C9orf72 FTLD/ALS-associated Gly-Ala dipeptide repeat proteins cause neuronal toxicity and Unc119 sequestration

Abstract

Hexanucleotide repeat expansion in C9orf72 is the most common pathogenic mutation in patients with amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). Despite the lack of an ATG start codon, the repeat expansion is translated in all reading frames into dipeptide repeat (DPR) proteins, which form insoluble, ubiquitinated, p62-positive aggregates that are most abundant in the cerebral cortex and cerebellum. To specifically analyze DPR toxicity and aggregation, we expressed DPR proteins from synthetic genes containing a start codon but lacking extensive GGGGCC repeats. Poly-Gly-Ala (GA) formed p62-positive cytoplasmic aggregates, inhibited dendritic arborization and induced apoptosis in primary neurons. Quantitative mass spectrometry analysis to identify poly-GA co-aggregating proteins revealed a significant enrichment of proteins of the ubiquitin-proteasome system. Among the other interacting proteins, we identified the transport factor Unc119, which has been previously linked to neuromuscular and axonal function, as a poly-GA co-aggregating protein. Strikingly, the levels of soluble Unc119 are strongly reduced upon poly-GA expression in neurons, suggesting a loss of function mechanism. Similar to poly-GA expression, Unc119 knockdown inhibits dendritic branching and causes neurotoxicity. Unc119 overexpression partially rescues poly-GA toxicity suggesting that poly-GA expression causes Unc119 loss of function. In C9orf72 patients, Unc119 is detectable in 9.5 % of GA inclusions in the frontal cortex, but only in 1.6 % of GA inclusions in the cerebellum, an area largely spared of neurodegeneration. A fraction of neurons with Unc119 inclusions shows loss of cytosolic staining. Poly-GA-induced Unc119 loss of function may thereby contribute to selective vulnerability of neurons with DPR protein inclusions in the pathogenesis of C9orf72 FTLD/ALS.

Fig. 1

DPR species show differential aggregation properties in HEK293 cells. HEK293 cells were transfected with the five different DPR constructs (GA₁₇₅-GFP, GFP-GR₁₄₉, PR₁₇₅-GFP, PA₁₇₅-myc and GP₈₀-V5) or GFP as a control and analyzed 2 days later by GFP fluorescence or in case of PA₁₇₅-myc and GP₈₀-V5 by immunofluorescence using specific antibodies. DAPI was used as a nuclear marker. Cytoplasmic inclusions (white arrows) and nuclear inclusions (magenta arrows) are seen for GA₁₇₅-GFP, GFP-GR₁₄₉ and PR₁₇₅-GFP. Many dot-like and star-shaped GA₁₇₅-GFP inclusions co-localize with p62 (second column from the left). Right panels show close-ups of areas indicated in the merge column. Magnifications of intranuclear GA₁₇₅-GFP inclusions are shown in Fig. S2a. Negative control stainings are shown in Fig. S2b. Scale bar represents 15 µm for overview and 5 µm for close-up

Fig. 2

Poly-GA forms p62-positive aggregates in neurons. a Immunofluorescence of primary hippocampal neurons transduced with GA₁₄₉-GFP or GFP control lentivirus at day 6 in vitro for 15 days (DIV6 + 15). Immunostaining for p62 and the dendritic marker protein MAP2. DAPI was used a nuclear marker. Poly-GA forms p62-positive inclusions (arrow) in the soma and dendrites. Scale bar 15 µm. b Immunoblotting of primary cortical neurons transduced with GA₁₄₉-myc or GFP control lentivirus (DIV6 + 17) with the indicated antibodies. Poly-GA aggregates are stuck at the top of the gel (arrow). GA₁₄₉-myc induces upregulation of p62, but levels of TDP-43 and the synaptic marker protein PSD-95 are not affected. Three separate transductions are shown. c Filter trap assay of primary cortical neurons transduced with GA₁₄₉-myc or GFP (DIV6 + 17). Poly-GA aggregates are detected in the serial dilution of homogenates using anti-GA

Fig. 3

Poly-GA causes dendrite loss and induces apoptosis in primary neurons. a Cortical neurons were co-transfected with empty vector as control (Ctrl) or GA₁₄₉-myc together with GFP to outline cell morphology (DIV7 + 4). Scale bars represent 40 µm. b Dendritic complexity was measured using Sholl analysis by manually counting the number of dendrites crossing concentric circles around the soma. Poly-GA expression leads to significant reduction of dendritic branching. N = 3 with 40 cells analyzed per condition in each experiment, mean ± SEM. p < 0.001 for 12.5 µm radius, p < 0.0001 from 25 to 50 µm radius, p < 0.001 for 62.5 µm radius, p < 0.01 for 75 µm radius and p < 0.05 from 87.5 to 112.5 µm radius (two-way ANOVA). c Apoptosis in transduced neurons was analyzed using a fluorogenic assay to detect caspase 3/7 activation and a TUNEL assay to detect apoptotic DNA fragmentation (DIV6 + 17). Caspase 3/7 activity was increased 2.0-fold in GA₁₄₉-myc transduced cortical neurons. TUNEL-positive apoptotic cells (manually counted using the Fiji cell count plug-in) were increased by 2.5-fold in GA₁₄₉-myc transduced hippocampal neurons compared to control cells. Representative images of TUNEL stainings are shown in Fig. S2. DIV6 + 17. n = 3 experiments with 6 replicates each; mean ± SD, Student’s t test, ***p < 0.001. d Immunoblots of cortical neurons transduced with GA₁₄₉-myc constructs with or without start codon (DIV8 + 10). Replacing the ATG start codon in the synthetic GA₁₄₉-myc gene with a TAG stop codon prevents poly-GA expression and aggregation. Arrow indicates top of the gel. e LDH release assay detected neurotoxicity of GA₁₄₉-myc only in the presence of an ATG start codon in transduced cortical neurons (DIV8 + 14). One-way ANOVA with Tukey’s post-test. ***p < 0.001, n = 3 with six replicates in each experiment

Fig. 4

Unc119 specifically co-aggregates with poly-GA. a Quantitative proteomics of GFP immunoprecipitations from primary cortical neurons transduced with GFP or GA₁₄₉-GFP (DIV6 + 17). p62/Sqstm1 shows highest enrichment and statistical significance. Unc119 was identified by two unique peptides (GGGGTGPGAEPVPGASNR and LGPLQGK) and one peptide (YQFTPAFLR) shared with its homolog Unc119b. Full protein names are listed in Table 1. Upper panel illustrates distribution of quantified protein abundances binned for enrichment factors (x-axis below). Enrichment of ubiquitin-related and proteasomal proteins in the poly-GA interactome is highlighted in green. Lower panel depicts volcano plot showing poly-GA interacting proteins. False discovery rate (FDR) controlled statistical analysis identified 20 poly-GA interacting proteins compared to control (red dots). Dotted line depicts threshold for statistical significance. b Immunofluorescence of HEK293 cells co-transfected with GFP or GA₁₇₅-GFP and HA-Unc119 or empty vector control (ctrl). Staining with HA and GFP antibodies and DAPI as nuclear marker. Many GA₁₇₅-GFP inclusions show co-aggregation of HA-Unc119 (examples marked with arrows). Separate channels of these images are shown in Fig. S6. Scale bar 30 µm. c HEK293 cells were transfected with the five different poly-DPR constructs (GA₁₇₅-GFP, GFP-GR₁₄₉, PR₁₇₅-GFP, PA₁₇₅-myc and GP₈₀-V5) or Q₁₀₂-GFP and analyzed using GFP fluorescence and immunostaining of HA-Unc119, PA₁₇₅-myc and GP₈₀-V5 using specific antibodies 2 days later. HA-Unc119 co-aggregates only with GA₁₇₅-GFP (white arrows). DAPI (in blue) was used as a nuclear marker. Scale bar 20 µm

Fig. 5

Unc119 sequestration in neurons contributes to poly-GA toxicity. a Immunofluorescence of primary hippocampal neurons co-transduced with HA-Unc119 and either GFP or GA₁₄₉-GFP (DIV6 + 17). Arrows indicate examples of poly-GA inclusions showing co-aggregation of HA-Unc119. Scale bar 15 µm. b Immunoblot with the indicated antibodies in GFP or GA₁₄₉-myc transduced cortical neurons shows decreased levels of soluble Unc119 running at 27 kDa. Two separate transductions are shown (DIV6 + 17). c qPCR analysis of neurons transduced as in (b) shows no significant changes in Unc119 mRNA levels (mean ± SD, Student’s t test, DIV7 + 10). (d, e) Hippocampal neurons transfected with shRNA targeting Unc119 (shUnc) or a non-targeting control (shCtrl) together with GFP to outline cell morphology (DIV7 + 5). Dendritic branching was quantified by Sholl analysis. Unc119 knockdown reduced dendrite complexity significantly (p < 0.0001 for 12.5–62.5 µm radius and p < 0.001 for 75 µm radius, two-way ANOVA, n = 40 neurons per condition). Scale bar depicts 40 µm. f LDH release assay from cortical neurons co-transduced with either GFP or GA₁₄₉-GFP (GA) together with HA-Unc119 (Unc), shRNA targeting Unc119 (shUnc) or non-targeting shRNA (shCtrl) (DIV6 + 17). Note that Unc119 knockdown causes toxicity in GFP-transduced neurons, but does not increase poly-GA toxicity further. HA-Unc119 expression rescues GA₁₄₉-GFP toxicity. One-way ANOVA with Tukey’s post-test. **p < 0.01, ***p < 0.001, n = 3 with six replicates in each experiment

Fig. 6

Unc119 forms neuronal cytoplasmic inclusions in C9orf72 patients. a–g Immunohistochemistry for Unc119 in two C9orf72 mutation carriers (C9-1 and C9-2) and a control case (Ctrl-2) using antibody Unc119#1. a Whereas Unc119 is distributed throughout the cytosol in hippocampal cornu ammonis regions 3/4 (CA3/4) of a control case, a C9orf72 patient shows neuronal cytoplasmic inclusions. Scale bar represents 20 µm in overviews and 10 µm in close-up. b Unc119-positive inclusions have a similar shape as poly-GA inclusions. c, d In the superior frontal gyrus (SFG) Unc119-positive cytoplasmic inclusions are detectable in large neurons of mutations carriers. e–g) Further Unc119-positive neuronal cytoplasmic inclusions are found in the occipital cortex (OCX) and in the granular cell layers of the dentate gyrus (DG) and the cerebellum (CBL). In various areas, a fraction of cells with Unc119 inclusions shows a clear reduction of cytosolic Unc119 suggesting a redistribution of cytosolic Unc119 into aggregates (close-ups in b, d, f). Scale bars represent 10 μm. Counterstains in A-G were done with hemalum. h Filter trap assay detects insoluble Unc119 in 1 % SDS in the frontal cortex of mutation carriers, but not in healthy controls

Fig. 7

Unc119 co-aggregates with poly-GA, but not with TDP-43 in patients with C9orf72 mutation. Double immunofluorescence analysis of Unc119 with poly-GA or phosphorylated TDP-43 (pTDP-43) in C9orf72 mutation cases C9-1, C9-2 and C9-3. a In the superior frontal gyrus (SFG), a subset of poly-GA-positive neuronal cytoplasmic inclusions also contains Unc119. Redistribution of Unc119 compared to GA-negative cells can be seen in a fraction of co-aggregating cells (white arrows). b In the cerebellar granular cell layer (CBL) abundant cytoplasmic poly-GA inclusions are only rarely positive for Unc119 (white arrows). c As shown for the superior frontal gyrus, Unc119 (white arrow) and pTDP (red arrow) are not co-localized in the same cytoplasmic inclusions. Scale bars represent 10 μm for overviews and 5 µm for the close-up in the second column