higher degree of proficiency such as non-locomotor skills, locomotor, and mobility skills. Also, the majority of the respondents are mobile phone users which justified the adoption of mobile instruction and the development of an application for MCT. Moreover, indicated in this research was the preference of the intended users in terms of design and interface that led to the development of the mobile application and had specified the requirements based on the users' needs. The initial assessment of the developed application indicated a highly acceptable level of functional suitability, usability, and portability by the student-respondents. A field testing of the application may be recommended to determine its effectiveness in mastering the skills in MCT independently

Cell-Autonomous Degeneration of PCs in Chimeric Mice

<div><p>(A) Genotyping of chimeric mice. Upper gel: PCR to amplify a fragment of the <i>npc1</i> gene from <i>npc1</i>^−/−↔GFP, <i>npc1</i>^+/−↔GFP, and <i>npc1</i>^+/−mice results in <i>npc1⁻</i> (1,067 bp) and <i>npc1⁺</i> (947 bp) bands. Lower gel: The three genotypes can be distinguished by ApaLI digestion of the PCR products. The <i>npc1⁻</i> allele gives rise to 937- and 130-bp bands. The wild-type allele varies depending on the source: the BALB/c allele is digested to 477-, 340-, and 130-bp bands while the allele from the GFP mouse is digested to 817- and 130-bp bands. Note that the 477- and 340-bp bands are absent from the homozygous mutant chimera.</p> <p>(B) Quantification of <i>npc1</i>^−/− PC density in chimeric mice. For each mouse, the actual density observed (the average of the mean densities counted in each lobule) is compared to the number expected if no PC loss occurs (calculated from the density at 30 d in <i>npc1</i>^−/− mice and the chimerism percentage). Five sections separated by at least 100 μm were analyzed for each mouse. For the three mice sacrificed at 70 d, only lobules I–IX were included in the analysis because of the lack of degeneration in lobule X in <i>npc1</i>^−/− mice at this age. A value for the number of <i>npc1</i>^−/− PCs expected if loss does occur (calculated from the density at 70 d in <i>npc1</i>^−/− mice and the chimerism percentage) is also included for these three mice. No rescue of <i>npc1</i>^−/− PCs was observed for any of the chimeric mice (<i>p</i> < 0.0001 for each mouse comparing observed versus expected if no loss occurs).</p> <p>(C) Quantification of <i>npc1^−/−</i> PC density by lobule in C1.4 at 70 d. Observed and expected densities are shown for <i>npc1</i>^−/− PCs in each lobule. No clear rescue of <i>npc1</i>^−/− PCs was observed in any lobule (<i>p</i> > 0.05 for all lobules comparing observed versus expected if loss occurs, except II and III, where <i>p</i>-value is between 0.01 and 0.05; <i>p</i> < 0.001 for all lobules comparing observed versus expected if no loss occurs, except lobule X, where <i>p</i> > 0.05).</p> <p>(D and E) Images of lobules II and X taken from C1.4 at 70 d. GFP is green and Calbindin staining is red. In lobule II, the number of PCs is clearly reduced and of the five remaining PCs, four are wild-type (GFP-positive). In lobule X (where no degeneration occurs during the normal <i>npc1</i>^−/− life span), PC density is normal and the majority of PCs (23 of 32; 72%) are mutant, as expected based on <i>npc1</i>^−/− contribution to this mouse (67%).</p> <p>(F) Quantification of wild-type PC density in chimeric mice. Observed and expected densities are shown for wild-type PCs. For mice with more than 15% wild-type contribution, the density of wild-type PCs is similar to the number expected if these cells have not degenerated (<i>p</i> > 0.05 for each mouse comparing observed versus expected if no loss occurs, except C5.2, where <i>p</i> = 0.02, and C5.6, where <i>p</i> = 0.0005).</p> <p>(G) Quantification of wild-type PC density by lobules in C1.4 at 70 d. No loss of wild-type PCs was observed in any of the lobules (<i>p</i> > 0.05 for all lobules comparing observed versus expected if no loss occurs; <i>p</i> < 0.001 for all lobules comparing observed versus expected if loss occurs, except for lobule X, where <i>p</i> > 0.05).</p></div

Other Histological Characteristics in the <i>npc1</i> Mutant and Chimeric Mice

<div><p>(A–C) Microglia characteristics. Sections from 50-d-old <i>npc1</i> mutants were stained with anti-Calbindin (green) to visualize PCs and anti-F4/80 (red) to mark microglia. (A) Very few microglia are present in lobule X, where no PC degeneration has yet occurred. (B) Lobule VIII demonstrates an infiltration of microglia in the granule cell layer and white matter tract. (C) In lobule II, nearly all PCs are gone and numerous microglia are present throughout the cerebellum. The dashed line indicates the edge of the granule cell layer.</p> <p>(D) Microglia infiltration is not sufficient to induce PC degeneration. C1.4 demonstrates numerous microglia marked by anti-F4/80 (red) in all layers of the cerebellum, including immediately adjacent to wild-type (GFP-positive) PCs (arrows). Despite this close approximation, no loss of wild-type PCs was detected by cell counting. The GFP transgene is apparently poorly expressed in microglia, as all microglia appear GFP-negative regardless of their genotype.</p> <p>(E) Mutant PCs are lost even when surrounded by wild-type Bergmann glia. A stretch of the PC layer in C4.8 is shown where no <i>npc1^−/−</i> (GFP-positive) PCs remain despite the presence of numerous wild-type (GFP-negative) glia (arrowheads) marked by anti-S100β (red). The space occupied by two wild-type PCs is indicated (arrows) with two <i>npc1^−/−</i> (GFP-positive) glia directly underneath.</p> <p>(F) Wild-type PCs do not degenerate even when surrounded by mutant Bergmann glia. Three wild-type PCs (GFP-positive; arrows) in C1.4 have not degenerated despite only mutant glia (arrowheads) being in the immediate vicinity.</p> <p>(G) Oligodendrocytes in chimeric mice are a mixture of wild-type and mutant cells. Oligodendrocyte cell bodies are stained with anti-CC1 (red). Wild-type (GFP-positive; arrows) and <i>npc1^−/−</i> (GFP-negative; arrowheads) oligodendrocytes are interspersed in C1.4.</p> <p>Bar = 50 μm for (A), (D), and (G); 10 μm for (E) and (F).</p></div

Features of PC Loss in <i>npc1</i> Mice

<div><p>(A) Sections from the cerebellar vermis of a 70-d-old <i>npc1^−/−</i> mouse were stained with anti-Calbindin (green) to visualize PCs and 7AAD (red) for nuclei. Although there are scant PCs remaining in lobule II, the PCs in lobule X have not decreased.</p> <p>(B) Quantification of progressive anterior-to-posterior PC loss. PC densities from 30-d <i>npc1</i>^+/+ and 30-, 50-, and 70-d <i>npc1</i>^−/− mice were quantified from five sections from two mice each. Error bars show standard deviation.</p> <p>(C) Bergmann glia visualized in 70-d <i>npc1</i>^+/+ and <i>npc1</i>^−/− mice using anti-S100β (green) have normal morphology. Note that the glial cell bodies near the PC layer and the radial processes extending into the molecular layer are intact in the <i>npc1</i>^−/− mouse despite the loss of PCs.</p> <p>(D) Oligodendrocyte cell bodies stained with anti-CC1 (green) have a similar distribution and morphology in the cerebellar white matter of 70-d <i>npc1^−/−</i> and wild-type mice. TOTO-3 (red) was used as a nuclear counterstain. Bar = 5 μm.</p> <p>(E) Axonal spheroids in <i>npc1</i>^−/− mice. Spheroids (arrows), seen as Calbindin-positive (red) swellings surrounded by myelin (anti-myelin basic protein; green), are numerous in the <i>npc1^−/−</i> cerebellum (50-d-old, lobule X shown) but are not seen in the wild-type control. Bar = 5 μm.</p> <p>(F) Sections from wild-type and <i>npc1</i> mice were stained with filipin to visualize free cholesterol. PCs from <i>npc1</i> mice show an accumulation of intracellular cholesterol at 30 d regardless of their lobular location. Images are oriented such that the PCs (arrowheads) are in the center of the field, with the molecular layer above and the inner granule cell layer below.</p></div

Models of PC Degeneration in <i>npc1</i> Mice

<p>Four basic mechanisms are depicted. (1) The accumulation of cholesterol, sphingolipids, or other molecules within PCs lacking Npc1 could be toxic. (2) Loss of Npc1 function could cause a block in trafficking that leads to a localized subcellular deficiency in lipids and/or proteins. (3) Other cells, most likely glia, could produce a secreted factor, such as apoE-cholesterol or a trophic factor required for PC survival, whose export is reduced or blocked without Npc1 function. (4) Any cell type in the <i>npc1</i> mouse could produce toxic metabolites, such as released lysosomal hydrolases or beta amyloid, that could kill surrounding cells or particularly susceptible cell types regardless of genotype.</p