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

Persistence is the most characteristic attribute of long-term memory (LTM). To understand LTM, we must understand how memory traces persist over time despite the short-lived nature and rapid turnover of their molecular substrates. It is widely accepted that LTM formation is dependent upon hippocampal de novo protein synthesis and Brain-Derived Neurotrophic Factor (BDNF) signaling during or early after acquisition. Here we show that 12 hr after acquisition of a one-trial associative learning task, there is a novel protein synthesis and BDNF-dependent phase in the rat hippocampus that is critical for the persistence of LTM storage. Our findings indicate that a delayed stabilization phase is specifically required for maintenance, but not formation, of the memory trace. We propose that memory formation and memory persistence share some of the same molecular mechanisms and that recurrent rounds of consolidation-like events take place in the hippocampus for maintenance of the memory trace.

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

Frontotemporal lobar degeneration (FTLD) is the diagnostic term for a group of clinically, genetically, and neuropathologically heterogeneous neurodegenerative disorders characterized by prominent atrophy of the frontal and anterior temporal lobes. FTLD is the second most common neurodegenerative cause of dementia after Alzheimer's Disease (AD) under age 65.1,2 The most prevalent clinical form of FTLD is frontotemporal dementia, which primarily manifests as changes in social and personal behavior, including disinhibition, and a progressive disorder of language.3 Affected individuals can develop a movement disorder including Parkinsonism and/or motor neuron disease (MND). The neuropathology of FTLD can be broadly divided into those with tau-positive inclusions known as tauopathies, and those with ubiquitin-

Information storage in the brain is a temporally graded process involving different memory types or phases. It has been assumed for over a century that one or more short-term memory (STM) processes are involved in processing new information while long-term memory (LTM) is being formed. It has been repeatedly reported that LTM requires de novo RNA synthesis around the time of training. Here we show that LTM formation of a one-trial inhibitory avoidance training in rats, a hippocampal-dependent form of contextual fear conditioning, depends on two consolidation periods requiring synthesis of new mRNAs. By injecting the RNA polymerase II inhibitors 5,6-dichloro-1-beta-d-ribofuranosylbenzimidazole or alpha-amanitin into the CA1 region of the dorsal hippocampus at various times before and after training, we found that hippocampal gene expression is critical in two time windows: around the time of training and 3-6 hr after training. Interestingly, these two periods of sensitivity to transcriptional inhibitors are similar to those observed using the protein synthesis inhibitor anisomycin. These findings underscore the parallel dependence of LTM formation of contextual fear on mRNA and protein synthesis in the hippocampus and suggest that the two time periods of anisomycin-induced amnesia depend at least in part on new mRNA synthesis.

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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.