TAR DNA-Binding Protein 43 and Disrupted in Schizophrenia 1 Coaggregation Disrupts Dendritic Local Translation and Mental Function in Frontotemporal Lobar Degeneration

Abstract

Background: Neurodegenerative diseases involving protein aggregation often accompany psychiatric symptoms. Frontotemporal lobar degeneration (FTLD) associated with TAR DNA-binding protein 43 (TDP-43) aggregation is characterized by progressive neuronal atrophy in frontal and temporal lobes of cerebral cortex. Furthermore, patients with FTLD display mental dysfunction in multiple behavioral dimensions. Nevertheless, their molecular origin for psychiatric symptoms remains unclear.

Methods: In FTLD neurons and mouse models with TDP-43 aggregates, we examined coaggregation between TDP-43 and disrupted in schizophrenia 1 (DISC1), a key player in the pathology of mental conditions and its effects on local translation in dendrites and psychiatric behaviors. The protein coaggregation and the expression level of synaptic proteins were also investigated with postmortem brains from patients with FTLD (n = 6).

Results: We found cytosolic TDP-43/DISC1 coaggregates in brains of both FTLD mouse model and patients with FTLD. At the mechanistic levels, the TDP-43/DISC1 coaggregates disrupted the activity-dependent dendritic local translation through impairment of translation initiation and, in turn, reduced synaptic protein expression. Behavioral deficits detected in FTLD model mice were ameliorated by exogenous DISC1 expression.

Conclusions: Our findings reveal a novel role of the aggregate-prone TDP-43/DISC1 protein complex in regulating local translation, which affects aberrant behaviors relevant to multiple psychiatric dimensions.

Keywords: Coaggregation; DISC1; FTLD; Local translation; Psychiatric behaviors; TDP-43.

Figure 1

DISC1 binds to and forms co-aggregates with TDP-43 in neurons. (A) TDP-43 interacts with DISC1 in vivo. DISC1 (m317C) or TDP-43 was immunoprecipitated from homogenates of wild-type mouse cerebral cortex, followed by western blotting (2B3 for DISC1). (B) DISC1 was immunoprecipitated (h598C) from healthy human temporal lobes, followed by western blotting (HM6-5 for DISC1). (C) The binding of DISC1 to TDP-43 is more dominant in cerebral cortex and striatum than that in cerebellum. DISC1 was immunoprecipitated with an anti-DISC1 antibody (m595C) from homogenates of wild-type mouse cerebral cortex, striatum or cerebellum, followed by western blotting (M49 for DISC1) (Left). Co-immunoprecipitated TDP-43 was normalized to immunoprecipitated DISC1 and the binding between DISC1 and TDP-43 is expressed as the relative ratio to cerebral cortex (Right) (n=4, F(2,9)=16.87, P=0.0009, one-way ANOVA; **P<0.01, Bonferroni’s multiple comparison test post hoc). Error bars represent S.E.M. (D) DISC1 binds to TDP-43, FMRP and FUS/TLS in an RNA-dependent manner. Mouse brain (wild-type) homogenates were treated with or without 200 μg/ml RNase A, followed by immunoprecipitation with an anti-DISC1 antibody (m317C) and western blotting (2B3 for DISC1). (E) Cultured cortical neurons were infected with a lentivirus expression vector encoding Venus-TDP-220C (green) and endogenous DISC1 was immunostained with an anti-DISC1 antibody (HM6-5) (red). Nuclei were stained with DAPI (blue) and dendrites were stained with an anti-MAP2 antibody (blue). A representative image is shown for the whole neuron (Top) and dendrite (Bottom), respectively. 85% of neurons contained TDP-220C aggregates both in soma and dendrites, and 91% of TDP-220C aggregates were DISC1-positive (n=216). Scale bar represents 5 μm.

Figure 2

DISC1 binds to initiation factors and regulates translation initiation. (A) The polysome gradient analysis using lysates of cultured cortical neurons that were treated with or without 30 mM EDTA prior to centrifugation. A representative absorption profile of sucrose gradient at 254 nm is shown (left). The inset shows the overall view of the absorption profile. Fractions were collected and analyzed by western blotting with indicated antibodies (M49 for DISC1) (right). DISC1 showed the co-migration with polysomes both before and after the EDTA treatment. “inp” denotes input. (B) DISC1 binds to eIF4G, eIF2α and eIF1 in the 40S ribosome fraction. DISC1 was immunoprecipitated (m595C) from 40S ribosome fractions purified from wild-type mouse cerebral cortex, followed by western blotting. The right top panel shows the absorption profile of sucrose gradient at 254 nm and the collected fractions were analyzed by western blotting with an anti-RS6 antibody. (C) Ribosome-free (Free), 40S, 60S, 80S monosome and polysome fractions were obtained from N2a cell lysates by sucrose gradient centrifugation. mRNA levels of β-actin and PSD95 in each fraction were analyzed by RT-qPCR. The percentages of mRNA distribution in Free + 40S + 80S and monosome + polysome fractions are shown. (n=3, β-actin: F(3,8)=19.85, P=0.0005; PSD95: F(3,8)=18.68, P=0.0006, one-way ANOVA, *P<0.05, ***P<0.001 Bonferroni’s multiple comparison test post hoc). (D) In vitro translation using N2a cell lysates. In vitro transcribed mRNA encoding Gaussian luciferase (Gluc) driven by the m7GpppG cap structure was added to cell lysates and incubated for 5 hours followed by measurement of luciferase activity. The luciferase activity of in vitro translated Gluc is expressed as the relative ratio to control cells (n=4, F(2,9)=36.46, P<0.0001, one-way ANOVA; *P<0.05, ***P<0.001, Bonferroni’s multiple comparison test post hoc). Throughout the figures, error bars represent S.E.M.

Figure 3

Local translation in dendrites is regulated by DISC1 in neurons. (A) The isolated synaptosomal fraction was subjected to western blotting with an anti-puromycin antibody for detection of puromycin-incorporated newly synthesized proteins. The signal intensities of puromycin-labeled polypeptides were normalized to those of GAPDH and then expressed as the relative ratio of control neurons (right) (n=4, F(5,18)=28.06, P<0.0001, one-way ANOVA; ***P<0.001, Bonferroni’s multiple comparison test post hoc). (B) Neurons were immunostained with anti-puromycin (gray) and anti-MAP2 (red) antibodies. The fluorescent intensities in MAP2-labeled dendrites were measured and normalized by those in 5 mM KCl treated-control neurons (right). (n=19,18,25,21 for Scramble RNAi + 5 mM KCl, DISC1 RNAi + 5 mM KCl, Scamble RNAi + 55 mM KCl and DISC1 RNAi + 55 mM KCl respectively. F(3,79)=74.17, P<0.0001, one-way ANOVA; ***P<0.001, Bonferroni’s multiple comparison test post hoc). Scale bar represents 25 μM. (C) Total mRNA levels in the synaptosomal fraction were not affected by DISC1 knockdown. A synaptosomal fraction was isolated from neurons in which indicated lentivirus was infected. Total mRNA levels in the synaptosomal fraction were measured by a fluorometer and normalized to total RNA levels. The levels of total mRNA relative to those in control neurons are shown (n=3, F(2,6)=0.5678, P=0.5945, one-way ANOVA; Bonferroni’s multiple comparison test post hoc). (D) The protein levels in the synaptosomal fraction were examined by the knockdown of DISC1. The levels of synaptic proteins relative to those in control (Scramble RNAi) neurons are shown (right) (n=4 for CaMKIIα, n=3 for Shank3 and n=6 for rest of genes, NR1: F(2,15)=176.7, P<0.0001, PSD95: F(2,15)=5.386 P=0.0173, GluR2: F(2,15)=30.71 P<0.0001, Shank3: F(2,6)=11.22 P=0.0094, mGluR1/5: F(2,15)=7.155, P=0.0066, one-way ANOVA; *P<0.05, ***P<0.001, Bonferroni’s multiple comparison test post hoc). Throughout the figures, error bars represent S.E.M.

Figure 4

Co-aggregation of TDP43 and DISC1 impairs DISC1-mediated local translation. (A) Indicated synaptic proteins in the synaptosomal fraction of cultured cortical neurons infected with indicated lentivirus were detected by western blotting (top). The levels of synaptic proteins relative to those in control neurons are shown (bottom), (n=3 for NR2B n=4 for rest of genes, NR1: F(2,9)=15.60, P=0.0012; PSD95: F(2,9)=41.39, P<0.0001; GluR2: F(2,9)=16.17, P=0.0010; NR2B: F(2,9)=10.50, P=0.0110, one-way ANOVA; *P<0.05, **P<0.01, ***P<0.001, Bonferroni’s multiple comparison test post hoc). (B) Co-expression of DISC1 restored the decrease in neural stimulation-dependent protein synthesis by TDP-220C aggregation. Cultured cortical neurons were stimulated with 55 mM KCl, followed by the treatment with 10 μg/ml puromycin. The puromycin-labeled proteins in the isolated synaptosomal fraction were detected by western blotting with an anti-puromycin antibody. The signal intensities of puromycin-incorporated polypeptides were normalized to those of GAPDH and then expressed as the relative ratio of control neurons (right) (n=4, F(5,18)=18.99, P<0.0001, one-way ANOVA; *P<0.05, **P<0.01, Bonferroni’s multiple comparison test post hoc). Throughout the figures, error bars represent S.E.M.

Figure 5

TDP-43 aggregates sequestered endogenous DISC1 in FTLD patient brain. (A) DISC1 and TDP-43 are mislocalized and co-aggregated in the cytosol in FTLD patient brains. DISC1 and TDP-43 were immunostained using anti-DISC1 antibody (h598C) and anti-TDP43 antibodies, respectively, in control (Control, top panels) and FTLD patient brains (FTLD, bottom panels). Brain samples from three different FTLD patients were stained. In neurons containing TDP-43 aggregates, 89% of TDP-43 aggregates were DISC1-positive (n=27). Other images immunostained with another anti-DISC1 antibody (HM6-5) are shown in Supplemental Figure S8D. Scale bar represents 5 μm. (B–D) Insoluble DISC1 levels were increased while soluble DISC1 levels were decreased in FTLD patient brains. (B) 125 μg of the homogenates of temporal lobes from control (n=5) and FTLD (n=5) subjects were incubated with 2% sarkosyl and amounts of insoluble proteins were analyzed by the filter-trap assay, followed by immunoblotting with indicated antibodies (h598C for DISC1). The levels of each genes relative to those in control are also shown (below) (TDP43: t=7.833, ***P<0.0001; pSer409/410: t=12.928, ***P<0.0001; DISC1: t=4.051, **P=0.0037, unpaired two-tailed t-tes). (C) The homogenates of temporal lobe from control (n=6) and FTLD (n=5) subjects were incubated with 2% sarkosyl and then centrifuged at 100,000×g. The resulting sarkosyl-insoluble pellets and the input were analyzed by western blotting with indicated antibodies (h598C for DISC1). Arrowheads indicate the band position of each monomeric protein. The levels of each genes relative to those in control are shown (right) (TDP43: t=3.246, **P=0.0098; pSer409/410: t=3.415, **P=0.0077; DISC1: t=3.253, **P=0.0099, unpaired two-tailed t-test). (D) The homogenates of temporal lobe from control (n=6) and FTLD (n=5) subjects were incubated with 2% sarkosyl and then centrifuged at 100,000×g. The resulting sarkosyl-soluble supernatans and the input were analyzed by western blotting with an anti DISC1 antibody (h598). The signal intensities of DISC1 in supernatants were once normalized to those in the input and then expressed as the relative ratio of control brains (right). (t=5.798, ***P=0.0003, unpaired two-tailed t-test). (E) Homogenates of temporal lobes from control (n=5) and FTLD patient brains (n=5) were subjected to western blotting with indicated antibodies (left). GAPDH was used as a loading control. The signal intensities of each gene relative to those in control brains are shown (right) (NR2B: t=2.689, *P=0.0275; PSD95: t=2.404, *P=0.0429; mGlulR1: t=2.369, *P=0.0453; GluR2: t=2.964, *P=0.0181, unpaired two-tailed t-test). Throughout the figures, error bars represent S.E.M.

Figure 6

Psychiatric symptoms in TDP-220C mice are rescued by exogenous DISC1 expression. (A to D) AAV encoding indicated genes was stereotaxically injected into mouse prefrontal cortex. (A) Indicated synaptic proteins in the synaptosomal fraction were detected by western blotting (left). The levels of synaptic proteins relative to those in control neurons are shown (right) (n=4, NR2B: F(3,12)=5.231, P=0.0154; GluR2: F(3,12)=25.59, P<0.0001; PSD95: F(3,12)=4.819, P=0.0199; SV2: F(3,12)=0.9337, P=0.4545, one-way ANOVA; *P<0.05, **P<0.01, ***P<0.001, Bonferroni’s multiple comparison test post hoc). (B) In the open field test, TDP-220C mice showed hyperactivity, which was restored by the co-expression of DISC1 (left). The time spent in a center region was not altered between all mice groups (right). n=17, 17, 17, 18 for EGFP (white), TDP-220C+EGFP (black), TDP-220C+DISC1 (dark gray), DISC1+EGFP (light gray) mice, respectively (F(3,65)=6.371, P=0.0007, one-way ANOVA, *P<0.05, **P<0.01, ***P<0.001, Bonferroni’s multiple comparison test post hoc). (C) In the social interaction test, TDP-220C mice were less interested in novel stranger mice, which was rescued by the co-expression of DISC1. During the first encounter, all mice group spent more time in the area with first stranger mice (S1) compared to empty cage (E). During the second encounter, EGFP control mice spent more time with novel, unfamiliar mice (S2) than with familiar mice (F) whereas TDP-220C mice spend equal time with unfamiliar and familiar mice. This behavior was normalized by co-expression of DISC1. n=23, 23, 21, 24 for EGFP (white), TDP-220C+EGFP (black), TDP 220C+DISC1 (dark gray), DISC1+EGFP (light gray) mice, respectively (EGFP: F(5,132)=9.048, P<0.0001; TDP-220C+EGFP: F(5,132)=4.875, P=0.0004; TDP 220C+DISC1: F(5,120)=3.666, P=0.004; DISC1+EGFP: F(5,138)=19.95, P<0.0001, one-way ANOVA, *P<0.05, **P<0.01, ***P<0.001, Bonferroni’s multiple comparison test post hoc). (D) Measurement of forearm grip strength. Grip strength was comparable between all mice groups. n=, 12, 11, 12, 12 for EGFP (white), TDP-220C+EGFP (black), TDP 220C+DISC1 (dark gray), DISC1+EGFP (light gray) mice, respectively. No statistical differences were detected (F(3,39)=2.796, P=0.0528, one-way ANOVA). Throughout the figures, error bars represent S.E.M.