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The Pathological Phenotypes of Human TDP-43 Transgenic Mouse Models Are Independent of Downregulation of Mouse Tdp-43

  • Ya-Fei Xu,

    Affiliation Department of Neuroscience, Mayo Clinic Jacksonville, Jacksonville, Florida, United States of America

  • Mercedes Prudencio,

    Affiliation Department of Neuroscience, Mayo Clinic Jacksonville, Jacksonville, Florida, United States of America

  • Jaime M. Hubbard,

    Affiliation Department of Neuroscience, Mayo Clinic Jacksonville, Jacksonville, Florida, United States of America

  • Jimei Tong,

    Affiliation Department of Neuroscience, Mayo Clinic Jacksonville, Jacksonville, Florida, United States of America

  • Ena C. Whitelaw,

    Affiliation Department of Neuroscience, Mayo Clinic Jacksonville, Jacksonville, Florida, United States of America

  • Karen Jansen-West,

    Affiliation Department of Neuroscience, Mayo Clinic Jacksonville, Jacksonville, Florida, United States of America

  • Caroline Stetler,

    Affiliation Department of Neuroscience, Mayo Clinic Jacksonville, Jacksonville, Florida, United States of America

  • Xiangkun Cao,

    Affiliation Department of Neuroscience, Mayo Clinic Jacksonville, Jacksonville, Florida, United States of America

  • John Song,

    Affiliation Department of Neuroscience, Mayo Clinic Jacksonville, Jacksonville, Florida, United States of America

  • Yong-Jie Zhang

    Zhang.Yongjie@mayo.edu

    Affiliation Department of Neuroscience, Mayo Clinic Jacksonville, Jacksonville, Florida, United States of America

The Pathological Phenotypes of Human TDP-43 Transgenic Mouse Models Are Independent of Downregulation of Mouse Tdp-43

  • Ya-Fei Xu, 
  • Mercedes Prudencio, 
  • Jaime M. Hubbard, 
  • Jimei Tong, 
  • Ena C. Whitelaw, 
  • Karen Jansen-West, 
  • Caroline Stetler, 
  • Xiangkun Cao, 
  • John Song, 
  • Yong-Jie Zhang
PLOS
x

Abstract

Tar DNA binding protein 43 (TDP-43) is the major component of pathological deposits in frontotemporal lobar degeneration with TDP-43 inclusions (FTLD-TDP) and in amyotrophic lateral sclerosis (ALS). It has been reported that TDP-43 transgenic mouse models expressing human TDP-43 wild-type or ALS-associated mutations recapitulate certain ALS and FTLD pathological phenotypes. Of note, expression of human TDP-43 (hTDP-43) reduces the levels of mouse Tdp-43 (mTdp-43). However, it remained unclear whether the mechanisms through which TDP-43 induces ALS or FTLD-like pathologies resulted from a reduction in mTdp-43, an increase in hTDP-43, or a combination of both. In elucidating the role of mTdp-43 and hTDP-43 in hTDP-43 transgenic mice, we observed that reduction of mTdp-43 in non-transgenic mice by intraventricular brain injection of AAV1-shTardbp leads to a dramatic increase in the levels of splicing variants of mouse sortilin 1 and translin. However, the levels of these two abnormal splicing variants are not increased in hTDP-43 transgenic mice despite significant downregulation of mTdp-43 in these mice. Moreover, further downregulation of mTdp-43 in hTDP-43 hemizygous mice, which are asymptomatic, to the levels equivalent to that of mTdp-43 in hTDP-43 homozygous mice does not induce the pathological phenotypes observed in the homozygous mice. Lastly, the number of dendritic spines and the RNA levels of TDP-43 RNA targets critical for synapse formation and function are significantly decreased in symptomatic homozygous mice. Together, our findings indicate that mTdp-43 downregulation does not lead to a loss of function mechanism or account for the pathological phenotypes observed in hTDP-43 homozygous mice because hTDP-43 compensates for the reduction, and associated functions of mTdp-43. Rather, expression of hTDP-43 beyond a certain threshold leads to abnormal metabolism of TDP-43 RNA targets critical for neuronal structure and function, which might be responsible for the ALS or FTLD-like pathologies observed in homozygous hTDP-43 transgenic mice.

Introduction

Tar DNA-binding protein 43 (TDP-43) is the principal component of ubiquitinated inclusions in frontotemporal lobar degeneration with TDP-43-positive inclusions (FTLD-TDP) and amyotrophic lateral sclerosis (ALS) [1], [2]. Mutations in TARDBP, the gene encoding TDP-43, are associated with sporadic and familial ALS, indicating that TDP-43 can directly cause neurodegeneration [3], [4], [5], [6], [7]. Normally, TDP-43 localizes to the nucleus; however, a substantial loss of nuclear TDP-43 is observed in neurons bearing aberrant cytoplasmic TDP-43 inclusions in disease [1], [2]. The mechanisms through which TDP-43 proteins mediate neurodegeneration remains unclear, though it is believed to be attributable to either a toxic gain of function, a loss of function, or a combination of both.

Previous studies have explored the TDP-43 loss of function hypothesis in vitro and in vivo [8], [9], [10], [11]. Wu and colleagues disrupted the expression of mouse Tdp-43 (mTdp-43) in vivo and observed that mice developed early embryonic lethality, suggesting an important role for TDP-43 in development [8]. Conditional downregulation of mTdp-43 in mouse spinal cord led to the development of ALS-like phenotypes supporting the hypothesis that loss of TDP-43 function is a major cause of neurodegeneration in ALS [9]. Several other studies demonstrated the potential contribution of TDP-43 deficiency to disease pathogenesis [10], [12], while our group and others have demonstrated that overexpression of the human TDP-43 (hTDP-43) protein, either wild-type (hTDP-43WT) or mutant hTDP-43, leads to pathological phenotypes consistent with certain TDP-43 proteinopathies. These phenotypes may include some of the following: increased ubiquitination, truncation, aggregation and phosphorylation of TDP-43, cytoplasmic TDP-43 inclusions, neuronal degeneration, motor dysfunction, learning and memory deficits, and mitochondrial abnormalities [13], [14], [15], [16], [17], [18], [19]. Moreover, we [14], [15] and others [12] have observed that expression of hTDP-43 protein in transgenic mice decreases the mRNA levels of endogenous mouse Tardbp. Additionally, recent studies demonstrate that TDP-43 autoregulates itself to decrease its expression [20], suggesting that hTDP-43 may thus decrease mouse Tardbp levels through this mechanism. However, it remains unclear whether the reduction in mTdp-43 (loss of function) or the overexpression of hTDP-43 (gain of function), or combined events are responsible for the ALS or FTLD-like pathologies observed in hTDP-43 transgenic mice.

To address the aforementioned question, we first generated an antibody that specifically detects mTdp-43 protein, and confirmed that downregulation of mouse Tardbp mRNA observed in our hTDP-43 transgenic mice results in significant reduction of mTdp-43 protein. We found that this reduction in mTdp-43 does not increase the levels of splicing variants known to be inhibited by TDP-43. In particular, the levels of mouse Ex17b-containing sortilin 1 (mSort1+Ex17b) and Ex5-deleted translin (mTsn_ΔEx5) mRNAs are significantly upregulated when mTdp-43 protein is reduced in non-transgenic (NT) mice but not in hTDP-43 mice, suggesting that hTDP-43 has similar biological functions to that of mTdp-43 in vivo. Moreover, a further reduction of mTdp-43 in asymptomatic hemizygous hTDP-43 (hTDP-43hemi) mice to the levels equivalent to that of symptomatic homozygous hTDP-43 (hTDP-43homo) mice did not lead to the development of pathological phenotypes indicating that downregulation of mTdp-43 is not sufficient to induce the pathological phenotypes observed in hTDP-43homo mice. Finally, we provide evidence that high expression levels of hTDP-43 beyond a certain threshold lead to alterations in TDP-43 RNA substrates critical for maintaining neuronal structure and function and this, in turn, might cause neuronal dysfunction and the development of pathological phenotypes.

Results

The Protein Levels of Mouse Tdp-43 are Reduced in Human TDP-43 Transgenic Mice

Recently, we and others have reported that the mRNA levels of mTdp-43 are significantly decreased in hTDP-43 transgenic mice [12], [14], [15]; however, it had remained unclear whether levels of mTdp-43 protein are similarly decreased in hTDP-43 transgenic mice. As such, we generated a novel antibody that specifically recognizes the mTdp-43 protein, since both mTdp-43 and hTDP-43 proteins are indistinguishable in size by Western blot techniques and there are no commercially available TDP-43 antibodies that recognize the mouse but not the human TDP-43 protein. In doing so, we immunized rabbits with a synthetic peptide corresponding to residues 203–221 of mTdp-43 protein (Fig. 1A). The resulting sera were then affinity purified and the specificity of the resulting mTdp-43 antibody was characterized by Western blot. We observed that our mTdp-43 antibody recognizes mTdp-43 protein from mouse brain lysates of non-transgenic (NT) and hTDP-43 mice, but not the hTDP-43 protein present in human neuroblastoma M17 cells (Fig. 1A). Moreover, incubation of the peptide used for the generation of this antibody with mouse brain lysates blocked the detection of mTdp-43 protein but did not interfere with the ability of human specific TDP-43 antibodies to detect hTDP-43 protein in hTDP-43 mice (Fig. 1A), further verifying the specificity of the antibody. By using this new antibody, we examined the protein levels of mTdp-43 in NT, hTDP-43hemi and hTDP-43homo transgenic mice. The levels of mTdp-43 protein in hTDP-43hemi and hTDP-43homo mice respectively decreased to ∼55% and 45% of NT mice levels (Fig. 1B and C), suggesting that hTDP-43 overexpression downregulates the levels of mTdp-43, but that the downregulation is modest once hTDP-43 expression levels exceed a certain threshold. We also observed downregulation of mouse Tardbp mRNA and mTdp-43 protein levels in both hemizyogous and homozygous hTDP-43M337V mice (data not shown). Of importance, TDP-43M337V and TDP-43WT display similar biological activity in autoregulating the levels of TDP-43 in vivo, suggesting the ALS-associated M337V mutation does not lead to loss of function.

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Figure 1. The protein levels of mouse Tdp-43 are reduced in human TDP-43 transgenic mice.

(A) Generation and characterization of an antibody that specifically detects mouse Tdp-43 (mTdp-43 antibody). Schematic representation of mouse TDP-43 protein and the amino acid residues used as immunizing peptide. Western blot analyses of lysates from mouse brain of non-transgenic (NT) and human TDP-43 transgenic (Tg) mouse, and human M17 neuroblastoma cells. Before proceeding with the staining protocol, mTdp-43 antibody is neutralized with immunizing peptide. The neutralized antibody is then used side-by-side as a control for the antibody alone. Our results showed that our mTdp-43 antibody can specifically recognize mTdp-43 in both NT and hTDP-43 Tg mice brain, but cannot recognize human TDP-43 in human sample. Human TDP-43 antibody (hTDP-43) was used to confirm the expression of human TDP-43 in samples. (B) Compared to NT mice, significant reduction of mTdp-43 protein was observed in both TDP-43 hemizygous and homozygous mice brain. Data shown are the means ± SEM of 5 mice per group; *p<0.05, **p<0.001, as assessed by one-way ANOVA with Tukey’s posthoc analysis.

https://doi.org/10.1371/journal.pone.0069864.g001

Expression of hTDP-43 Rescues the Abnormal Splicing Variants Induced by mTdp-43 Downregulation in Human TDP-43 Transgenic Mice

One of the well characterized functions of TDP-43 is its ability to regulate the splicing of its RNA targets, and loss of mTdp-43 has been shown to result in the generation of abnormal splicing variants of some of these targets in cells and in vivo [20], [21]. Since the levels of mTdp-43 protein are significantly decreased in our hTDP-43 transgenic mice (Fig. 1), we sought to determine whether there is loss of mTdp-43 function in these mice by evaluating the ability of mTdp-43 to regulate splicing of two of its RNA targets: sortilin 1 (Sort1) [20], [21] and translin (Tsn) [20]. Consistent with previous reports [20], [21], downregulation of mTdp-43 in mouse N2a neuroblastoma cells by small inference RNA (siRNA) significantly increased the levels of the abnormal splicing variants: mSort1+Ex17b (sortilin 1 including exon 17b) and mTsn_ΔEx5 (translin with exon 5 deletion) (siTardbp, Fig. 2A). To further quantify this data, we designed primers for quantitative real time PCR (qRT-PCR) to specifically measure the levels of mouse Tardbp, mSort1+Ex17b and mTsn_ΔEx5. Compared to cells treated with a control siRNA (siCtrl), the levels of mSort1+Ex17b and mTsn_ΔEx5 increased 3.7- and 5.2-fold, respectively, when mTdp-43 levels are reduced (siTardbp, Fig. 2B–D). Then, by using those primers, we measured the levels of mSort1+Ex17b and mTsn_ΔEx5 in hTDP-43 transgenic mice. Surprisingly, while mTdp-43 protein is significantly decreased in hTDP-43 transgenic mice (Fig. 1B–C), the levels of mSort1+Ex17b and mTsn_ΔEx5 splicing variants were not increased compared to NT mice (Fig. 2E and F). Similar results regarding the levels of mSort1+Ex17b and mTsn_ΔEx5 were observed in hTDP-43M337V transgenic mice (Fig. S1D, E).

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Figure 2. Expression of hTDP-43 rescues the abnormal splicing variants induced by mTdp-43 downregulation in human TDP-43 transgenic mice.

(A–D) Knockdown of mTdp-43 induces abnormal splicing of TDP-43 RNA targets mouse sortilin 1 (Sort1) and translin (Tsn) in N2a cells. (A) Knockdown of mTdp-43 in N2a cell line using small inference RNA (siRNA) leads to generation of abnormal splicing variants mSort1+Ex17b and mTsn_ΔEx5, indicating loss of mTdp-43 function. (B–D) Quantitative PCR (q-PCR) is used to quantify the mRNA levels of mTdp-43, mSort1+Ex17b and mTsn_ΔEx5. The levels of mSort1+Ex17b and mTsn are significantly increased when Tdp-43 is depleted. (E–F) The mRNA levels of mSort1+Ex17b (E) and mTsn_ΔEx5 (F) are not increased in hTDP-43 hemizygous and homozygous mice brain. In contrast, the levels of these two abnormal splicing variants are reduced dramatically in hTDP-43 transgenic mice. Data shown are the means ± SEM of 5 mice per group; **p<0.001, as assessed by one-way ANOVA with Tukey’s posthoc analysis.

https://doi.org/10.1371/journal.pone.0069864.g002

To determine whether downregulation of mTdp-43 in vivo can increase the levels of mSort1+Ex17b and mTsn_ΔEx5 splicing variants, we performed intraventricular brain injections of adeno-associated virus type 1 (AAV1)-shRNA targeting mTdp-43 in neonatal mice expressing only mTdp-43 (NT mice) or mTdp-43 and hTDP-43 (hTDP-43hemi mice). Note that efficient downregulation of mTdp-43 protein and RNA was achieved in both NT and hTDP-43hemi transgenic mice (Fig. 3A–C). Co-expression of EGFP with shRNA confirmed the transduction efficiency is comparable among treated groups (Fig. 3A). Moreover, downregulation of mTdp-43 in NT mice dramatically increased the levels of mSort1+Ex17b and mTsn_ΔEx5 (Fig. 3D, E). However, compared to hTDP-43hemi mice transduced with a control shRNA (shCtrl), the levels of mSort1+Ex17b and mTsn_ΔEx5 were not increased after further reduction of mTdp-43 in shTardbp-treated hTDP-43hemi mice. Similar results were observed in hTDP-43M337V mice (Fig. S1). These data suggest that human TDP-43 protein expressed in these mice is inhibiting the formation of the mSort1+Ex17b and mTsn_ΔEx5 mouse splicing isoforms.

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Figure 3. Intraventricular injection of AAV1-shTardbp in neonatal mouse brain results in loss of TDP-43 function in non-transgenic mice, but not human TDP-43 hemizygouse transgenic mice.

(A–C) Compared to AAV1-shCtrl, intraventricular injection of AAV1-shTardbp reduces the levels of mTdp-43 protein (A and B) and mRNA (C) in brains of both non-transgenic (NT) and hTDP-43WT hemizygous mice. Of note, co-expression of EGFP with shRNA confirmed the transduction efficiency is comparable among treated groups (A). Moreover, the mTdp-43 protein and RNA levels between AAV1-shTardbp injected hemizygous mice and homozygous mice are not significantly different. (D–E) Injection of AAV1-shTardbp significantly increases the mRNA level of mSort1+Ex17b (D) and mTsn_ΔEx5 (E) in the brain of NT mice, but not in hTDP-43WT hemizygous mice. There is no significant difference of mSort1+Ex17b and mTsn_ΔEx5 RNA levels between AAV1-shTardbp injected hemizygous mice and homozygous mice. Data shown are the means ± SEM of 3–5 mice per group; **p<0.001, N.S. no significance, as assessed by One-way ANOVA with Tukey’s posthoc analysis.

https://doi.org/10.1371/journal.pone.0069864.g003

A Reduction in the Levels of Mouse Tdp-43 does not Account for the Pathological Phenotypes Observed in Human TDP-43 Homozygous Transgenic Mice

We have previously reported that hTDP-43homo mice develop certain ALS or FTLD-like pathologies; however, hTDP-43hemi mice are phenotypically indistinguishable from NT mice [14], [15]. Compared to hTDP-43hemi, hTDP-43homo mice have lower mTdp-43 protein levels, but higher hTDP-43 protein levels (Fig. 1). To investigate whether the lower levels of mTdp-43 in hTDP-43homo mice are responsible for the phenotypic differences between hTDP-43homo and hTDP-43hemi mice, we treated hTDP-43hemi mice with AAV-shTardbp and compared their phenotype to that of hTDP-43homo mice (see Fig. 3A–C). Since hTDP-43homo mice developed pathological phenotypes at 1 month of age [14], [15], we harvested hTDP-43hemi mice 1 month after administration of AAV-shTardbp. Compared to shCtrl-treated hTDP-43hemi mice, administration of AAV1-shTardbp significantly decreased the mRNA and protein levels of mTdp-43 to the levels equivalent to that of hTDP-43homo mice (Fig. 3A–C). At 1 month old, hTDP-43homo mice develop ALS and FTLD-like pathologies such as cytoplasmic eosinophilic aggregates (Fig. 4A), phosphorylated TDP-43 inclusions (Fig. 4B), and abnormal mitochondrial aggregation (Fig. 4C). These pathological features were absent in both AAV1-shTardbp injected hTDP-43hemi mice (Fig. 4D–F) and NT mice (data not shown). In addition, shTardbp-treated hTDP-43hemi mice did not develop any motor deficits or early mortality that is characteristic of hTDP-43homo mice (data not shown). Overall our data suggest that a decrease in mTdp-43 protein in hTDP-43 transgenic mice is not responsible for the observed phenotype in these mice. Rather, it does seem that the human TDP-43 may be driving these events.

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Figure 4. Further downregulation of mTdp-43 in the brain of hTDP-43 hemizygous mice do not induce pathologies observed in hTDP-43 homozygous mice.

(A–B) Hematoxylin and eosin staining (H&E) shows that eosinophilic aggregates in neurons from the brain sections of 1 month old hTDP-43 homozygous mice (A) are not observed in AAV1-shTardbp injected hTDP-43 hemizygous mice (B). (C–D) Phosphorylated TDP-43 aggregates are only observed in the neurons of the brain sections of 1 month old hTDP-43 homozygous mice (C), but not in AAV1-shTardbp injected hTDP-43 hemizygous mice (D). (E–F) COX-IV immunoreactivity illustrates densely stained mitochondria aggregates in neurons from the brain sections of 1 month old hTDP-43 homozygous mice (E), but not in AAV1-shTardbp injected hTDP-43 hemizygous mice (F). Scale bars: 100 µm.

https://doi.org/10.1371/journal.pone.0069864.g004

High Levels of Human TDP-43 Decrease the RNA Levels of Synaptic Proteins and Reduce the Number of Dendritic Spines in Vivo

Given our data indicate that hTDP-43 compensates for loss of mTdp-43 in hTDP-43homo mice to a certain point, beyond which the overexpression of hTDP-43 induces toxicity, we sought to investigate whether the highly abundant hTDP-43 protein present in these mice would alter the levels of certain TDP-43 mRNA targets and consequently account for the TDP-43 associated pathologies in hTDP-43homo mice. To this end, we measured the levels of several neuronal TDP-43 RNA targets that are critical for neuronal survival and function [22]. We measured the following RNA targets: bassoon (Bsn), discs large homolog 1 (Dlg1), discs large homolog 2 (Dlg2), discs large homolog 4 (Dlg4, also called Psd-95), segment polarity protein dishevelled homolog (DVL-1), growth associated protein 43 (Gap-43), synaptosomal-associated protein 25 (Snap-25), synaptotagmin 1 (Syt1), and synaptotagmin 7 (Syt7). Among these, the mRNA levels of Psd-95, Bsn, Gap-43, and Syt7 were found to be dramatically reduced in hTDP-43homo mice compared to NT mice and hTDP-43hemi mice (Fig. 5A). Since the proteins encoded by these TDP-43 RNA targets are known to play important roles in synaptic function and formation of dendritic spines [23], [24], we evaluated the number of dendritic spines in hTDP-43homo compared to NT mice. We observed that the number of dendritic spines is significantly reduced in hTDP-43homo mice (Fig. 5B, C). Of note, the RNA levels of these synaptic proteins and the number of dendritic spines are not reduced in AAV1-shTardbp injected hTDP-43hemi mice (Fig. S2A–C), indicating that expression of hTDP-43 over in excess of a certain level, rather than downregulation of mTdp-43, causes abnormal metabolism of TDP-43 mRNA targets in hTDP-43 transgenic mouse models.

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Figure 5. Alternation of RNA levels of synaptic proteins and dendritic spines in hTDP-43WT homozygous mice.

(A) Compared with non-transgenic (NT) mice, the RNA levels of TDP-43 targets PSD-95, Bsn, GAP-43 and Syt7 are significantly decreased in TDP-43 homozygous mice, but not in TDP-43 hemizygous mice. (B–C) The numbers of dendritic spine are dramatically reduced in the brain of TDP-43 hemozygous mice compared to NT mice. Data shown are the means ± SEM of 3–5 mice per group; **p<0.001, as assessed by one-way ANOVA with Tukey’s posthoc analysis or Two-tailed student’s t test to compare measures among 2 groups.

https://doi.org/10.1371/journal.pone.0069864.g005

Discussion

Generation of transgenic mice is a widely used research technique to model human diseases, given transgenic mice recapitulate key pathological features of disease and also serve as valuable tools to explore the role of disease-causing molecules (e.g. TDP-43). More importantly, the models provide opportunities to evaluate potential therapeutic strategies for translational research. For instance, mutations in genes encoding amyloid precursor protein (APP), presenilins (PSEN1 and PSEN2) or microtubule associated protein tau (MAPT) have been overexpressed in animals to make Alzheimer’s disease (AD) mouse models, which are characterized by the production and accumulation of amyloid-β into plaques or of hyperphosphorylated tau into neurofibrillary tangles [25], [26]. Moreover, mutations in the SOD1 gene have also been used to generate transgenic mouse models, which develop the progressive loss of motor neurons, muscle weakness and atrophy, and eventual death similar to the pathological phenotypes in ALS [27].

In 2006, TDP-43 was identified as the principal protein component of pathological inclusions in FTLD-TDP and ALS [1], [2]. Mutations in the TARDBP gene have also been found in sporadic and familiar ALS patients, further supporting that alterations of TDP-43 directly cause ALS [3], [4], [5], [6], [7]. Since then, several transgenic mouse models that either over-express human wild-type TDP-43 or ALS-associated TDP-43 mutations have been generated and characterized [12], [13], [14], [15], [16], [17]. Briefly, these mouse models share very similar pathological phenotypes such as ubiquitin accumulation, TDP-43 fragmentation, astrogliosis, microgliosis, axonal degeneration, neuronal loss, motor function impairment, and shortened lifespan [12], [13], [14], [15], [16], [17], [18], [19]. In some of those models, phosphorylated TDP-43 inclusions are rarely observed in the brain and spinal cord [12], [14], [15], [28].

Given that TDP-43 plays critical roles in RNA metabolism, such as splicing, transport and transcription [29], excessive levels of hTDP-43 in transgenic models might result in the disruption of RNA metabolism. Interestingly, several studies revealed that TDP-43 protein binds to the 3′-UTR of its mRNA, and auto-regulates its levels through combined mechanisms [20], [30], [31]. We, as well as others, have observed that the mRNA levels of mTdp-43 are significantly decreased in hTDP-43 transgenic mice [12], [14], [15], [20]. Furthermore, it has been shown that downregulation of mTdp-43 alters splicing events of multiple mRNAs [20], and that conditional downregulation of mTdp-43 in vivo leads to ALS-like phenotypes including kyphosis, motor dysfunction, muscle atrophy, and astrocytosis [9]. These data suggest that the development of disease in hTDP-43 transgenic mice may result from loss of mTdp-43 function; however, whether the overexpression of high levels of hTDP-43 in mice accounts for any of the disease-associated phenotypes remained unknown.

In the present study, we used our hTDP-43 transgenic mouse models to determine whether the disease phenotypes that develop in these mice result from high hTDP-43 protein levels or by the downregulation of endogenous mTdp-43 protein. To help us address this question, we generated an antibody that specifically detects mTdp-43 to measure protein levels in vivo. By using this antibody, we observed that mTdp-43 protein is significantly decreased in hTDP-43 mice compared to NT mice, suggesting that hTDP-43 may be regulating mTdp-43 expression through downregulation of mouse Tardbp RNA. This idea is consistent with previous findings that elucidated TDP-43′s autoregulation ability [20], [30], [31].

To further determine whether the lower levels of mTdp-43 protein in our hTDP-43 transgenic mice result in loss of mTdp-43 function, we examined the levels of abnormal splicing variants of two of its RNA targets: Sort1, which encodes multi-ligand type 1 transmembrane receptor [32], and Tsn, encoding a recombinant hotspot DNA-binding protein [33]. When mTdp-43 levels are decreased in NT mice, mRNA levels of mSort1 containing exon 17b (99 base pairs of intron 17, mSort1+Ex17b) and mTsn lacking exon 5 (mTsn_ΔEx5) increase dramatically. These two opposite splicing patterns upon mTdp-43 downregulation (exon inclusion in mSort1 and exon exclusion in mTsn) make our assay more comprehensive. Of note, the levels of mSort1+Ex17b and mTsn_ΔEx5 are not increased in hTDP-43 transgenic mice, despite the fact that these mice present lower levels of mTdp-43 protein than in AAV1-shTardbp injected NT mice, which do show a significant increase in these aberrant splicing isoforms. Moreover, further reduction of mTdp-43 in hTDP-43hemi mice to the levels equivalent to that of hTDP-43homo mice do not lead to an increase in mSort1+Ex17b nor mTsn_ΔEx5 isoforms. As a result, our data indicate that loss of mTdp-43 function in hTDP-43 mice is compensated by the expression of hTDP-43. These results are not surprising since mouse and human TDP-43 proteins are 96% homologous [28]. We have reported that homozygous transgenic mice of both hTDP-43WT and hTDP-43M337V develop phenotypes characteristic of FTLD and ALS diseases. However, hTDP-43hemi transgenic mice are virtually indistinguishable from NT controls [14], [15]. Compared to the hTDP-43homo mice hTDP-43hemi mice have ∼75% hTDP-43 protein or ∼25% less hTDP-43 protein. As such, we hypothesize that expression of hTDP-43 over a certain threshold will disrupt the processing of TDP-43 RNA targets that will consequently cause the neuropathologies observed in hTDP-43homo mice. While we were unable to detect differences in TDP-43 RNA targets known to be altered by mTdp-43 downregulation, we found significant changes in TDP-43 RNA targets that are critical for neuronal function and survival in hTDP-43 mice overexpressing high levels of hTDP-43. In particular, the mRNA levels of synaptic markers Psd-95, Bsn, Gap-43, and Syt7 are significantly decreased in hTDP-43homo mice, but not in hTDP-43hemi mice. Moreover, the number of dendritic spines in the hippocampus of hTDP-43homo mice is also significantly decreased compared to NT. More important, the mRNA levels of the above mentioned targets and the number of dendritic spines are not altered in AAV1-shTardbp treated hTDP-43hemi mice, excluding the possibility that low levels of mTdp-43 are responsible for the synaptic dysfunction in hTDP-43homo mice. Together, our results suggest that expression of total levels of TDP-43 protein (human and mouse) above 2.5 fold of endogenous mouse Tdp-43 in NT mice might disrupt RNA metabolism of several synaptic targets [14], [15], [17], [19], [34], consequently leading to neuronal dysfunction in mice.

In conclusion, although expression of hTDP-43 reduces the levels of mTdp-43 in hTDP-43 transgenic mice, this reduction does not lead to loss of mTdp-43 function because hTDP-43 compensates for functional losses in mTdp-43. Moreover, expression of hTDP-43 above a certain threshold disrupts RNA metabolism, especially of genes critical for neuronal survival and function, which consequently causes the pathological phenotypes observed in hTDP-43homo mice. In addition, there is no difference between wild-type and M337V hTDP-43 proteins regarding their ability to regulate RNA splicing. Our findings suggest that the current hTDP-43 transgenic mouse models might not be the most suitable tools to investigate loss of mTdp-43 function. Knock-in transgenic mouse models expressing either wild-type hTDP-43 or ALS-associated TDP-43 mutations at similar levels to mTdp-43 should be generated to understand the roles of TDP-43 in disease pathogenesis.

Materials and Methods

TDP-43 Transgenic Mice

The hTDP-43 transgenic mice expressing wild-type or M337V mutant hTDP-43 have been previously reported [14], [15]. The intraventricular injection of virus in mouse brain was performed according to a previous report [35]. Briefly, 2 µl of AAV1-shTardbp or AAV1-shControl virus were injected into the lateral ventricles of postnatal day 0 mice. EGFP is co-expressed with ShRNA to monitor transduction efficiency. One month after injection, the mice were harvested for RT-PCR, Western blot and immunochemistry analyses. This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Mayo Clinic Institutional Animal Care and Use Committee (Protocol number A34210). Mice were terminally anesthetized with sodium pentobarbital, and all efforts were made to minimize suffering.

Generation of Mouse Specific Tdp-43 Antibody

The polyclonal mTdp-43 antibody is produced by using synthetic peptide corresponding to residues 203–221 of mTDP-43 (TAEELQQFFCQYGEVVDVF) to immunize rabbits. The resulting sera were affinity purified and the specificity of the antibody was characterized using Western blot.

Quantitative Real-time PCR (qRT-PCR)

TRIzol (Invitrogen, Carlsbad, CA) and Pure Link™ RNA Mini Kit (Invitrogen, Carlsbad, CA) were used for RNA extraction. A total of 3 µg of RNA were used to synthesize cDNA using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems). The qRT- PCR was performed using an HT7900 analyzer (Applied Biosystems). Each 5 µl reaction contained: 2 µl of cDNA diluted 1∶20, 0.5 µl primer mix (1 µM for each primer), and 2.5 µl SYBR green (Invitrogen). The qRT-PCR program was as follows: 50°C 2min, 95°C 2min, 40 cycles of 95°C 15s and 60°C 1min. For dissociation curves, a dissociation stage of 95°C 15s, 60°C 15s was added at the end of the program. Tardbp, mSort1+Ex17b and mTsn_ΔEx5 mRNA values were normalized to Gapdh values, an endogenous transcript control. The data was analyzed by using Software RQ Manager 1.2 (Applied Biosystems). The primers used were: Gapdh: 5′-CATGGCCTTCCGTGTTCCTA-3′ and 5′-CCTGCTTCACCACCTTCTTGAT-3′; Tardbp: 5′-GGGGCAATCTGGTATATGTTG-3′ and 5′-TGGACTGCTCTTTTCACTTTCA-3′; mSort1+Ex17b: 5′-AAATCCCAGGAGACAAATGC-3′ and 5′-GAGCTGGATTCTGGGACAAG-3′; mTsn_ΔEx5∶5′GGTCTTCCTGGCAGCATTTG-3′ and 5′-TTGACAGACAGCCTCGATGC-3′. To quantify the RNA levels of TDP-43 synaptic targets in human TDP-43 transgenic mice, we also performed q-PCR. There primers used were: Psd-95∶5′-CGCTACCAAGATGAAGACACG-3′ and 5′-CAATCACAGGGGGAGAATTG-3′; Bsn: 5′-TGTGGCTTTAACCCAACACC-3′ and 5′-TTTGGCAGTTCAGACAGAGC-3′; Dlg1∶5′-TCATTCTCATATCTCACCAATAAAGC-3′ and 5′-CAGGGGTCACAGGGACAA-3′; Dlg2∶5′-TCAACTCCCTACCCCCACTA-3′ and 5′-GCAGTACTGTGCTGGGAATG-3′; Dvl1∶5′-CCATGGACCAGGACTTCG-3′ and 5′-GGCAACTTGGCATTGTCAT-3′; Gap-43∶5′-AGAGGATGCTGCCACCAA-3′ and 5′-GGCTTCGTCTACAGCGTCTTT-3′; Syt7∶5′-GAGGCTTGGACATGAAATCC-3′ and 5′-TCCGAAAGCCCTAATACCAG-3′; Snap25∶5′-GCTCCTCCACTCTTGCTACC-3′ and 5′-CAGCAAGTCAGTGGTGCTTC-3′; Syt1∶5′-AAGGAGATTCCAAAAGGAACAA-3′ and 5′-TTTTGGTTCAAGCGGAATG-3′.

For semi-quantitative PCR, 10 µl of 1∶40 diluted cDNA was used for PCR amplification in a final volume of 20 µl using Taq polymerase (Qiagen). PCR cycling conditions were as follows: 94°C, 3min; 94°C, 30s; 60°C, 30s; 72°C, 30s (10 cycles); 94°C, 30s; 50°C, 30s; 72°C, 30s (20 cycles); 72°C, 10min. PCR products were electrophoresed on 2% agarose gels. The primers used were: Sort1∶5′-CAAATGCCAAGGTGGGATGAA-3′ and 5′-TTGAATCCAAAGCCTCTACGCC-3′; Tsn: 5′-CCCGAGAGGCTGTTACAGAG-3′ and 5′-CCTCGGATGGAAAGGTCATA-3′.

Western Blotting

Tissues were homogenized at 10 ml/g (volume/weight) in lysis buffer (50 mM Tris-HCl, pH 7.4, 300 mM NaCl, 1% Triton X-100, 5 mM EDTA, 2% SDS, PMSF, and protease and phosphatase inhibitors). Following centrifugation, protein concentration of the supernatant was assessed by BCA assay (Pierce, Rockford, IL). Following Western blotting, membranes were incubated with rabbit polyclonal mouse specific Tdp-43 antibody that we have generated(1∶1000), mouse monoclonal human specific TDP-43 antibody (1∶1000, 2E2-D3, Novus Biologicals), rabbit polyclonal GFP antibody (1∶1000, Invitrogen), and mouse monoclonal Gapdh antibody (1∶10000, Biodesign) overnight at 4°C. Following incubation with an appropriate secondary antibody, immunoreactivity was visualized by ECL and exposure to film.

Immunohistochemistry

Tissues were embedded in paraffin, sectioned (5 µm thick) and mounted on glass slides. Sections were deparaffinized in xylene and rehydrated in a graded series of alcohol, followed by dH2O. Antigen retrieval was performed in a dH2O steam bath for 30 min. Tissues were immunostained with cytochrome oxidase subunit IV (Cox-IV; 1∶3000; Abcam) and pS403/S404-phosphorylated TDP-43 (1∶2000; Cosmo Bio) antibodies, using the DAKO Autostainer (Dako Auto Machine Corporation) and the DAKO EnVision+ HRP system. DAKO Liquid DAB Substrate–Chromogen system was the chromogen. After immunostaining, sections were briefly counterstained with hematoxylin to visualize cell nuclei and coverslipped. Paraffin-embedded sections were also stained with hematoxylin and eosin.

DiI-labeling

Mice were deeply anesthetized with a mixture of ketamine (100 mg/kg, i.p.) and xylazine (10 mg/kg, i.p.) and perfused with 4% paraformaldehyde (PFA). Brains were removed and put into 4% PFA for additional 20 minutes and then washed overnight in PBS at 4°C. Vibratome sections of 200 µM were prepared and immersed in PBS at 4°C. The Helios Gene Gun System (165–2431; Bio-Rad, Hercules, CA) was used to deliver gold (1.0 µm) and tungsten particles coated with lipophilic dyes into perfusion-fixed brain slices. Preparations of the DiOlistic bullets were performed according the methods described previously [36], [37]. A series of images with interval of 0.5 µm in the Z direction was taken with confocal microscope with 63×oil-immersion lens and image stacks were then generated by Image J software. The stacked images were then used to count dendritic spines by using Metamorph software. Dendritic spines of hippocampus were counted by scrolling through the z plane about 10 µm length of each segment. The number of spines in each segment was counted and the data was expressed at dendritic spines/10 µm.

AAV1 production

AAV1-shTardbp and AAV1-shControl was prepared by standard methods. Briefly, AAV vectors expressing shTardbp and shControl under the control of the cytomegalovirus enhancer/chicken β-actin promoter, a woodchuck post-transcriptional regulatory element, and the bovine growth hormone, poly (A), were generated by plasmid transfection with helper plasmids in HEK293T cells. All plasmids were sequence verified as described above. Forty-eight hours after transfection, the cells were harvested and lysed in the presence of 0.5% sodium deoxycholate and 50 U/ml Benzonase (Sigma, St. Louis, MO) by freeze thawing, and the virus was isolated using a discontinuous iodixanol gradient. The genomic titer of each virus was determined by qRT-PCR.

Statistics

One-way ANOVA with Tukey’s posthoc analysis were used to compare measures among 3 groups. Two-tailed student’s t test was used to compare measures among 2 groups. For data presentation, normalized values were averaged and presented as mean ± standard error of mean (SEM). Values of p<0.05 were considered statistically significant.

Supporting Information

Figure S1.

Intraventricular injection of the neonatal mouse brain with AAV1-shTardbp can further decrease mTdp-43 levels in the brain of hTDP-43M337V transgenic mice, but cannot cause loss of mTdp-43 function. (A–B) Intraventricular injection of AAV1-shTardbp decreases mTdp-43 protein levels (A–B) and RNA levels (C) in the brain of both non-transgenic (NT) and hTDP-43M337V hemizygous mice. There are no significant difference of the protein RNA levels of mTdp-43 between AAV1-shTDP-43 injected hemizygous mice and homozygous mice. (D–E) Injection of AAV1-shTardbp significantly increases the mRNA levels of mSort1+Ex17b (D) and mTsn_ ΔEx5 in the brain of NT mice, but not in hTDP-43M337V hemizygous mice. mSort1+Ex17b and mTsn_ΔEx5 RNA levels between AAV1-shTardbp injected hemizygous mice and homozygous mice are not significantly different. Data shown are the means ± SEM of 3–5 mice per group; **p<0.001, N.S. no significance, as assessed by one-way ANOVA with Tukey’s posthoc analysis.

https://doi.org/10.1371/journal.pone.0069864.s001

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Figure S2.

No alternations of RNA of synaptic proteins and dendritic spines in hTDP-43WT hemizygous mice injected with AAV1-shTardbp. (A) Intraventricular injection of AAV1-shTardbp in the brain of hTDP-43WT hemizygous mice does not alter the RNA levels of PSD-95, Bsn, GAP-43 and Syt7. (B–C) Intraventricular injection of AAV1-shTardbp in hTDP-43WT hemizygous mice does not decrease dendritic spine number in the brain. Data shown are the means ± SEM of 3–5 mice per group; **p<0.001, as assessed by one-way ANOVA with Tukey’s posthoc analysis.

https://doi.org/10.1371/journal.pone.0069864.s002

(TIFF)

Acknowledgments

We thank Dr. Leonard Petrucelli for valuable discussions and suggestions during manuscript preparation. We also acknowledge expert technical assistance of Monica Castanedes-Casey, Linda Rousseau, and Virginia Phillips for histology.

Author Contributions

Conceived and designed the experiments: YX YZ. Performed the experiments: YX YZ JMH JT ECW JS KJW XC. Analyzed the data: YX MP YZ. Wrote the paper: YX MP CS YZ.

References

  1. 1. Arai T, Hasegawa M, Akiyama H, Ikeda K, Nonaka T, et al. (2006) TDP-43 is a component of ubiquitin-positive tau-negative inclusions in frontotemporal lobar degeneration and amyotrophic lateral sclerosis. Biochem Biophys Res Commun 351: 602–611.
  2. 2. Neumann M, Sampathu DM, Kwong LK, Truax AC, Micsenyi MC, et al. (2006) Ubiquitinated TDP-43 in frontotemporal lobar degeneration and amyotrophic lateral sclerosis. Science 314: 130–133.
  3. 3. Kabashi E, Valdmanis PN, Dion P, Spiegelman D, McConkey BJ, et al. (2008) TARDBP mutations in individuals with sporadic and familial amyotrophic lateral sclerosis. Nat Genet 40: 572–574.
  4. 4. Sreedharan J, Blair IP, Tripathi VB, Hu X, Vance C, et al. (2008) TDP-43 mutations in familial and sporadic amyotrophic lateral sclerosis. Science 319: 1668–1672.
  5. 5. Rutherford NJ, Zhang YJ, Baker M, Gass JM, Finch NA, et al. (2008) Novel mutations in TARDBP (TDP-43) in patients with familial amyotrophic lateral sclerosis. PLoS Genet 4: e1000193.
  6. 6. Yokoseki A, Shiga A, Tan CF, Tagawa A, Kaneko H, et al. (2008) TDP-43 mutation in familial amyotrophic lateral sclerosis. Ann Neurol 63: 538–542.
  7. 7. Gitcho MA, Baloh RH, Chakraverty S, Mayo K, Norton JB, et al. (2008) TDP-43 A315T mutation in familial motor neuron disease. Ann Neurol 63: 535–538.
  8. 8. Wu LS, Cheng WC, Hou SC, Yan YT, Jiang ST, et al. (2010) TDP-43, a neuro-pathosignature factor, is essential for early mouse embryogenesis. Genesis 48: 56–62.
  9. 9. Wu LS, Cheng WC, Shen CK (2012) Targeted depletion of TDP-43 expression in the spinal cord motor neurons leads to the development of amyotrophic lateral sclerosis-like phenotypes in mice. J Biol Chem 287: 27335–27344.
  10. 10. Chiang PM, Ling J, Jeong YH, Price DL, Aja SM, et al. (2010) Deletion of TDP-43 down-regulates Tbc1d1, a gene linked to obesity, and alters body fat metabolism. Proc Natl Acad Sci U S A 107: 16320–16324.
  11. 11. Fiesel FC, Voigt A, Weber SS, Van den Haute C, Waldenmaier A, et al. (2010) Knockdown of transactive response DNA-binding protein (TDP-43) downregulates histone deacetylase 6. EMBO J 29: 209–221.
  12. 12. Igaz LM, Kwong LK, Lee EB, Chen-Plotkin A, Swanson E, et al. (2011) Dysregulation of the ALS-associated gene TDP-43 leads to neuronal death and degeneration in mice. J Clin Invest 121: 726–738.
  13. 13. Wils H, Kleinberger G, Janssens J, Pereson S, Joris G, et al. (2010) TDP-43 transgenic mice develop spastic paralysis and neuronal inclusions characteristic of ALS and frontotemporal lobar degeneration. Proc Natl Acad Sci U S A 107: 3858–3863.
  14. 14. Xu YF, Gendron TF, Zhang YJ, Lin WL, D’Alton S, et al. (2010) Wild-type human TDP-43 expression causes TDP-43 phosphorylation, mitochondrial aggregation, motor deficits, and early mortality in transgenic mice. J Neurosci 30: 10851–10859.
  15. 15. Xu YF, Zhang YJ, Lin WL, Cao X, Stetler C, et al. (2011) Expression of mutant TDP-43 induces neuronal dysfunction in transgenic mice. Mol Neurodegener 6: 73.
  16. 16. Tsai KJ, Yang CH, Fang YH, Cho KH, Chien WL, et al. (2010) Elevated expression of TDP-43 in the forebrain of mice is sufficient to cause neurological and pathological phenotypes mimicking FTLD-U. J Exp Med 207: 1661–1673.
  17. 17. Wegorzewska I, Bell S, Cairns NJ, Miller TM, Baloh RH (2009) TDP-43 mutant transgenic mice develop features of ALS and frontotemporal lobar degeneration. Proc Natl Acad Sci U S A 106: 18809–18814.
  18. 18. Cannon A, Yang B, Knight J, Farnham IM, Zhang Y, et al. (2012) Neuronal sensitivity to TDP-43 overexpression is dependent on timing of induction. Acta Neuropathol 123: 807–823.
  19. 19. Shan X, Chiang PM, Price DL, Wong PC (2010) Altered distributions of Gemini of coiled bodies and mitochondria in motor neurons of TDP-43 transgenic mice. Proc Natl Acad Sci U S A 107: 16325–16330.
  20. 20. Polymenidou M, Lagier-Tourenne C, Hutt KR, Huelga SC, Moran J, et al.. (2011) Long pre-mRNA depletion and RNA missplicing contribute to neuronal vulnerability from loss of TDP-43. Nat Neurosci.
  21. 21. Prudencio M, Jansen-West KR, Lee WC, Gendron TF, Zhang YJ, et al. (2012) Misregulation of human sortilin splicing leads to the generation of a nonfunctional progranulin receptor. Proc Natl Acad Sci U S A 109: 21510–21515.
  22. 22. Sephton CF, Cenik C, Kucukural A, Dammer EB, Cenik B, et al. (2011) Identification of neuronal RNA targets of TDP-43-containing ribonucleoprotein complexes. J Biol Chem 286: 1204–1215.
  23. 23. El-Husseini AE, Schnell E, Chetkovich DM, Nicoll RA, Bredt DS (2000) PSD-95 involvement in maturation of excitatory synapses. Science 290: 1364–1368.
  24. 24. Nikonenko I, Boda B, Steen S, Knott G, Welker E, et al. (2008) PSD-95 promotes synaptogenesis and multiinnervated spine formation through nitric oxide signaling. J Cell Biol 183: 1115–1127.
  25. 25. Gotz J, Ittner LM (2008) Animal models of Alzheimer’s disease and frontotemporal dementia. Nat Rev Neurosci 9: 532–544.
  26. 26. Lithner CU, Hedberg MM, Nordberg A (2011) Transgenic mice as a model for Alzheimer’s disease. Curr Alzheimer Res 8: 818–831.
  27. 27. Peviani M, Caron I, Pizzasegola C, Gensano F, Tortarolo M, et al. (2010) Unraveling the complexity of amyotrophic lateral sclerosis: recent advances from the transgenic mutant SOD1 mice. CNS Neurol Disord Drug Targets 9: 491–503.
  28. 28. Tsao W, Jeong YH, Lin S, Ling J, Price DL, et al. (2012) Rodent models of TDP-43: recent advances. Brain Res 1462: 26–39.
  29. 29. Buratti E, Baralle FE (2010) The multiple roles of TDP-43 in pre-mRNA processing and gene expression regulation. RNA Biol 7: 420–429.
  30. 30. Avendano-Vazquez SE, Dhir A, Bembich S, Buratti E, Proudfoot N, et al. (2012) Autoregulation of TDP-43 mRNA levels involves interplay between transcription, splicing, and alternative polyA site selection. Genes Dev 26: 1679–1684.
  31. 31. Ayala YM, De Conti L, Avendano-Vazquez SE, Dhir A, Romano M, et al. (2011) TDP-43 regulates its mRNA levels through a negative feedback loop. EMBO J 30: 277–288.
  32. 32. Nykjaer A, Willnow TE (2012) Sortilin: a receptor to regulate neuronal viability and function. Trends Neurosci.
  33. 33. Aoki K, Suzuki K, Sugano T, Tasaka T, Nakahara K, et al. (1995) A novel gene, Translin, encodes a recombination hotspot binding protein associated with chromosomal translocations. Nat Genet 10: 167–174.
  34. 34. Swarup V, Phaneuf D, Bareil C, Robertson J, Rouleau GA, et al. (2011) Pathological hallmarks of amyotrophic lateral sclerosis/frontotemporal lobar degeneration in transgenic mice produced with TDP-43 genomic fragments. Brain 134: 2610–2626.
  35. 35. Passini MA, Watson DJ, Vite CH, Landsburg DJ, Feigenbaum AL, et al. (2003) Intraventricular brain injection of adeno-associated virus type 1 (AAV1) in neonatal mice results in complementary patterns of neuronal transduction to AAV2 and total long-term correction of storage lesions in the brains of beta-glucuronidase-deficient mice. J Virol 77: 7034–7040.
  36. 36. Benediktsson AM, Schachtele SJ, Green SH, Dailey ME (2005) Ballistic labeling and dynamic imaging of astrocytes in organotypic hippocampal slice cultures. J Neurosci Methods 141: 41–53.
  37. 37. Gan WB, Grutzendler J, Wong WT, Wong RO, Lichtman JW (2000) Multicolor “DiOlistic” labeling of the nervous system using lipophilic dye combinations. Neuron 27: 219–225.