Journal: Autophagy

Article Title: Autophagy inhibition promotes SNCA/alpha-synuclein release and transfer via extracellular vesicles with a hybrid autophagosome-exosome-like phenotype

doi: 10.1080/15548627.2017.1395992

Figure Lengend Snippet: ALP inhibition enhances SNCA release. (A, B) ICC showing increased expression and homogeneous SNCA distribution in SNCA-overexpressing H4 cells (SNCA H4 compared to CTR H4) and neurons (SNCA-tg compared to WT). TUBB3 or GFP were used to visualize cellular outline. Scale bar: 10 µm. SNCA expression was also examined by WB analysis of cell lysate from H4 cells (C) or neurons (D). SNCA is not detectable in CTR H4, therefore quantification was not applicable. SNCA-tg neurons overexpress the protein by 2.5 fold compared to WT (*p = 0.024, N = 3). ACTB or TUBB3 were used as loading controls. (E) Dot blot analysis of SNCA H4 conditioned medium (CM) shows that relative total extracellular SNCA levels increase by treatment with the ALP inhibitors Baf and CQ over the corresponding Veh, which is plotted as a dotted line. (For Baf: *p<0.05, with 2 nM N = 7, 10 nM N = 6, 20 and 200 nM N = 5, one sample t test; for 50 µM CQ: **p<0.008, N = 4, one sample t test.) (F) Total extracellular SNCA was assessed by ultra-sensitive ELISA, and values were normalized to total cell protein of the corresponding lysate. With 20 nM Baf or 50 µM CQ, an approximately 3-fold SNCA increase over Veh was measured (for Baf: **p = 0.002, N = 3; for CQ: *p = 0.013, N = 4). (G) The ratio of extracellular:intracellular SNCA was calculated from the ELISA measurements. Baf (20 nM) and CQ (50 µM) increased by approximately 4 fold the percent SNCA amount present extracellularly over intracellularly. (For Baf: ***p = 0.0003, N = 3; for CQ: ** p = 0.007, N = 4.)

Article Snippet: Nuclei were stained with 4’6’-diamidino-2-phenylindol (DAPI, 1:10,000; Sigma-Aldrich, D8417) for 5 min. SNCA (human) (15G7, 1:200; Alexis-Axxora, ALX-804-258) or SNCA (human and rodent) (Clone 42/α-Synuclein, 1:200; BD Transduction Laboratories, 610787), SQSTM1 (1:700; MBL, PM045), TUBB3 (1:1000; Promega, G7121), and MAP2 (Clone AP-20, 1:500; Sigma Aldrich, M1406) antibodies were used.

Techniques: Inhibition, Expressing, Dot Blot, Enzyme-linked Immunosorbent Assay

Journal: Biomaterials

Article Title: A customizable microfluidic platform for medium-throughput modeling of neuromuscular circuits

doi: 10.1016/j.biomaterials.2019.119537

Figure Lengend Snippet: Formation of functional motor units using MFPs with PEMA-BME (A–B) Quantification of (A) days required for MN axons to grow through the micro-channels and (B) number of MN axons per image. Error bars indicate SEM. * and *** indicate p < 0.05 and 0.001, respectively, according to unpaired T-test with Welch’s correction. (C) Immunostaining for TUBB3 of MNs axons exiting the micro-channels when cultured as indicated. Scale bar = 50 μm. (D) Immunostaining of MNs and myotubes for TUBB3 and MY-32, respectively, when cultured in MFPs with PEMA-BME for 21 days. Scale bar = 50 μm. (E) Phase contrast image showing axons of MNs exiting micro-channels and attaching to myotubes in the peripheral compartment. (F) Immunostaining of neuromuscular junctions (NMJs) using the MN marker SMI-32 as well as fluorescently conjugated BTX, which labels acetylcholine receptors. Scale bar = 5 μm. (G) Schematic of optogenetic set up. MNs and myotubes were cultivated in the PEMA-BME MFP, and MNs were infected with a lentivirus carrying CatCh-EYFP under a Synapsin promotor (SynP). (H) 10 days after infection cultures were illuminated with 200 ms blue light (475 nm) pulses every 5 s (0.2 Hz). Contraction was quantified by change in pixel. Myotubes showed contraction in response to light pulses. Contraction was not recorded without stimulation. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: Subsequently, the primary antibodies (mouse anti tubulin beta 3 (TUBB3) (1:1000, Covance), monoclonal Anti-Myosin (MY-32) (1:500, Sigma), microtubule-associated protein 2 (MAP2) (1:5000, Abcam), neurofilament heavy polypeptide (SMI-32) (1:500, Millipore), choline Acetyltransferase (CHAT) (1:400, Millipore), Islet-1 (1:1500, Abcam), titin (T11) (1:1000, Bio-Techne), sarcomeric alpha Actinin antibody (EA-53) (1:200, GeneTex) were applied overnight at 4 °C in 0.1% BSA in PBS.

Techniques: Functional Assay, Immunostaining, Cell Culture, Marker, Infection

Journal: Biomaterials

Article Title: A customizable microfluidic platform for medium-throughput modeling of neuromuscular circuits

doi: 10.1016/j.biomaterials.2019.119537

Figure Lengend Snippet: Derivation of MNs and skeletal myotubes from iPSCs (A) Schematic of MN differentiation protocol. MNPs = motor neuron progenitors. (B) Immunostaining of iPSC-derived MNs for the neuronal markers MAP2 and TUBB3 and the MN markers CHAT, SMI-32 and Islet-1. Scale bar = 50 μm. (C) Quantification of MN differentiation efficiency using Islet-1 immunostaining. n = 3. Error bars show SEM. (D) Multi-electrode array recording of MNs showing spontaneous activity (base line), which can be blocked using 1 μM tetrodotoxin (TTX) treatment and recovered by washing out (E) Quantification of action potential frequency shows significant effect of TTX, which proves electrical activity of neurons. (F) Schematic of skeletal myotube differentiation protocol. Bars indicated SEM. ** and *** indicate p < 0.01 and 0.001, respectively, according to t -test. (G) Quantitative RT-PCR showing increased expression of the indicated skeletal myotube markers. Bars indicated SEM. * and ** indicate p < 0.05 and 0.01, respectively, according to one-way ANOVA and Dunnett’s multiple comparisons test. (H) Myotube contraction after contraction optogenetic stimulation using ChR2-EYFP with 20 ms blue light pulses (475 nm) at 0.2 Hz. (I) Skeletal myotubes differentiated from myoblasts show skeletal muscle marker expression (MY-32, titin, α-actinin) and striation. Scale bar = 25 μm. (J) Skeletal myotubes display acetylcholine receptor clusters visualized with fluorescently conjugated α-bungarotoxin (BTX). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: Subsequently, the primary antibodies (mouse anti tubulin beta 3 (TUBB3) (1:1000, Covance), monoclonal Anti-Myosin (MY-32) (1:500, Sigma), microtubule-associated protein 2 (MAP2) (1:5000, Abcam), neurofilament heavy polypeptide (SMI-32) (1:500, Millipore), choline Acetyltransferase (CHAT) (1:400, Millipore), Islet-1 (1:1500, Abcam), titin (T11) (1:1000, Bio-Techne), sarcomeric alpha Actinin antibody (EA-53) (1:200, GeneTex) were applied overnight at 4 °C in 0.1% BSA in PBS.

Techniques: Immunostaining, Derivative Assay, Activity Assay, Quantitative RT-PCR, Expressing, Marker

Journal: Biomaterials

Article Title: A customizable microfluidic platform for medium-throughput modeling of neuromuscular circuits

doi: 10.1016/j.biomaterials.2019.119537

Figure Lengend Snippet: Motor neurons and skeletal myotubes have different adhesion requirements (A) Schematic of the CNS compartment inside one experimental unit of the MFP. (B) Immunostaining for TUBB3 of MNs inside the CNS compartment. Scale bar = 100 μm. Axons enter the micro-channels towards the peripheral compartment. Scale bar = 25 μm. (C) Axons of MNs exit the micro-channels into the peripheral compartment four days (4Ds) after plating in the CNS compartment. Scale bar = 50 μm. (D–E) Number of days that (D) skeletal myotubes and (E) MNs were attached on glass cover slips functionalized as indicated. (F) Size of clusters formed by MNs in MFPs using glass cover slips functionalized as indicated. Bars in all graphs indicate SEM. Green bars indicate the positive control condition. Black bars indicate coatings resulting in cell detachment. Blue bars indicate conditions selected for further testing. * and ** indicate p < 0.05 and 0.01, respectively, according to ordinary one-way ANOVA and Dunnett’s multiple comparisons test. (G) Immunostaining for TUBB3 of MNs in the CNS compartment using the indicated condition. Scale bar = 100 μm. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: Subsequently, the primary antibodies (mouse anti tubulin beta 3 (TUBB3) (1:1000, Covance), monoclonal Anti-Myosin (MY-32) (1:500, Sigma), microtubule-associated protein 2 (MAP2) (1:5000, Abcam), neurofilament heavy polypeptide (SMI-32) (1:500, Millipore), choline Acetyltransferase (CHAT) (1:400, Millipore), Islet-1 (1:1500, Abcam), titin (T11) (1:1000, Bio-Techne), sarcomeric alpha Actinin antibody (EA-53) (1:200, GeneTex) were applied overnight at 4 °C in 0.1% BSA in PBS.

Techniques: Immunostaining, Positive Control

Journal: Biomaterials

Article Title: A customizable microfluidic platform for medium-throughput modeling of neuromuscular circuits

doi: 10.1016/j.biomaterials.2019.119537

Figure Lengend Snippet: PEMA-BME and PEMA-cRGD-Laminin are suitable coatings for culturing MNs and skeletal myotubes (A–B) Quantification of how long (A) myotubes and (B) MNs were attached when cultured as indicated. (C–D) Immunostaining of (C) myotubes for MY-32 (MyHC) (scale bar = 100 μm), which marks skeletal muscle, and (D) titin and α-actinin (scale bar = 25 μm) which marks sarcomeres which were cultured on glass cover slips functionalized as indicated. (E) Immunostaining of MNs for TUBB3, which were cultured on glass cover slips functionalized as indicated. Scale bar = 100 μm. (F) Quantification of myotubes after 21 days of culture as indicated. (G–H) Quantification of (G) cluster size and (H) axonal growth of MNs when cultured as indicated. Bars in all graphs indicate SEM. Green bars indicate the positive control condition. Black bars indicate coatings resulting in cell detachment. Blue bars indicate conditions selected for further testing. * and **** indicate p < 0.05 and 0.0001, respectively, according to ordinary one-way ANOVA and Dunnett’s multiple comparisons test. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: Subsequently, the primary antibodies (mouse anti tubulin beta 3 (TUBB3) (1:1000, Covance), monoclonal Anti-Myosin (MY-32) (1:500, Sigma), microtubule-associated protein 2 (MAP2) (1:5000, Abcam), neurofilament heavy polypeptide (SMI-32) (1:500, Millipore), choline Acetyltransferase (CHAT) (1:400, Millipore), Islet-1 (1:1500, Abcam), titin (T11) (1:1000, Bio-Techne), sarcomeric alpha Actinin antibody (EA-53) (1:200, GeneTex) were applied overnight at 4 °C in 0.1% BSA in PBS.

Techniques: Cell Culture, Immunostaining, Positive Control