TAR DNA-binding protein 43 (TDP-43), encoded by the TARDBP gene on chromosome 1, is a highly conserved, ubiquitously expressed nuclear protein implicated in repression of gene transcription, inhibition of exon splicing, and interactions with splicing factors and nuclear bodies (1, 2). Recently, we identified TDP-43 as the disease protein forming insoluble aggregates in the central nervous system of patients with frontotemporal lobar degeneration (FTLD) 2 and amyotrophic lateral sclerosis (ALS). Since FTLD patients often develop motor neuron disease consistent with ALS and since ALS patients can also develop cognitive impairment and FTLD, the presence of TDP-43 neuropathology in both disorders provides a molecular link connecting FTLD and ALS as a clinicopathological spectrum of the same neurodegenerative disorder (i.e. TDP-43 proteinopathy) (3-6). FTLD includes a group of clinically, genetically and neuropathologically heterogeneous neurodegenerative disorders that account for ϳ20% of presenile dementia (7-9). Although neurodegenerative tauopathies account for about 40% of familial and sporadic FTLD cases, TDP-43 is the major disease protein found within the ubiquitin-positive, tau-and ␣-synculein-negative inclusions that account for the majority of the FTLD cases (designated as FTLD-U) (4, 10). TDP-43 inclusions are also present in the spinal cord and brain of sporadic and familial ALS cases with the notable exception of familial ALS due to SOD-1 mutations (3-6).TDP-43 neuropathology in FTLD-U and ALS is characterized by cytoplasmic, neuritic, and nuclear inclusions in neurons and glia (4, 11-13). We showed previously that the presence of cytoplasmic TDP-43 aggregates in disease neurons is accompanied by a dramatic clearance of normal TDP-43 staining, suggesting a redistribution of TDP-43 from the entire nucleus to a focal point adjacent to the nucleus (4, 13-15). Moreover, normal TDP-43 is found to be condensed as intranuclear inclusions mainly in familial FTLD with granulin (GRN) mutations and a rare disease linked to valosin-containing protein mutations (4, 14). Here we model TDP-43 cytoplasmic, neuritic, and nuclear inclusions in cultured cells and demonstrate that perturbation of endogenous TDP-43 trafficking between the nucleus and the cytoplasm leads to aggregate formation. Furthermore, the expression of mutant TDP-43 with defective nuclear localization (⌬NLS) or nuclear export signals (⌬NES) perturbs endogenous TDP-43 trafficking and recapitulates the unique TDP-43 pathologies that are signatures of the FTLD-U and ALS spectrum of disease. Our data implicate altered TDP-43 trafficking as a pathogenic mechanism underlying FTLD-U and ALS. EXPERIMENTAL PROCEDURESConstructs-cDNA encoding human TDP-43 (accession number NM 007375) in the plasmid pENTR-221 was obtained from Invitrogen. The addition of a Myc epitope tag to the

IntroductionFrontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS) are characterized by the presence of ubiquitin-positive inclusions (1). These inclusions are found in the brain and spinal cord of ALS patients as well as in patients with a major subtype of FTLD designated FTLD-TDP because these inclusions were shown to be comprised of the TAR-DNA binding protein 43 (TDP-43) (2). Since (a) cognitive abnormalities or dementia consistent with FTLD are increasingly recognized in ALS patients, (b) some FTLD patients develop MND, and (c) cytoplasmic TDP-43 aggregates are found in the brain and spinal cord of both ALS and FTLD-TDP patients, TDP-43 pathology appears to define a single neurodegenerative disorder (TDP-43 proteinopathy) with a spectrum of clinical manifestations (3-5). The importance of TDP-43 in the pathogenesis of these diseases is supported by the presence of autosomal dominant mutations in the TARDBP gene associated with ALS and FTLD (6).Human TDP-43 (hTDP-43) is a highly conserved and ubiquitously expressed 414-amino acid nuclear protein that binds to both DNA and RNA (7,8). TDP-43 is implicated in repression of gene transcription, regulation of exon splicing, and nuclear body functions (for a summary see recent reviews, refs. 4 and 6). Pathological TDP-43 can be abnormally cleaved, phosphorylated, and ubiquitinated, and most TDP-43 aggregates are mislocalized outside the nucleus within the cytoplasm or neurites (2). Interestingly,

The disease protein in frontotemporal lobar degeneration with ubiquitin-positive inclusions (FTLD-U) and amyotrophic lateral sclerosis (ALS) was identified recently as the TDP-43 (TAR DNA-binding protein 43), thereby providing a molecular link between these two disorders. In FTLD-U and ALS, TDP-43 is redistributed from its normal nuclear localization to form cytoplasmic insoluble aggregates. Moreover, pathological TDP-43 is abnormally ubiquitinated, hyperphosphorylated, and N-terminally cleaved to generate C-terminal fragments (CTFs). However, the specific cleavage site ( 4 with ubiquitin-positive, tau-negative inclusions (FTLD-U) with or without motor neuron disease as well as sporadic and the majority of familial amyotrophic lateral sclerosis (ALS) cases (1, 2). Human TDP-43 is encoded by the TARDBP gene on chromosome 1. It is a 414-amino acid nuclear protein with two highly conserved RNA recognition motifs (RRM1 and RRM2) and a C-terminal tail with a typical glycine-rich region that mediates protein-protein interactions, including interactions with other heterogeneous ribonucleoprotein (hnRNP) family members such as hnRNP A1, A2/B1, and A3 (3). Thus, TDP-43 is a ubiquitously expressed RNA/DNA-binding protein that also interacts with other nuclear proteins such as splicing factors. As such, TDP-43 is implicated in repression of gene transcription, regulation of exon splicing, and the functions of nuclear bodies (4 -9).Pathological TDP-43 accumulates as insoluble aggregates in the central nervous system neurons and glia of patients with FTLD-U and ALS (1). Moreover, FTLD-U patients can develop ALS, and ALS patients often suffer from a dementia consistent with FTLD-U (10). We therefore proposed that these diseases are part of a clinicopathological spectrum of the same neurodegenerative process collectively referred to as TDP-43 proteinopathy (1, 2). TDP-43 inclusions are present as cytoplasmic, neuritic, or nuclear inclusions, and affected neurons show a dramatic depletion of normal nuclear 11,12). To mimic this nuclear clearance and to model the sequestration of endogenous TDP-43 into cytoplasmic aggregates, we overexpressed TDP-43 with mutated nuclear localization signals (⌬NLS-TDP-43) in cultured cells that showed a reduction in endogenous nuclear TDP-43 and accumulations of insoluble cytoplasmic aggregates (13). Moreover, overexpression of

Growth inhibitory proteins in the central nervous system (CNS) block axon growth and regeneration by signaling to Rho, an intracellular GTPase. It is not known how CNS trauma affects the expression and activation of RhoA. Here we detect GTP-bound RhoA in spinal cord homogenates and report that spinal cord injury (SCI) in both rats and mice activates RhoA over 10-fold in the absence of changes in RhoA expression. In situ Rho-GTP detection revealed that both neurons and glial cells showed Rho activation at SCI lesion sites. Application of a Rho antagonist (C3–05) reversed Rho activation and reduced the number of TUNEL-labeled cells by ∼50% in both injured mouse and rat, showing a role for activated Rho in cell death after CNS injury. Next, we examined the role of the p75 neurotrophin receptor (p75NTR) in Rho signaling. After SCI, an up-regulation of p75NTR was detected by Western blot and observed in both neurons and glia. Treatment with C3–05 blocked the increase in p75NTR expression. Experiments with p75NTR-null mutant mice showed that immediate Rho activation after SCI is p75NTR dependent. Our results indicate that blocking overactivation of Rho after SCI protects cells from p75NTR-dependent apoptosis.

Inclusion body myopathy associated with Paget's disease of bone and frontotemporal dementia (IBMPFD) is a dominantly inherited degenerative disorder caused by mutations in the valosin-containing protein (VCP7) gene. VCP (p97 in mouse, TER94 in Drosophila melanogaster, and CDC48 in Saccharomyces cerevisiae) is a highly conserved AAA ϩ (ATPases associated with multiple cellular activities) ATPase that regulates a wide array of cellular processes. The mechanism of IBMPFD pathogenesis is unknown. To elucidate the pathogenic mechanism, we developed and characterized a Drosophila model of IBMPFD (mutant-VCP-related degeneration). Based on genetic screening of this model, we identified three RNA-binding proteins that dominantly suppressed degeneration; one of these was TBPH, the Drosophila homolog of TAR (trans-activating response region) DNA-binding protein 43 (TDP-43). Here we demonstrate that VCP and TDP-43 interact genetically and that disease-causing mutations in VCP lead to redistribution of TDP-43 to the cytoplasm in vitro and in vivo, replicating the major pathology observed in IBMPFD and other TDP-43 proteinopathies. We also demonstrate that TDP-43 redistribution from the nucleus to the cytoplasm is sufficient to induce cytotoxicity. Furthermore, we determined that a pathogenic mutation in TDP-43 promotes redistribution to the cytoplasm and enhances the genetic interaction with VCP. Together, our results show that degeneration associated with VCP mutations is mediated in part by toxic gain of function of TDP-43 in the cytoplasm. We suggest that these findings are likely relevant to the pathogenic mechanism of a broad array of TDP-43 proteinopathies, including frontotemporal lobar degeneration and amyotrophic lateral sclerosis.

Pathological TDP-43 is the major disease protein in frontotemporal lobar degeneration characterized by ubiquitin inclusions (FTLD-U) with/without motor neuron disease (MND) and in amyotrophic lateral sclerosis (ALS). As Guamanian parkinsonism-dementia complex (PDC) or Guamanian ALS (G-PDC or G-ALS) of the Chamorro population may present clinically similar to FTLD-U and ALS, TDP-43 pathology may be present in the G-PDC and G-ALS. Thus, we examined cortical or spinal cord samples from 54 Guamanian subjects for evidence of TDP-43 pathology. In addition to cortical neurofibrillary and glial tau pathology, G-PDC was associated with cortical TDP-43 positive dystrophic neurites and neuronal and glial inclusions in gray and/or white matter. Biochemical analyses showed the presence of FTLD-U-like insoluble TDP-43 in G-PDC, but not in Guam controls (G-C). Spinal cord pathology of G-PDC or G-ALS was characterized by tau positive tangles as well as TDP-43 positive inclusions in lower motor neurons and glial cells. G-C had variable tau and negligible TDP-43 pathology. These results indicate that G-PDC and G-ALS are associated with pathological TDP-43 similar to FTLD-U with/without MND as well as ALS, and that neocortical or hippocampal TDP-43 pathology distinguishes controls from disease subjects better than tau pathology. Finally, we conclude that the spectrum of TDP-43 proteinopathies should be expanded to include neurodegenerative cognitive and motor diseases, affecting the Chamorro population of Guam.

Inactivation of Rho promotes neurite growth on inhibitory substrates and axon regeneration in vivo.Here, we compared axon growth when neuronal cell bodies or injured axons were treated with a cell-permeable Rho antagonist (C3-07) in vitro and in vivo. In neurons plated in compartmented cultures, application of C3-07 to either cell bodies or distal axons promoted axonal growth on myelinassociated glycoprotein substrates. In vivo, an injection of C3-07 into the eye promoted regeneration of retinal ganglion cell (RGC) axons in the optic nerve after microcrush lesion. Delayed application of C3-07 promoted RGC growth across the lesion scar. Application of C3-07 completely prevented RGC cell death for 1 week after axotomy. To investigate the mechanism by which Rho inactivation promotes RGC growth, we studied slow axonal transport. Reduction in slow transport of cytoskeletal proteins was observed after axotomy, but inactivation of Rho did not increase slow axonal transport rates. Together, our results indicate that application of a Rho antagonist at the cell body is neuroprotective and overcomes growth inhibition but does not fully prime RGCs for active growth.

TAR DNA-binding protein-43 (TDP-43) is a highly conserved, ubiquitously expressed nuclear protein that was recently identified as the disease protein in frontotemporal lobar degeneration with ubiquitin-positive inclusions (FTLD-U) and amyotrophic lateral sclerosis (ALS). Pathogenic TDP-43 gene (TARDBP) mutations have been identified in familial ALS kindreds, and here we report a TARDBP variant (A90V) in a FTLD/ALS patient with a family history of dementia. Significantly, A90V is located between the bipartite nuclear localization signal sequence of TDP-43 and the in vitro expression of TDP-43-A90V led to its sequestration with endogenous TDP-43 as insoluble cytoplasmic aggregates. Thus, A90V may be a genetic risk factor for FTLD/ALS because it predisposes nuclear TDP-43 to redistribute to the cytoplasm and form pathological aggregates.