Morphological characteristics of motor neurons do not determine their relative susceptibility to degeneration in a mouse model of severe spinal muscular atrophy

Spinal muscular atrophy (SMA) is a leading genetic cause of infant mortality, resulting primarily from the degeneration and loss of lower motor neurons. Studies using mouse models of SMA have revealed widespread heterogeneity in the susceptibility of individual motor neurons to neurodegeneration, but the underlying reasons remain unclear. Data from related motor neuron diseases, such as amyotrophic lateral sclerosis (ALS), suggest that morphological properties of motor neurons may regulate susceptibility: in ALS larger motor units innervating fast-twitch muscles degenerate first. We therefore set out to determine whether intrinsic morphological characteristics of motor neurons influenced their relative vulnerability to SMA. Motor neuron vulnerability was mapped across 10 muscle groups in SMA mice. Neither the position of the muscle in the body, nor the fibre type of the muscle innervated, influenced susceptibility. Morphological properties of vulnerable and disease-resistant motor neurons were then determined from single motor units reconstructed in Thy.1-YFP-H mice. None of the parameters we investigated in healthy young adult mice - including motor unit size, motor unit arbor length, branching patterns, motor endplate size, developmental pruning and numbers of terminal Schwann cells at neuromuscular junctions - correlated with vulnerability. We conclude that morphological characteristics of motor neurons are not a major determinant of disease-susceptibility in SMA, in stark contrast to related forms of motor neuron disease such as ALS. This suggests that subtle molecular differences between motor neurons, or extrinsic factors arising from other cell types, are more likely to determine relative susceptibility in SMA

Corrigendum to “Proteoglycan 4 regulates macrophage function without altering atherosclerotic lesion formation in a murine bone marrow-specific deletion model.” [Atherosclerosis 274 (July 2018) 120–127]

The purpose of this research was to establish whether the implementation of adequate internal control procedures will enhance the financial management of tourist services MSEs in Lima, taking into account that the critical point for any losses caused by various factors, refer to absence of proper supervision of the implementation of internal control in the collection, influencing so many times in total liquidity. The research design was non-experimental, correlational with mixed approach (qualitative and quantitative), regarded as applied research because of the practical scope, applications underpinned by standards and technical tools of information gathering. We used a sample of 44 people involved in the conduct of business for services performed for different customers in general and companies accounted for 11 representative, who answered a questionnaire designed for the diagnosis, formulation and revision of strategies. The results and analysis of the research showed that there is an adequate internal control partially impossible, the fulfillment of the main objectives of all MSEs are immersed in this field.El propósito de la presente investigación fue establecer si la adecuada implementación de los procedimientos de control interno optimizará la gestión financiera en las Mypes de servicios turísticos en Lima Metropolitana, teniendo en cuenta que el punto crítico de las pérdidas ocasionadas por diversos factores, se refieren a la inexistencia de una la correcta supervisión de la implementación del control interno en las cobranzas, influyendo muchas veces en forma total en su liquidez. El diseño de la investigación fue de tipo no experimental, correlacional con enfoque mixto (cualitativo-cuantitativo), considerada como investigación aplicada, debido a los alcances prácticos, aplicativos sustentada por normas e instrumentos técnicos de recopilación de información. Se utilizó una muestra compuesta por 44 personas, involucradas en el desarrollo de las labores de servicios realizados a diversos clientes en general y que correspondió a 12 empresas representativas, quienes respondieron un cuestionario diseñado para el diagnóstico, formulación y revisión de estrategias. Los resultados y el análisis de la investigación demostraron que existe un inadecuado control interno que imposibilita de forma parcial, el cumplimiento de los objetivos principales de toda Mype inmersa en este rubro

Corrigendum to: Proteoglycan 4 regulates macrophage function without altering atherosclerotic lesion formation in a murine bone marrow-specific deletion model (vol 274, pg 120, 2018)

Biopharmaceutic

Proteoglycan 4 regulates macrophage function without altering atherosclerotic lesion formation in a murine bone marrow-specific deletion model

Background and aims: Proteoglycan 4 (Prg4) has a high structural similarity with the established atherosclerosis-modulating proteoglycan versican, but its role in atherogenesis is still unknown. Therefore, the impact of Prg4 deficiency on macrophage function in vitro and atherosclerosis susceptibility in vivo was investigated. Methods: The presence and localization of Prg4 was studied in atherosclerotic lesions. Furthermore, the effect of Prg4 deficiency on macrophage foam cell formation, cholesterol efflux and lipopolysaccharide (LPS) response was determined. Finally, susceptibility for atherosclerotic lesion formation was investigated in bone marrow-specific Prg4 knockout (KO) mice. Results: Prg4 mRNA expression was induced 91-fold (p<0.001) in murine initial atherosclerotic lesions and Prg4 protein co-localized with human lesional macrophages. Murine Prg4 KO macrophages showed increased foam cell formation (+2.1-fold, p<0.01). In parallel, the expression of the cholesterol efflux genes ATP-binding cassette transporter A1 and scavenger receptor type B1 was lower (−35%, p<0.05;-40%, p<0.05) in Prg4 KO macrophages. This translated into an impaired cholesterol efflux to high-density lipoprotein (−13%, p<0.001) and apolipoprotein A1 (−8%, p<0.05). Furthermore, Prg4 KO macrophages showed an impaired LPS-induced rise in TNFα secretion as compared to wild-type controls (−31%, p<0.001), indicating a reduced inflammatory response. Combined, these pro- and anti-atherogenic effects did not translate into a significant difference in atherosclerotic lesion formation upon bone marrow-specific deletion of Prg4 in low-density lipoprotein receptor KO mice. Conclusions: Prg4 is present in macrophages in both murine and human atherosclerotic lesions and critically influences macrophage function, but deletion of Prg4 in bone marrow-derived cells does not affect atherosclerotic lesion development

Reconstruction of entire single motor units from Thy.1-YFP-H mice.

<p>A – Representative example of a low magnification confocal montage showing YFP-expressing motor neurons innervating the LALr muscle from a Thy.1-YFP-H mouse. Whole mount muscles were dissected and incubated with TRITC-conjugated α-bungarotoxin to label motor endplates (red). B – An example trace of one motor unit from the LALr shown in panel A. A total of 105 entire motor unit reconstructions were produced for subsequent analyses of motor unit morphology.</p

Differential susceptibility to degeneration between motor neurons innervating anatomically distinct muscles in a mouse model of severe SMA.

<p>A – Schematic illustration of the anatomical locations of the LALr, LALc, AAL, AS, and IS muscles in the mouse, collectively known as the cranial muscles (Figure adapted from Murray et al., 2010). B – Schematic illustration of the anatomical locations of the TVA and TS muscles in the abdominal and thoracic walls of the mouse. C – Schematic illustration of the anatomical locations of the TA, EDL and GS muscles in the hind limb of the mouse. D−F – Representative confocal micrographs showing differing levels of synaptic pathology at neuromuscular junctions in P5 <i>Smn−/−;SMN2</i> mice (green = neurofilament and SV2; red = bungarotoxin-labelled acetylcholine receptors). D shows an example of a healthy, fully occupied motor endplate. E shows an example of a partially occupied motor endplate, where the motor nerve terminal (green) has retracted from the majority of the motor endplate. F shows an example of a vacant motor endplate where the nerve terminal has completely retracted from the motor endplate. Scale bars = 5 µm. G – Bar chart (mean±SEM) showing the percentage of fully occupied endplates in healthy littermate controls (white bars; N = 3 mice) and <i>Smn−/−;SMN2</i> mice (coloured bars; N = 3 mice). Mean values were used to rank the muscles from low vulnerability (yellow) to high vulnerability (red). This colour coding system has been applied to subsequent figures in order to distinguish muscles with vulnerable and disease-resistant motor neurons.</p

Further analysis of motor unit branching patterns revealed no influence on the susceptibility to degeneration in SMA

<p>. A – Representative examples of individual branching diagrams from single motor units innervating the range of vulnerable and disease-resistant muscles analysed. Note the similarities in overall branching patterns in all examples shown. B – Bar charts (mean±SEM) showing the percentage of branch points per branching order in motor units innervating the range of vulnerable and disease-resistant muscles analysed. Once again, note the similarity in distribution of branch orders in all examples shown.</p

The number of terminal Schwann cells at the neuromuscular junction does not influence the relative susceptibility of motor neurons in SMA.

<p>A/B – Example confocal micrographs showing immunohistochemically labelled terminal Schwann cells (S100; green) at neuromuscular junctions in the LALr (A) and AAL (B) muscles. Nuclei were labelled with TOPRO-3 (blue) and motor endplates were labelled with bungarotoxin (red). The bottom left panel of A and B shows a merge of all three individual channels. Images were acquired on a confocal microscope using sequential capture to ensure no bleed-through from one channel to the next. The arrows in A show a single motor endplate (in the BTX channel), with clear terminal Schwann cell cytoplasm above it (in the S100 channel), but with two nuclei present within the S100 footprint (TO-PRO3 channel). This NMJ was therefore assessed to have 2 associated terminal Schwann cells. The arrows in B show two distinct motor endplates (in the BTX channel), each with clear terminal Schwann cell cytoplasm above it (in the S100 channel), but with only one nucleus present within the S100 footprint (TO-PRO3 channel) at each NMJ. These NMJs were therefore assessed to have 1 associated terminal Schwann cell each. Scale bars = 5 µm. C – Bar chart (mean±SEM) showing the mean number of terminal Schwann cells (tSCs) per NMJ in a range of muscles from P5 healthy littermate control mice (N = 3 mice). D – Scatter plot showing the number of tSCs per NMJ in each muscle examined, plotted against the relative vulnerability of motor neurons innervating that muscle. Statistical analysis showed that there was no significant correlation between the number of tSCs per NMJ and the susceptibility of the motor neuron in SMA (P>0.05, Spearman correlation analysis).</p

Motor unit branching patterns do not influence susceptibility to degeneration in SMA.

<p>A/B – Bar chart (mean±SEM; A) and scatter plot (B) showing the number of branch points in motor units from a range of vulnerable (red bars in A) and disease-resistant (yellow bars in A) muscles. No significant correlation was found between the number of branch points and the relative susceptibility of a motor neuron (P>0.05, Spearman correlation analysis; N≥3 mice per muscle).</p

Muscle fibre twitch types of muscles analysed from <i>Smn−/−;SMN2</i> mice.

<p><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052605#pone-0052605-t001" target="_blank">Table 1</a>. Table showing the muscle fibre type for muscles collected from <i>Smn−/−;SMN2</i> mice (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052605#pone-0052605-g001" target="_blank">Figure 1G</a>). Muscles are ranked in order of vulnerability to SMA, from low at the top to high at the bottom. Muscle fibre type is based on data from previously published studies <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052605#pone.0052605-Murray1" target="_blank">[10]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052605#pone.0052605-Murray3" target="_blank">[23]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052605#pone.0052605-Lionikas1" target="_blank">[25]</a>.</p