Journal: Cells
Article Title: Emerging Role of miR-21-5p in Neuron–Glia Dysregulation and Exosome Transfer Using Multiple Models of Alzheimer’s Disease
doi: 10.3390/cells11213377
Figure Lengend Snippet: Characterization reveals successfully differentiated and maturated neurons, while Neu-AD cell lines show alterations in intracellular and exosomal miRNAs, which include the simultaneous elevation of miR-21 and miR-124. ( A ) Schematic representation of iPSC differentiation into induced neurons (Neu), as detailed in Materials and Methods. ( B ) iPSC-derived neurons express the typical neuronal markers synapsin-1, MAP-2, Tau, SV-2, PSD-95 and F-actin. ( C ) Representative images of synaptic puncta in neurons, with co-localization of synapsin-1 and PSD-95, and ( D ) quantification of individual and double-labeled puncta. Nuclei were counterstained with Hoechst. ( E ) Comparative number of cells expressing MAP-2, Tau, PSD-95 and SV-2 protein markers in neurons from AD and Ctrl donors. ( F ) Quantification of the selected inflamma-miRNAs in Neu-AD cell lines and ( G ) in their derived exosomes expressed as the binary logarithm of fold change vs. Neu-Ctrl cells (dotted line). Samples from a male patient (in blue dots) and a female patient (in pink dots) were distinguished. Scale bars correspond to 40 µm in ( A , B ) and to 20 µm in ( C ). Results are mean ± SEM from at least 7 independent experiments. Two-tailed Student’s t -test: * p < 0.05, Neu-AD vs. Neu-Ctrl; ## p < 0.01, male (n = 4) vs. female (n = 3) samples. Neu, iPSC-induced neurons; AD, Alzheimer’s disease; Ctrl, control; EXO, exosomes. SV-2, synaptic vesicle protein 2; PSD-95, postsynaptic density protein; MAP-2, microtubule-associated protein-2; F-actin, filamentous actin; NDM, neural differentiation media; LDN, LDN193189 dihydrochloride; SB, SB431542; FGF, fibroblast growth factor; EGF, epidermal growth factor; NIM, neural induction media; BDNF, brain-derived neurotrophic factor; GDNF, glial-cell-derived neurotrophic factor; ULA, ultra-low-attachment plates; PO, poly-L-ornithine; L, laminin; D, day.
Article Snippet: For neuronal maturation, NPSs were dissociated with StemPro ® Accutase (Thermo Fisher Scientific) and plated on Poly-ornithine + Laminin-coated dishes in Neuron Induction Media (NIM) consisting of DMEM-F12 and Neurobasal (1:1), 1% N2, 2% B27 without vitamin A, 1% L-Glutamax and 0.5% penicillin/streptomycin (50 IU/50 mg/mL) (all from Invitrogen, Waltham, MA, USA), supplemented with 20 ng/mL of neuronal growth factors BDNF and GDNF (both from Peprotech), for 1 month prior to experiments. iPCS-derived midbrain dopaminergic neurons expressed Tau and microtubule-associated protein 2 (MAP-2), as well as pre- and post-synaptic markers [ ].
Techniques: Derivative Assay, Labeling, Expressing, Two Tailed Test, Control
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