Altered distributions of Gemini of coiled bodies and mitochondria in motor neurons of TDP-43 transgenic mice

Keywords: ALS, RNA metabolism, frontotemporal lobar degeneration with ubiquitinated inclusions, fused in sarcoma/translocated in liposarcoma, survival motor neuron

Growth Retardation, Muscle Weakness, and Death in TDP-43 Transgenic Mice.

Several lines (W1, W2, and W3) of mice expressing wild-type human TDP-43 (hTDP-43) were generated using the Thy1.2 promoter (Fig. S1), which is capable of driving expression postnatally in neurons, including motor neurons. TDP-43 mice exhibited retardation of development when compared with nontransgenic littermates (Fig. 1A). The severities of this phenotype correlated with the copy number of the transgene: Mice from line W1, with the highest transgene copy number (Fig. S1), were markedly smaller than their nontransgenic littermates (Fig. 1A) and die within 3 wk of age, whereas mice derived from W2 and W3, two lines harboring lower numbers of transgenes (Fig. S1), exhibited growth retardation to a lesser extent (Fig. 1A), and most of those mice grew to adulthood. Interestingly, male TDP-43 mice exhibited ≈20% reduction in body weight at 4 wk of age when compared with nontransgenic male littermates (Fig. 1B). We observed that transgenic males derived from both W2 and W3 lines exhibited a more severe phenotype when compared with transgenic female littermates, an outcome that appears to be related to a higher (2- to 3-fold) accumulation of TDP-43 in transgenic males (Fig. 1 C and D). Male TDP-43 mice abruptly developed severe tremor, abnormal reflex of hindlimbs (Fig. S1), and gait abnormalities within a short time window, ranging from postnatal day 14 to day 18. Female TDP-43 mice did not show such significant reduction in body weight and developed fine tremor only after 3 mo of age. Because transgenic mice from lines W2 and W3 exhibited similar behavioral phenotypes and pathology in addition to comparable levels of transgene expression, we focused our subsequent analyses using line W3.

Abnormal Distribution of Mitochondria in Motor Neurons of TDP-43 Transgenic Mice.

Consistent with our observation of motor deficits in these lines of mice, histological analyses revealed eccentric nuclei with abnormal eosinophilic aggregates in cell bodies of motor neurons in spinal cord (Fig. 2 A and B and Fig. S2A) and brainstem. To investigate whether the transgenic product was a component of the eosinophilic aggregates, sections of spinal cord and brainstem were stained with antibodies recognizing specifically human TDP-43. Although human TDP-43 can be localized to nuclei of motor neurons, we failed to observe TDP-43 immunoreactivity associated with these cytoplasmic inclusions in mice ranging from 3 wk (Fig. 2 C–E) to 3 mo of age. Interestingly, some of the human TDP-43-immunoreactive nuclei were eccentric, suggesting the presence of abnormal cytoplasmic aggregates in these motor neurons (Fig. 2E, arrow). Moreover, human TDP-43 often was localized to two prominent intranuclear structures associated with eccentric nuclei in motor neurons (Fig. 2E, arrowheads). Increased ubiquitin immunoreactivity was present in motor neurons of spinal cord (Fig. 2 F and G) and brainstem, particularly in those neurons with eccentric nuclei, indicating the presence of cytoplasmic inclusions (Fig. 2G, arrows). However, ubiquitin immunoreactivity was more intensive in the nuclear as compared with the cytoplasmic compartment, suggesting that the cytoplasmic inclusions may not be composed of highly ubiquitinated proteins. To ascertain the composition of these cytoplasmic aggregates, we performed immunocytochemical analyses using a series of markers for various organelles. Markers of endoplasmic reticulum and Golgi were not associated with these cytoplasmic aggregates (Fig. S2B), whereas several mitochondrial markers including mitochondrial chaperonin HSP60 (Fig. 2I) and mitochondrial voltage-dependent anion channel (Fig. S3) localized to these cytoplasmic inclusions in many motor neurons expressing human TDP-43, strongly indicating that these eosinophilic aggregates are composed, in part, of accumulations of mitochondria. Ultrastructural analysis of motor neurons from 3-wk-old TDP-43 mice confirmed these observations. Mitochondria are normally evenly distributed within the cell bodies of neurons (Fig. 2J), but motor neurons from TDP-43 mice displayed cytoplasmic inclusions composed of massive accumulation of mitochondria (Fig. 2 K and L). These observations suggest that elevated levels of TDP-43 impact on the intracellular trafficking of organelles and consequently lead to abnormal distributions of mitochondria in motor neurons.

To observe directly the distributions of mitochondria in different compartments of motor neurons, TDP-43 mice were cross-bred to Thy1-mitoCFP mice, in which a subpopulation of mitochondria are fluorescently labeled with CFP in neurons (14). As expected, CFP-labeled mitochondria marked motor neurons and were visualized in processes in the ventral horns of nontransgenic mice (Fig. 3A). In contrast, mtCFP;TDP-43 compound mice showed mitochondria clustered within inclusions of motor neurons (Fig. 3B, arrows). The observations that mitochondria mainly accumulated within inclusions of cell bodies and were sparsely distributed in neuronal processes in the mtCFP;hTDP-43 compound mice suggest the possibility that trafficking of organelle, particularly mitochondria, is impaired in these nerve cells. If this is the case, nerve terminals of these mice may be deficient in mitochondria. Consistent with this speculation, we observed a marked reduction of mitochondria (as indicated by the CFP signal intensity) at nerve terminals of neuromuscular junctions (NMJ) in mtCFP;TDP-43 compound mice (Fig. 3C). In muscle sections of control mice, the normal juxtaposition of the pre- and postsynaptic terminals is reflected in the clustering of acetylcholine receptors (AChR) in a multiperforating pattern characteristic of normal NMJ (Fig. 3D Top and Fig. S4). In contrast, in the NMJ of mtCFP;TDP-43 mice, AChRs form a plaque-like pattern (Fig. 3D Bottom and Fig. S4) similar to that described in a mouse model of spinal muscular atrophy (SMA), a motor neuron disease of infancy and childhood (15). Such abnormalities are associated with abnormal synaptic transmission in these SMA mice (15). These observations suggest the possibility that synaptic transmission is altered at the NMJ of TDP-43 mice. This interpretation is consistent with the weakness and reduction in size of muscle fibers observed in the TDP-43 mice (Fig. 3E). Quantitative analysis revealed ≈20% reduction in cross-sectional area of muscle fibers in TDP-43 mice as compared with that of nontransgenic littermate controls (Fig. 3E), indicating that impaired transmission at the NMJ may underlie weakness observed in these TDP-43 mice.

TDP-43 Regulates SMN-Associated GEMs in Motor Neurons.

To determine how elevated levels of TDP-43 in the nucleus lead to motor neuron dysfunction, we asked whether aspects of TDP-43-related nuclear functions are altered in motor neurons of TDP-43 mice. Immunocytochemical analysis of TDP-43 in motor neurons with cytoplasmic inclusions revealed a striking abnormal localization of TDP-43 in the nuclear compartment that is usually associated with two conspicuous intranuclear aggregates (Fig. 2E and Fig. S5). To begin to identify the protein components of these TDP-43-containing nuclear inclusions, we costained spinal cord sections of TDP-43 mice using antisera directed against a variety of nuclear markers along with a human TDP-43-specific antibody. Although we did not observe the colocalization of TDP-43 with ubiquitin in these nuclear inclusions (Fig. 4A, arrowheads), we discovered that TDP-43-immunoreactive nuclear aggregates contained FUS/TLS, an RNA-binding protein (Fig. 4B, arrowheads, and Fig. S6) recently linked to cases of ALS (9, 10). In addition, the central cores of these TDP-43 nuclear inclusions associate with SC35 (Fig. 4C, arrowheads), a marker of non-snRNP (small nuclear ribonucleoprotein) splicing speckles (16). These results suggest that increased levels of TDP-43 induce its association with FUS/TLS and SC35, proteins involved in RNA metabolism.

To examine whether elevated levels of TDP-43 impact on pathways that are involved in RNA splicing, we assessed the distributions of the SMN complex in relation to GEMs and to Cajal bodies, two nuclear structures containing high concentrations of the SMN protein (17, 18), which is linked to SMA (19–21). The SMN complex is part of a large multimeric protein assembly essential for biogenesis of snRNPs required for pre-messenger RNA splicing (22). Although previous studies of TDP-43 and SMN showed colocalization of these two proteins in the nucleus of transient transfected cells (23), we failed to detect colocalization of TDP-43 with SMN-associated GEMs in motor neurons of TDP-43 mice. Rather, our immunocytochemical analysis using SMN antibody showed a significant increase in the number of GEM bodies in motor neurons of TDP-43 mice (Fig. 4D, arrows). The nontransgenic motor neurons usually harbor two GEMs. In the transgene-expressing motor neurons, the number of GEMs varies widely from one to eight. The number is significantly high in neurons with eccentric nuclei and cytoplasmic inclusions (Fig. 4D). However, such neurons account for 5–30% of total neurons in cervical or lumbar spinal cord. We observe on average three or four GEMs in TDP-43-expressing motor neurons (Fig. 4F). Because GEM bodies dynamically shuttle between the nucleolus and the nucleoplasm (24, 25), we examined the distributions of GEMs in motor neurons of TDP-43 mice. Double immunofluorescence staining of GEMs (using SMN antisera) and the nucleoli (using antisera specific for fibrillarin) disclosed that whereas GEMs are normally distributed with one or two discretely associated with the nucleolus in neurons of nontransgenic mice, SMN is present diffusely within the entire nucleolus and SMN-containing GEMs are confined to the perinucleolar region of motor neurons in TDP-43 mice (Fig. 4E). Similar results were observed using sections derived from spinal cords of three transgenic mice and three littermate controls. The integrity of GEMs was confirmed by the identification of these same nuclear structures with other essential components of GEMs, including Gemin 2 and Gemin 8.

To further examine the role of TDP-43 in motor neurons, we used a complementary loss-of-function approach to delete the gene encoding TDP-43. Because TDP-43 is required for embryogenesis (26, 27), we generated and characterized conditional knockout mice with floxed Tardbp alleles (28). Immunofluorescence analysis revealed, in addition to localization of SMN in the cytoplasm, two prominent SMN-containing GEMs as expected in each spinal motor neuron of control mice (Fig. 4G Upper). However, we failed to find such SMN-containing GEMs in motor neurons of conditional Tardbp knockout mice, although SMN can be localized in the cytoplasm (Fig. 4G Lower). To confirm that GEMs failed to form in the nucleus of neurons lacking TDP-43, we immunostained spinal cord sections using antisera against other components of GEMs. Whereas both Gemin 2 and Gemin 8 can be localized to GEMs in motor neurons of wild-type mice (Fig. 4 H and I Upper), no GEMs were identified using these markers in conditional Tardbp knockout mice (Fig. 4 H and I Lower), indicating that TDP-43 is required for the formation of GEMs in the nucleus of motor neurons. Taken together, results from both loss- and gain-of-function studies of TDP-43 converge to support the idea that TDP-43 is critical for the generation of SMN-containing GEMs, and that alteration of TDP-43 could impact on pathways that control RNA splicing.

Identification of Potential Downstream Targets of TDP-43 in Spinal Cord.

Our findings implicating a role of TDP-43 in the regulation of RNA metabolism led us to hypothesize that alteration in RNA transcription and splicing may occur in our TDP-43 mice. We therefore asked whether perturbations in RNA metabolism are observed in spinal cords of TDP-43 mice using a transcriptome-wide differential RNA expression (RNA-Seq) approach (29, 30). cDNA libraries generated from mRNA extracted from spinal cords of three 3-wk-old TDP-43 mice (line W3) and three nontransgenic littermates were sequenced. We identified 313 genes with changes in splicing pattern [P value < E-10; Gene Expression Omnibus (GEO) database, accession no. GSE22351]; 2,017 genes were differentially expressed (P value < E-10; GEO accession no. GSE22351). Some of these genes, such as glial fibrillar acidic protein (GFAP), are expressed exclusively in nonneuronal cells. This is not a surprise, because increased astrogliosis was observed in 3-wk-old TDP-43 mice (Fig. S7). Because the transgene is driven by the neuron-specific Thy1.2 promoter, we believe that the primary pathogenic events occur within neurons of TDP-43 mice. Interestingly, the top 30 affected genes (expressed in neurons) showed perturbations in both differential expression and alternative splicing (see list in Tables S1 and S2, respectively), and many of these genes are involved in the regulation of cellular architecture. For example, neurofilament mRNAs are decreased significantly in TDP-43 mice. Given the importance of neurofilament proteins in determining axonal caliber and its roles in ALS (31), we determined whether down-regulation of members of this gene family had functional consequences in TDP-43 mice. Consistent with the reduction of levels of these mRNAs, the protein levels of NF-M and NF-L were significantly decreased in TDP-43 mice (Fig. 5 A and B). Importantly, analysis of the ventral roots from the lumbar region of spinal cords revealed a decrease in the number of large-caliber axons in TDP-43 transgenic mice, an observation consistent with the reduced levels of NF proteins (Fig. 5 D and E). Taken together, these results provide additional support for the roles of TDP-43 in regulation of RNA splicing and RNA transcription of a subset of genes, including those members of the neurofilament family.

Generation of Human TDP-43 Transgenic Mice.

The complete human TDP-43 cDNA was subcloned into a mouse Thy1.2 expression cassette and subsequently injected into C57BL/6;SJL hybrid mouse embryos by the Transgenic Facility at The Johns Hopkins University School of Medicine. Integration of the transgene into the mouse genome was determined by Southern blot analysis. A 200-bp human TARDBP DNA probe, which shares 92% homology to mouse Tardbp gene, was used in the Southern blot to identify both the endogenous gene and transgene. The signal intensities were measured with Bio-Rad Quantity One and compared between transgene and endogenous Tardbp to assess the copy number of the transgene. Three founders were identified and bred with C57BL/6 mice. The copy number was assessed on F1 mice of each founder. The animal use protocol was approved by the Animal Care and Use Committee of the Johns Hopkins Medical Institutions.

SDS/PAGE and Immunoblotting.

Mouse tissues were homogenized in RIPA buffer with 1% SDS, resolved by 4–12% bis-Tris gel (Invitrogen), and transferred onto PVDF membrane (Millipore). Immunoblotting was performed using the following antibodies: mouse-anti-TDP-43 mAb (clone 2E2-D3; Novus Biologicals), rabbit anti-TDP-43 pAb (Proteintech Group), mouse-anti-actin (Sigma), mouse-anti-neurofilament light chain mAb (clone NR4; Sigma), and mouse-anti-neurofilament 160 kDa (clone NN18; Millipore).

Immunohistochemistry.

Mice under anesthetization by an i.p. injection of 15% chloral hydrate were perfused transcardially with PBS (pH 7.4) and subsequently by 4% paraformaldehyde in phosphate buffer (pH 7.4). Organs were removed, postfixed in the same fixative overnight, and embedded in paraffin blocks. Sagittal sections of brains and cross-sections of spinal cords (10 μm) were used for immunohistochemical analysis. The following antibodies were used: mouse-anti-TDP-43 mAb (clone 2E2-D3) and rabbit-anti-ubiquitin (Dako). The immunoreactivity was visualized by a Vectastain Elite ABC Kit (Vector Laboratories) and diaminobenzidine.

Immunofluorescence.

Tissues fixed by 4% paraformaldehyde in phosphate buffer (pH 7.4) were cryoprotected by 30% sucrose in PBS (pH 7.4). Frozen sections (10 μm) of spinal cord tissues from three or more nontransgenic and transgenic mice, respectively, were processed for double immunofluorescence using the following antibodies: mouse-anti-TDP-43 mAb (clone 2E2-D3), rabbit-anti-TDP-43 pAbs (Proteintech Group; Abcam), rabbit-anti-FUS pAb (Proteintech Group), rabbit-anti-fibrillarin pAb, mouse-anti-germin2, mouse-anti-SC35 mAbs (Abcam), rabbit-anti-HSP60 pAb (Cell Signaling), mouse-anti-SMN, germin8 mAbs (Sigma). Alexa Fluor 488 and 594 (Invitrogen) were used as secondary antibodies to visualize proteins. Sections were mounted and visualized by an Olympus IX71 fluorescence microscope or Zeiss 510 Meta confocal microscope.

Electron Microscopy.

Anesthetized mice were perfused with 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) and postfixed with 4% paraformaldehyde with 2% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4) overnight. Tissues were then washed in PBS, dehydrated, and embedded in Epon. Thick (1 μm) and thin (100 nm) sections were stained, respectively, with toluidine blue and lead citrate/uranyl acetate. The images were obtained using a Hitachi 7600 transmission electron microscope.

Visualization of Mitochondria in Spinal Motor Neurons and Nerve Terminals.

Thy1-hTDP-43 transgenic mice were mated with Thy1-mtCFP transgenic mice (The Jackson Laboratory, no. 006617). Double transgenic mice were identified by tail biopsy genotyping of hTDP-43 and CFP. mtCFP mice were used as the control for double transgenic mice. Anesthetized mice were transcardially perfused with 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). Spinal cord and tibialis anterior muscle were isolated, postfixed in the same fixative for 4 h, and cryoprotected in 30% sucrose in PBS (pH 7.4) overnight. The spinal cord tissues were cut into cross-sections at 10-μm thickness and muscle tissues were cut longitudinally at 50 μm. Mouse-anti-TDP-43 mAb was applied to identify the transgene-expressing neurons in spinal cord of hTDP-43 transgenic mice. Alexa Fluor 594 was used as the secondary antibody. The muscle sections were incubated with Alexa Fluor 594-conjugated α-bungrotoxin (Invitrogen) at 1:1,000 dilution and subsequently washed with PBS three times in 1 h. Sections were mounted and visualized by Zeiss 510 Meta confocal microscope.

Histology.

Muscle tissues were dissected and flash-frozen in freezing isopentane. Cryosections were cut at 10-μm thickness and stained with hematoxylin and eosin. Myofiber area was measured using ImageJ software (W.S. Rasband, National Institutes of Health, Bethesda, MD; http://rsb.info.nih.gov/ij). Quantification of axonal caliber in the ventral roots of lumbar spinal cord sections, which were stained with toluidine blue, was performed using ImageJ software. The frequency distribution of axonal calibers was plotted (nontransgenic, n = 3 mice, 3,981 axons; transgenic, n = 3 mice, 1,988 axons).

Statistical Analysis.

Biochemical and morphological data were analyzed by Excel and GraphPad Prism using unpaired Student's t tests. The quantitative data in this study were expressed as the mean ± SEM.

Supporting Information

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Supplementary Materials