3′UTR Length-Dependent Control of SynGAP Isoform α2 mRNA by FUS and ELAV-like Proteins Promotes Dendritic Spine Maturation and Cognitive Function

FUS is an RNA-binding protein associated with frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS). Previous reports have demonstrated intrinsic roles of FUS in synaptic function. However, the mechanism underlying FUS’s regulation of synaptic morphology has remained unclear. We found that reduced mature spines after FUS depletion were associated with the internalization of PSD-95 within the dendritic shaft. Mass spectrometry of PSD-95-interacting proteins identified SynGAP, whose expression decreased after FUS depletion. Moreover, FUS and the ELAV-like proteins ELAVL4 and ELAVL1 control SynGAP mRNA stability in a 3′UTR length-dependent manner, resulting in the stable expression of the alternatively spliced SynGAP isoform α2. Finally, abnormal spine maturation and FTLD-like behavioral deficits in FUS-knockout mice were ameliorated by SynGAP α2. Our findings establish an important link between FUS and ELAVL proteins for mRNA stability control and indicate that this mechanism is crucial for the maintenance of synaptic morphology and cognitive function

RNP2 of RNA Recognition Motif 1 Plays a Central Role in the Aberrant Modification of TDP-43

<div><p>Phosphorylated and truncated TAR DNA-binding protein-43 (TDP-43) is a major component of ubiquitinated cytoplasmic inclusions in neuronal and glial cells of two TDP-43 proteinopathies, amyotrophic lateral sclerosis and frontotemporal lobar degeneration. Modifications of TDP-43 are thus considered to play an important role in the pathogenesis of TDP-43 proteinopathies. However, both the initial cause of these abnormal modifications and the TDP-43 region responsible for its aggregation remain uncertain. Here we report that the 32 kDa C-terminal fragment of TDP-43, which lacks the RNP2 motif of RNA binding motif 1 (RRM1), formed aggregates in cultured cells, and that similar phenotypes were obtained when the RNP2 motif was either deleted from or mutated in full-length TDP-43. These aggregations were ubiquitinated, phosphorylated and truncated, and sequestered the 25 kDa C-terminal TDP-43 fragment seen in the neurons of TDP-43 proteinopathy patients. In addition, incubation with RNase decreased the solubility of TDP-43 in cell lysates. These findings suggest that the RNP2 motif of RRM1 plays a substantial role in pathological TDP-43 modifications and that it is possible that disruption of RNA binding may underlie the process of TDP-43 aggregation.</p></div

Ubiquitination of TDP-43 CTFs.

<p>(A) Immunocytochemistry of NSC34 cells expressing Wt, dNLS, TDP35, or TDP32. Cells were stained with anti-ubiquitin antibody (green), anti-V5 antibody (red), and DAPI (blue). Scale bar = 5 µm. (B) Percentage of cells with ubiquitin-positive aggregates. Error bars indicate SEM (n = 3). The percentage of TDP32-expressing cells containing ubiquitin-positive aggregates was significantly higher than that of cells expressing TDP35 (<i>p</i><0.05). (C) Immunoprecipitations with anti-ubiquitin antibody. The immunoreactivity of V5 was only detected in the TDP32 lane (arrow head). Asterisk indicates non-specific signal. (D) Immunoprecipitations with anti-V5 antibody. The ubiquitin-positive smear band was increased in the lane of TDP32 compared with that of TDP35. Asterisks indicate a heavy or light chain of IgG.</p

Disruption of the RNP2 motif leads to ubiquitin-positive aggregates of TDP-43.

<p>(A) Structures of Wt, ΔRNP2, and mtRNP2. (B) Immunocytochemistry of NSC34 cells expressing Wt, ΔRNP2, or mtRNP2. Cells were stained with anti-ubiquitin antibody (green), anti-V5 antibody (red), and DAPI (blue). Scale bar = 5 µm. (C) Percentage of cells with ubiquitin-positive aggregates. Error bars indicate SEM (n = 3). The percentage of mtRNP2 and ΔRNP2-expressing cells containing ubiquitin-positive aggregates was significantly higher than that of Wt-expressing cells (<i>p</i><0.01 and <i>p</i><0.001, respectively). (D) Immunoprecipitations with anti-ubiquitin antibody. The V5-positive smear band was evident in the mtRNP2 lane. (E) Immunoprecipitations with anti-V5 antibody. The ubiquitin-positive smear band was increased in the mtRNP2 lane compared with that of the Wt.</p

Phosphorylation and insolubilization of TDP32.

<p>(A) Immunocytochemistry of NSC34 cells expressing Wt, dNLS, TDP35, or TDP32. Cells were stained with anti-pTDP-43 antibody (green), anti-V5 antibody (red), and DAPI (blue). Scale bar = 5 µm. (B) Percentage of cells with pTDP-43-positive aggregates. Error bars indicate SEM (n = 3). The percentage of TDP32-expressing cells containing pTDP-43-positive aggregates was significantly higher than that of TDP35 (<i>p</i><0.01). (C and D) Immunoblots of RIPA-soluble and -insoluble fractions from HEK293 cells expressing TDP35 and TDP32. The amount of insoluble TDP32 was higher than that of TDP35 (C). TDP32 in the RIPA-insoluble fraction was detected with anti-pTDP-43 antibody (D).</p

Altered tau isoform ratio caused by loss of FUS and SFPQ function leads to FTLD-like phenotypes

Fused in sarcoma (FUS) and splicing factor, prolineandglutamine-rich (SFPQ) are RNA binding proteinsthat regulate RNA metabolism. We found that alternativesplicing of the Mapt gene at exon 10, whichgenerates 4-repeat tau (4R-T) and 3-repeat tau(3R-T), is regulated by interactions between FUSand SFPQ in the nuclei of neurons. HippocampusspecificFUS- or SFPQ-knockdown mice exhibitfrontotemporal lobar degeneration (FTLD)-like behaviors,reduced adult neurogenesis, accumulationof phosphorylated tau, and hippocampal atrophywith neuronal loss through an increased 4R-T/3R-Tratio. Normalization of this increased ratio by 4R-Tspecificsilencing results in recovery of the normalphenotype. These findings suggest a biological linkamong FUS/SFPQ, tau isoform alteration, andphenotypic expression, which may function in theearly pathomechanism of FTLD

Phosphorylation and insolubilization of RNP2-disrupted TDP-43.

<p>(A) Immunocytochemistry of NSC34 cells expressing ΔRNP2 or mtRNP2. Cells were stained with anti-pTDP-43 antibody (green), anti-V5 antibody (red), and DAPI (blue). Scale bar = 5 µm. (B) Percentage of cells with pTDP-43-positive aggregates. Error bars indicate SEM (n = 3). The percentage of mtRNP2 and ΔRNP2-expressing cells containing pTDP-43-positive aggregates was significantly higher than that of Wt-expressing cells (<i>p</i><0.01 and <i>p</i><0.01, respectively). (C and D) Immunoblots of RIPA-soluble and -insoluble fractions from HEK293 cells expressing Wt or mtRNP2. The amount of insoluble mtRNP2 was higher than that of Wt (C). mtRNP2 in the RIPA-insoluble fraction was detected with anti-pTDP-43 antibody (D).</p

Intracellular localizations of TDP-43 lacking the NLS and of CTFs of TDP-43.

<p>(A) Structures of wild-type (Wt), NLS-disrupted mutant (dNLS) and CTFs (TDP35, TDP32 and TDP25). (B) Immunoblots of the cytosol and nuclear fractions from HEK293 cells expressing Wt, dNLS, TDP35, TDP32, or TDP25.</p