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Altered Distributions of Gemini of Coiled Bodies and Mitochondria in Motor Neurons of TDP-43 Transgenic Mice

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Altered Distributions of Gemini of Coiled Bodies and Mitochondria in Motor Neurons of TDP-43 Transgenic Mice Altered distributions of Gemini of coiled bodies and mitochondria in motor neurons of TDP-43 transgenic mice Xiu Shana,b,1, Po-Min Chianga,b,1, Donald L. Pricea,b,c,d, and Philip C. Wonga,b,c,2 aDepartment of Pathology, bDivision of Neuropathology, cDepartment of Neuroscience, and dDepartment of Neurology, The Johns Hopkins University School of Medicine, Baltimore, MD 21205-2196 Edited* by Richard L. Huganir, The Johns Hopkins University School of Medicine, Baltimore, MD, and approved August 4, 2010 (received for review March 16, 2010) TAR DNA-binding protein-43 (TDP-43), a DNA/RNA-binding protein possibility that altered RNA metabolism or RNA processing may involved in RNA transcription and splicing, has been associated with underlie and contribute to motor neuron degeneration (2). the pathophysiology of neurodegenerative diseases, including ALS. Despite recent advances in development of TDP-43 transgenic However, the function of TDP-43 in motor neurons remains unde- models in mice (11, 12) and flies (13), no experimental evidence fined. Here we use both gain- and loss-of-function approaches to is currently available to support the view that TDP-43 partic- determine roles of TDP-43 in motor neurons. Mice expressing hu- ipates in pathways that regulate RNA processing in motor neu- man TDP-43 in neurons exhibited growth retardation and prema- rons. To begin to address this issue, we generated mice either ture death that are characterized by abnormal intranuclear lacking endogenous TDP-43 or expressing human TDP-43 in inclusions composed of TDP-43 and fused in sarcoma/translocated neurons, including motor neurons. Here we provide evidence to in liposarcoma (FUS/TLS), and massive accumulation of mitochon- support TDP-43 in regulating the physiology of motor neurons, dria in TDP-43-negative cytoplasmic inclusions in motor neurons, including those that impact on the proper distributions of mi- lack of mitochondria in motor axon terminals, and immature neu- tochondria in the cytoplasm and of fused in sarcoma/trans- romuscular junctions. Whereas an elevated level of TDP-43 disrupts located in liposarcoma (FUS/TLS) and SMN-associated Gemini the normal nuclear distribution of survival motor neuron (SMN)- of coiled bodies (GEMs) in the nucleus. Together with results associated Gemini of coiled bodies (GEMs) in motor neurons, its from our TDP-43 conditional knockout mouse model, our find- absence prevents the formation of GEMs in the nuclei of these cells. ings implicate a critical role of TDP-43 in controlling the for- Moreover, transcriptome-wide deep sequencing analysis revealed mation of SMN-associated GEMs that may impact on RNA fi that a decrease in abundance of neuro lament transcripts contrib- metabolism in motor neurons. uted to the reduction of caliber of motor axons in TDP-43 mice. In concert, our findings indicate that TDP-43 participates in pathways Results critical for motor neuron physiology, including those that regulate TDP-43 Growth Retardation, Muscle Weakness, and Death in Transgenic NEUROSCIENCE the normal distributions of SMN-associated GEMs in the nucleus and Mice. Several lines (W1, W2, and W3) of mice expressing wild-type mitochondria in the cytoplasm. human TDP-43 (hTDP-43) were generated using the Thy1.2 promoter (Fig. S1), which is capable of driving expression post- ALS | RNA metabolism | frontotemporal lobar degeneration with natally in neurons, including motor neurons. TDP-43 mice ubiquitinated inclusions | fused in sarcoma/translocated in liposarcoma | exhibited retardation of development when compared with non- survival motor neuron transgenic littermates (Fig. 1A). The severities of this phenotype correlated with the copy number of the transgene: Mice from line dentified first as a regulator of HIV gene expression, TAR W1, with the highest transgene copy number (Fig. S1), were IDNA-binding protein (TDP-43) is a DNA/RNA-binding protein markedly smaller than their nontransgenic littermates (Fig. 1A) that contains two RNA-recognition motifs and a glycine-rich C- and die within 3 wk of age, whereas mice derived from W2 and – terminal domain thought to be important for mediating protein W3, two lines harboring lower numbers of transgenes (Fig. S1), protein interactions (1, 2). Although TDP-43 has been implicated exhibited growth retardation to a lesser extent (Fig. 1A), and most as a key factor regulating RNA splicing of human cystic fibrosis of those mice grew to adulthood. Interestingly, male TDP-43 mice transmembrane conductance regulator (CFTR) (3), Apolipopro- exhibited ≈20% reduction in body weight at 4 wk of age when tein A-II (4), and Survival Motor Neuron (SMN) (5), the im- compared with nontransgenic male littermates (Fig. 1B). We ob- portance of TDP-43 in the central nervous system had not been served that transgenic males derived from both W2 and W3 lines fi demonstrated until it was identi ed as a component of ubiquiti- exhibited a more severe phenotype when compared with trans- nated protein aggregates in cases of amyotrophic lateral sclerosis genic female littermates, an outcome that appears to be related to (ALS) and frontotemporal lobar degeneration with ubiquitinated inclusions (FTLD-U) (6). In both of these diseases, TDP-43 is depleted from the nuclei but accumulates in the ubiquitinated Author contributions: X.S., P.-M.C., and P.C.W. designed research; X.S. and P.-M.C. inclusions of affected neurons, suggesting that loss of normal performed research; X.S., P.-M.C., D.L.P., and P.C.W. analyzed data; and X.S., P.-M.C., function of TDP-43 as a nuclear protein or, alternatively, gain of D.L.P., and P.C.W. wrote the paper. a toxic function by TDP-43 aggregates play significant roles in the The authors declare no conflict of interest. pathogenesis of ALS and FTLD-U (1). Moreover, the identifi- *This Direct Submission article had a prearranged editor. cation of mutations in TDP-43 that are linked to both sporadic Freely available online through the PNAS open access option. and familial ALS (2, 7, 8) provides evidence that TDP-43 directly Data deposition: The data reported in this paper have been deposited in the Gene Ex- contributes to the pathogenesis of these neurodegenerative dis- pression Omnibus (GEO) database, www.ncbi.nlm.nih.gov/geo (accession no. GSE22351). orders. However, the exact mechanisms by which mutant TDP-43 1X.S. and P.-M.C. contributed equally to this work. contributes to ALS remain elusive. Interestingly, recent discov- 2To whom correspondence should be addressed. E-mail: [email protected]. FUS/TLS eries of mutations in , a gene that encodes another This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. RNA-binding protein, linked to ALS (9, 10) offer the intriguing 1073/pnas.1003459107/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1003459107 PNAS | September 14, 2010 | vol. 107 | no. 37 | 16325–16330 Downloaded by guest on September 28, 2021 Fig. 1. Early postnatal growth retardation in mice expressing wild-type TDP-43. (A) Decrease in size of wild-type TDP-43 transgenic (tg) male mice derived from independent founders W1 and W3; asterisks indicate, re- spectively, transgenic mice at 14 and 21 d of age. (B) Body weights of 4-wk- Fig. 2. Pathological abnormalities in spinal cords of 3-wk-old W3 TDP-43 old male TDP-43 transgenic mice from line W3 were compared with non- mice. (A and B) Hematoxylin and eosin staining reveals eosinophilic aggre- transgenic (ntg) littermates. Note significant reduction in body weights of gates in cell bodies of motor neurons in spinal cords of transgenic (tg) mice TDP-43 transgenic mice (ntg, n = 13; W3 tg, n = 15; P < 0.0001). Error bars (B, arrows); such structures were not identified in nontransgenic (ntg) lit- indicate SEM. (C) Accumulation of TDP-43 in spinal cords of W3 mice at 4 wk termates (A). Boxes at bottom right are enlarged micrographs showing of age. A mouse monoclonal antibody recognizing human-specific TDP-43 a healthy motor neuron (A) and a neuron bearing a large cytoplasmic ag- was used to determine the level of transgene expression, whereas a rabbit gregate marked by an arrowhead (B). (C–E) The antibody recognizing human antibody against both mouse and human TDP-43 was used to compare the TDP-43 specifically reveals that the transgene is extensively expressed in level of transgene expression with that of endogenous TDP-43. f, female; m, spinal cord neurons of transgenic mice (D and E); no immunoreactivity is male. (D) Densitometric analysis of TDP-43 protein levels in 4-wk-old W3 detected in nontransgenic mice (C). hTDP-43 is localized in the nucleus and mice using the rabbit antibody against both mouse and human TDP-43 forms intranuclear granular structures (E, arrowheads) in some neurons (female tg, n = 3; male tg, n = 6; ntg littermates, n = 4). Compared with that bearing cytoplasmic aggregates (E, arrow), which are identified by eosin of mouse endogenous TDP-43, the levels of human TDP-43 are, respectively, counterstain. (F and G) Immunohistochemical analysis with ubiquitin anti- 1.3- and 3.6-fold in 4-wk-old W3 transgenic females (P = 0.0011) and males body shows that the level of ubiquitination is elevated in the spinal cords of (P < 0.0001). Error bars indicate SEM. transgenic mice (G) when compared with that in nontransgenic mice (F). Arrows point to neurons with eccentric nuclei, indicating the presence of cytoplasmic aggregates, and those neurons are heavily stained with ubiq- a higher (2- to 3-fold) accumulation of TDP-43 in transgenic males C D TDP-43 uitin, particularly within the nuclear compartments. (H and I) Double im- (Fig. 1 and ). Male mice abruptly developed severe munofluorescence analyses using antisera against HSP60, a mitochondrial tremor, abnormal reflex of hindlimbs (Fig.
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