Background
Frontotemporal lobar degeneration (FTLD) is a devastating age-dependent neurodegenerative condition primarily associated with impairments in cognition and social behaviors, as well as personality changes and other clinical abnormalities [
1]. Frontotemporal dementia (FTD), a major clinical syndrome of FTLD, is now recognized as the most common form of early-onset age-dependent dementia before the age of 60 [
2]. Increasing clinical, pathological, and molecular evidence indicates that FTD and amyotrophic lateral sclerosis (ALS) are closely related conditions [
3].
In addition to pathogenic mutations in the microtubule binding protein tau [
4,
5], rare mutations in other genes also cause FTD, such as those encoding valosin-containing protein, an AAA-type ATPase associated primarily with the endoplasmic reticulum [
6], and CHMP2B, a component of the endosomal sorting complex required for transport III [
7]. Mutations in progranulin located on chromosome 17 also cause FTD in some patients without tau pathology [
8,
9]. Progranulin is a secreted molecule, and many pathogenic mutations lead to progranulin haploinsufficiency. The pathogenic mechanism of FTD caused by progranulin deficiency is not known, but one of the pathological hallmarks is tau-negative and ubiquitin-positive neuronal inclusions that contain TDP-43 and its fragments [
10‐
13]. Genetic mutations in
TDP-43 are responsible for some sporadic and familial amyotrophic lateral sclerosis (ALS) [
14‐
16], further reinforcing the notion that FTD and ALS are closely related conditions, referred to as TDP-43 proteinopathies [
17]. In healthy cells, TDP-43 mostly resides in the nucleus. In diseased neurons, however, TDP-43 is excluded from the nucleus and aggregates in the cytosol [
10]. Moreover, in axotomized motor neurons, TDP-43 expression is dramatically increased and becomes prominently localized in the cytosol [
18]. These findings raise the possibility that loss of the normal function of TDP-43, especially in the nucleus, contributes to the initiation or progression of disease.
TDP-43 is a ubiquitously expressed RNA-binding protein that contains two RNA recognition motifs, a glycine-rich region, a nuclear localization signal, and a nuclear export signal [
19]. It is not known which aspects of cellular physiology are regulated by TDP-43. TDP-43 is primarily localized in the nucleus at the active sites of transcription and cotranscriptional splicing in mammalian neurons [
20]. Indeed, limited experimental evidence indicates that TDP-43 is involved in transcription [
21] and splicing [
22,
23] and possibly in mRNA transport and local translation as well [
24]. TDP-43 and many other proteins form a large complex with Drosha [
25], but its possible involvement in the microRNA pathway remains to be further explored. To understand how TDP-43 contributes to the pathogenesis of FTD and ALS, it is essential to dissect its normal functions in postmitotic neurons.
TDP-43 is highly conserved at the amino acid level from flies to humans, suggesting an evolutionarily conserved function [
19,
23,
26]. To investigate the normal roles of TDP-43 in postmitotic neurons, we used dendrites of sensory neurons in the
Drosophila peripheral nervous system (PNS) as our assay system and performed both gain- and loss-of-function genetic studies. We also examined the functional significance of some genetic mutations in TDP-43 that are associated with ALS. Our findings support the notion that a TDP-43 loss-of-function mechanism may contribute to the pathogenesis of FTD and ALS.
Methods
Fly strains and genetics
All flies were raised on standard food medium and kept at 25°C.
dTDP-43 RNAi lines 38377 and 38379 were obtained from the Vienna
Drosophila RNAi Center (VDRC). The
dTDP-43Q 367Xmutant allele was identified from the Seattle
Drosophila TILLING Project (Fly-TILL, Fred Hutchinson Cancer Research Center) using specific tilling primers (Additional file
1).
dTDP-43Q 367X/
CyO, GFP flies were crossed with
Pin/CyO, GFP; Gal4221,
UAS-mCD8-GFP to establish the stock
dTDP-43Q 367X/
CyO, GFP; Gal4221,
UAS-mCD8-GFP/+. The
Gal4221 driver was used to label ddaE and ddaF neurons with mCD8-GFP and drive the expression of transgenes [
27]. To visualize dendritic phenotypes of ddaE and ddaF neurons in third instar larvae, we crossed
dTDP-43Q 367X/
CyO, GFP; Gal4221,
UAS-mCD8-GFP/+ flies with
dTDP-43Q 367X/
CyO,
GFP or
w1118 flies to generate
dTDP-43Q 367X/
dTDP-43Q 367X; Gal4221,
UAS-mCD8-GFP/+ or
dTDP-43Q 367X/+;
Gal4221, UAS-
mCD8-GFP/+ third instar larvae. For RNAi expression,
dTDP-43Q 367X/
CyO, GFP; Gal4221,
UAS-mCD8-GFP/+ flies were crossed with UAS-
dTDP-43 RNAi lines (VDRC 38377 and 38379) to generate
dTDP-43Q 367X/
+; Gal4221, UAS-
mCD8-GFP/38377, and dTDP-43Q 367X/
38379; Gal4221, UAS-
mCD8-GFP/+ third instar larvae for phenotypic analysis. For transgene overexpression,
Gal4221,
UAS-mCD8-GFP flies were crossed with
UAS-dTDP-43,, UAS-hTDP-43, UAS-hTDP-43-M337V, or
UAS-hTDP-43-Q331K transgenic lines. In the above experiments,
Gal4221,
UAS-mCD8-GFP/+ third larvae served as the control.
Generation of transgenic Drosophila lines
Full-length
hTDP-43 cDNA was cloned from HEK293 cells (provided by Dr. J.-A. Lee). To generate
UAS-hTDP-43,
UAS-hTDP-43-M337V, UAS-hTDP-43-Q331K, and UAS-hTDP-43- C-terminal fragment (amino acids 209-414) constructs, the corresponding primers (Additional file
1) were used to amplify DNA fragments, which were then cloned into the pUAST vector. To generate
UAS-dTDP-43 constructs, the full-length dTDP-43 coding sequence was amplified from the cDNA plasmid GH09868 (
Drosophila Genomics Resource Center) and cloned into the pUAST vector. These constructs were confirmed by sequencing and microinjected into wild-type (
w1118) embryos to generate transgenic lines.
Antibody production and western blot
Anti-dTDP-43 polyclonal antibody was generated by immunizing rabbits with a peptide fragment spanning amino acids 179-192 (Thermo Fisher Scientific). For protein expression analysis, adult flies were frozen in ethanol with dry ice and vortexed to remove heads. Approximately 30 heads from each genotype were homogenized in 50 μl of lysis buffer (0.137 M NaCl, 20 mM Tris, pH 8.0, 10% glycerol, 1% NP-40, 0.1% SDS, 0.1% sodium deoxycholate, 1 mM DTT, Pierce protease inhibitors and phosphatase inhibitors). Homogenate was heated at 65°C for 10 min and centrifuged at 4°C for 10 min at 13,000 rpm. Protein concentrations were determined using Bradford Assay (Bio-Rad).
Supernatant containing 10 μg of protein was separated on a 10% acrylamide SDS gel and transferred to a PVDF membrane (Bio-Rad) in a wet transfer system at 4°C for 60 min at 100 V. The membrane was incubated in blocking solution containing 5% milk in TBST (25 mM Tris-HCl, 137 mM NaCl, 3 mM KCl, pH 7.4, and 0.1% Tween-20) at 4°C overnight, with dTDP-43 antibody (1:1000 in blocking solution) at room temperature for 3 h, and finally with anti-rabbit HRP-conjugated secondary antibody (Jackson Immunoresearch; 1:10,000) for 1 h. The signal was visualized with chemiluminescent substrate (Supersignal West Pico, Pierce). For other western blot analyses, the primary antibodies were hTDP-43 antibody (1:1000; 10782-2-AP, Proteintech), and β-actin antibody (1:1500; Cell Signaling).
Quantitative RT-PCR (qRT-PCR) analysis
Total RNAs were extracted from adult heads with Trizol (Invitrogen) and used as templates to generate cDNAs with TaqMan reverse transcription reagent (Applied Biosystems). cDNAs were used as templates for qRT-PCR in a final volume of 25 μl. A standard curve was run in each PCR reaction. Individual values were normalized to the value of the gene encoding the ribosomal protein RP-49. Two pairs of primers were designed to detect dTDP-43 transcripts (Additional file
2). All reactions were performed three times. Relative mRNA expression was calculated using the standard curve method and the delta-delta Ct method.
Mosaic analysis with a repressible cell marker (MARCM)
MARCM analysis of sensory neurons in the
Drosophila PNS was performed as described [
28]. First, the
dTDP-43Q 367Xallele was recombined onto the chromosome containing
FRTG 13.
FRTG 13,
dTDP-43Q 367X/
CyO or
FRTG 13/
CyO male flies were crossed with
Gal4C 155,
UAS-mCD8-GFP, hs-FLP1; FRTG 13,
Gal80/CyO virgin females to generate
Gal4C 155,
UAS-mCD8-GFP, hs-FLP1/+; FRTG 13,
Gal80/FRTG 13,
dTDP-43Q 367Xand
Gal4C 155,
UAS-mCD8-GFP, hs-FLP1/+; FRTG 13,
Gal80/FRTG 13embryos, respectively. Embryos from these crosses were collected on grape agar plates for 3 h in a 25°C incubator. The embryos were aged for 3 h and heat-shocked in a 37°C water bath for 40 min to induce mitotic recombination. The embryos were then kept in a moisture chamber at 25°C for 3-4 days. Third instar larvae were collected, and larvae that contained a single mCD8::GFP-labeled dorsal cluster PNS neuron were selected under a Nikon fluorescence dissection microscope. Images of the dendritic morphology of single DA neurons were recorded with a confocal microscope (Nikon, D-Eclipse C1). The significance of differences in dendritic branching complexity was determined with Student's
t test.
Quantitative Analysis of dendritic ends of sensory neurons
The dendritic morphology of GFP-labeled dorsal sensory neurons was recorded with a confocal microscope (Nikon, D-Eclipse C1), and dendritic branches of ddaE or ddaF neurons in the A3 dorsal cluster were counted as described [
28]. Briefly, dendritic ends of DA neuron images were identified visually and highlighted with dots, which were counted with Adobe Photoshop software. The data were analyzed by Student's
t test.
Discussion
The pathological role of TDP-43 was first recognized by its presence in ubiquitin-positive but tau-negative inclusions in diseased neurons of FTD and ALS patients [
10,
11]. TDP-43 pathology has two characteristic features. First, TDP-43 is proteolytically processed, and phosphorylated C-terminal fragments of approximately 20-25 kDa are present in the insoluble inclusions [
10]. Indeed, ectopic expression of these fragments in cultured cells results in aggregation [
33]. Second, TDP-43 is transported from the nucleus, where it predominantly resides in healthy cells. These findings suggest that TDP-43 may contribute to neurodegeneration through both a toxic gain-of-function mechanism and a loss-of-function mechanism. These possibilities are not mutually exclusive. However, the precise roles of TDP-43 in postmitotic neurons remain largely unknown.
Since TDP-43 is highly conserved at the amino acid sequence level from flies to humans,
Drosophila offers a powerful model system to examine the endogenous functions of TDP-43. We obtained
dTDP-43 null mutant flies and found that dTDP-43 is required for normal viability, consistent with a study published during the preparation of our manuscript [
26].
TDP-43 knockout mice have not been reported yet. Considering the high degree of conservation between dTDP-43 and hTDP-43, it is possible that TDP-43 is also essential for normal development in mammals. At the cellular level, multiple lines of evidence from our current study indicate that TDP-43 promotes dendritic branching in postmitotic neurons. This conclusion was based on overexpression studies, RNAi knockdown, and genetic analysis of a
dTDP-43 null allele. TDP-43 seems to also regulate axonal structures at the
Drosophila neuromuscular junction (NMJ) [
26]. These findings indicate an essential role for TDP-43 in neuronal structural integrity.
In many neurodegenerative diseases, defects in synaptic connections are probably one of the earliest alterations preceding neurodegeneration [
34]. Recent imaging studies in human brains suggest that specific functional connectivity networks are compromised in specific neurodegenerative conditions [
35]. It is conceivable that loss of the normal nuclear function of TDP-43 in specific vulnerable neurons reduces dendritic complexity, which in turn leads to compromised neuronal connectivity in that specific neuronal circuitry. Thus, exclusion of TDP-43 from the nucleus through unknown pathways in diseased neurons may represent a loss-of-function mechanism that may contribute to the pathogenesis of FTD and ALS.
Drosophila is also an excellent model system for studying human disease proteins. We found that hTDP-43 also promotes dendritic branching in
Drosophila neurons, indicating a functional conservation. More importantly, two point mutations associated with ALS attenuated the dendrite-promoting activity of hTDP-43. Both are located in a C-terminal region that mediates protein-protein interactions [
36]. Thus, these mutations may compromise the normal functions of TDP-43 in neurons. It was reported that these missense mutations might also enhance the formation of TDP-43 aggregates [
37]. Thus, multiple pathogenic mechanisms may work in concert to promote disease initiation and/or progression.
Acknowledgements
We thank the Vienna Drosophila RNAi Center (VDRC) for providing dTDP-43 RNAi lines (38377 and 38379), the Seattle Drosophila TILLING Project for recovering TDP-43 truncation mutant Q367*, and Jin-A Lee for providing the human TDP-43 cDNA construct. We also thank S. Ordway for editorial assistance, S. Mitchell for administrative assistance, and lab members for comments. This work was supported by the NIH (F.-B.G.).
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
YL and FBG designed the experiments, and YL and JF performed the experiments. YL, JF, and FBG analyzed the data, and wrote the paper. All authors read and approved the final version of the manuscript.