Cell-to-Cell Transmission of Dipeptide Repeat Proteins Linked to C9orf72-ALS/FTD - PubMed (original) (raw)

Cell-to-Cell Transmission of Dipeptide Repeat Proteins Linked to C9orf72-ALS/FTD

Thomas Westergard et al. Cell Rep. 2016.

Abstract

Aberrant hexanucleotide repeat expansions in C9orf72 are the most common genetic change underlying amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). RNA transcripts containing these expansions undergo repeat-associated non-ATG translation (RAN-T) to form five dipeptide repeat proteins (DPRs). DPRs are found as aggregates throughout the CNS of C9orf72-ALS/FTD patients, and some cause degeneration when expressed in vitro in neuronal cultures and in vivo in animal models. The spread of characteristic disease-related proteins drives the progression of pathology in many neurodegenerative diseases. While DPR toxic mechanisms continue to be investigated, the potential for DPRs to spread has yet to be determined. Using different experimental cell culture platforms, including spinal motor neurons derived from induced pluripotent stem cells from C9orf72-ALS patients, we found evidence for cell-to-cell spreading of DPRs via exosome-dependent and exosome-independent pathways, which may be relevant to disease.

Keywords: ALS; C9orf72; DPR; FTD; cell-to-cell transmission; dipeptide repeat proteins; exosomes; propagation.

Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

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Figures

Figure 1

Figure 1. In vitro transmission of DPRs from NSC34 cells to cortical neurons and from cortical neurons to astrocytes

(A-C) Schematics of experimental workflow. (A) Lower panels: Transfected NSC34 cells transmitted DPRs to cortical neurons in a transwell system. All DPR species were detected in cortical neurons besides poly-PR. Nuclei were stained with DAPI (blue), green represents DPRs, red represents MAP2+ neurons. (B) Lower panels: Representative confocal images show transmission of all DPR species (GFP tagged) from cortical neurons to astrocytes. Cells were stained with DAPI (blue for nuclei), DPRs (GFP green), GFAP (red), and MAP2 (cyan). There was negligible MAP2 staining in GA, GR, and PR groups, suggestive of extensive neuronal death. (C) Lower panels: Cells were stained with DAPI (blue for nuclei), DPRs (GFP green), and GFAP (red) for astrocytes. Small or low fluorescent aggregates are marked by arrows. All bars=50 μm.

Figure 2

Figure 2. Anterograde and retrograde transmission of DPRs

(A, C) Schematics of the experimental design. (B) Transfected cortical neurons in chamber #1 transmitted DPRs to cortical neurons in chamber #2. (D) Conditioned medium of DPR-expressing NSC34 cells added to chamber #2 transmitted DPRs to cortical neurons in chamber #1. All DPRs were detected in cortical neurons, except for PR. DAPI (blue), DPRs (green), MAP2 (red). DPR aggregates are marked by arrows. Bars=50 μm.

Figure 3

Figure 3. Exosome-dependent and independent transmission of DPRs

(A) Representative dot-blot of the exosomal fraction isolated from conditioned media of NSC34 cells transfected with different DPR-encoding constructs. DPRs were detected by GFP immunostaining. Flotillin and TSG101 were used as markers of the exosomal fraction. GM130 was used as marker of other membrane vesicle types. (B) Experimental workflow to compare DPR transmission from the exosomal fraction and total conditioned media. (C) Left: Representative confocal images show transmission of DPRs to cortical neurons through conditioned media. Right: Representative images show transmission of DPRs, except PR, to cortical neurons through isolated exosomes. DAPI (blue), DPRs (green), MAP2 (red). DPR aggregates are marked by arrows. Bars=50 μm.

Figure 4

Figure 4. DPRs transmit in iPSC-derived spinal motor neurons

(A) Representative confocal images show transmission of sense DPR species in co-cultures of control and C9orf72 iPSC-derived spinal motor neurons (sMNs). DPR production was verified in C9orf72 sMNs (a, c, e). Both C9orf72 iPSC lines provide similar results. Control sMNs, identified by GFP, showed presence of GA, GR, and GP, which is not normally present (b, d, f). MAP2 (red), DPRs (cyan). (B) Representative confocal images show transmission of sense DPR species through conditioned media of C9orf72 sMNs. Green represents transduced control sMNs, red represents MAP2+ neurons, cyan represents DPRs. Bars=50 μm.

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