TDP-43, or TAR DNA-binding protein 43, is a pathological marker of a spectrum of neurodegenerative disorders, including amyotrophic lateral sclerosis and frontotemporal lobar degeneration with ubiquitinpositive inclusions. TDP-43 is an RNA/DNA-binding protein implicated in transcriptional and posttranscriptional regulation. Recent work also suggests that TDP-43 associates with cytoplasmic stress granules, which are transient structures that form in response to stress. In this study, we establish sorbitol as a novel physiological stressor that directs TDP-43 to stress granules in Hek293T cells and primary cultured glia. We quantify the association of TDP-43 with stress granules over time and show that stress granule association and size are dependent on the glycine-rich region of TDP-43, which harbors the majority of pathogenic mutations. Moreover, we establish that cells harboring wild-type and mutant TDP-43 have distinct stress responses: mutant TDP-43 forms significantly larger stress granules, and is incorporated into stress granules earlier, than wild-type TDP-43; in striking contrast, wild-type TDP-43 forms more stress granules over time, but the granule size remains relatively unchanged. We propose that mutant TDP-43 alters stress granule dynamics, which may contribute to the progression of TDP-43 proteinopathies.TAR DNA-binding protein 43 (TDP-43) is a highly conserved, ubiquitously expressed RNA-binding protein of the heterogeneous nuclear ribonucleoprotein (hnRNP) family (11,47,73). TDP-43 and other hnRNPs are multifunctional proteins that regulate gene expression in both the nucleus and the cytoplasm (47, 75). In the nucleus, TDP-43 binds singlestranded DNA and RNA (10,11,19,20,49,62) and can function as both a transcriptional repressor (1, 2, 62) and a splicing modulator (15,17,20,55). Specifically, TDP-43 regulates pre-mRNA splicing by binding mRNA with (UG) 6-12 sequences (19) and by recruiting other hnRNP proteins into repressive splicing complexes (10,18,55). However, as a nucleocytoplasmic shuttling protein (12), TDP-43 also has distinct cytoplasmic functions, including mRNA stabilization (74).Recent studies indicate that TDP-43 localizes to stress granules (SGs) in response to heat shock, oxidative stress, and chemical inducers of stress (23,33). SGs are dynamic cytoplasmic structures that are believed to act as sorting stations for mRNAs (5). SG composition and morphology differ according to stress and cell type (5, 39), but some core components are conserved. These core components include the RNA-binding protein TIAR (TIA-1 cytotoxic granule-associated RNA-binding protein-like 1) and the stalled translation initiation complex components eIF3 and eIF4G (44,45). In contrast, the incorporation of the RNA-binding proteins HuR and hnRNP A1 into SGs differs with the cell type and stress (5, 39). The physiological stressors that cause TDP-43 aggregates and SGs to form-and the cells in which this occurs-remain unresolved. Moreover, very little is known about the function of cytoplasmic TDP-43, a press...

The gene coding TDP-43, 2 or TAR DNA-binding protein 43 (Tardbp), is highly conserved throughout evolution and is found in all higher eukaryotic species including distant species Drosophila melanogaster, Xenopus laevis, and Caenorhabditis elegans (1, 2). In humans, Tardbp is located at the chromosomal locus 1p36.22 and is comprised of six exons, five of which encode a ubiquitously expressed, predominantly nuclear, 43-kDa protein that contains two RNA recognition motifs and a glycine-rich C-terminal domain, characteristic of the heterogeneous nuclear ribonucleoprotein class of proteins (3). The RNA recognition motif domains of TDP-43 are highly homologous among species; however, the glycine-rich sequence varies significantly among all species, reflecting species-specific functions in the different organisms.TDP-43 has been implicated in the regulation of gene transcription, pre-mRNA splicing, mRNA stability, and mRNA transport (4). It was first identified to bind the TAR DNA of the human immunodeficiency virus 1 long terminal repeat region. Both in vitro and in vivo experiments showed that TDP-43 represses human immunodeficiency virus 1 proviral gene expression (5). Later, it was shown to enhance exon skipping of the cystic fibrosis transmembrane conductance regulator exon 9 through binding to a (UG) m (U) n motif near the 3Ј splice site of the cystic fibrosis transmembrane conductance regulator intron 8 (6). TDP-43 was also shown to be involved in splicing of the apolipoprotein A-II (7) and survival of motor neuron (8) genes. In addition, TDP-43 has been implicated in regulation of mRNA biogenesis (9) and shown to be localized to sites of mRNA transcription and processing in neurons (10). As the glycine-rich domain of TDP-43 has been shown to mediate interactions with other heterogeneous nuclear ribonucleoprotein proteins, the low homology of this particular domain may afford a multitude of interactions that allows for diverse biological functions (11).TDP-43 has been identified as the primary protein of neuronal and glial inclusions of sporadic and familial frontotemporal lobar degeneration with ubiquitin positive inclusions (FTLD-U), as well as in sporadic and the majority of familial amyotrophic lateral sclerosis (ALS) cases (12, 13). TDP-43, normally observed in the nucleus, is found in pathological inclusions mostly in the cytoplasm and in some cases accumulates in dense deposits in the nucleus. The inclusions consist prominently of TDP-43 C-terminal fragments of ϳ20 -25 kDa. Both full-length and C-terminal fragments of TDP-43 undergo abnormal phosphorylation and ubiquitination in diseased states (13). More recently, TDP-43 inclusions are found in patients with Alzheimer and Parkinson diseases implying a common mechanism of TDP-43-related

SUMMARY Generating a balanced network of inhibitory and excitatory neurons during development requires precise transcriptional control. In the dorsal spinal cord, Ptf1a, a basic helix-loop-helix (bHLH) transcription activator, maintains this delicate balance by inducing homeodomain (HD) transcription factors such as Pax2 to specify the inhibitory lineage, while suppressing HD factors such as Tlx1/3 that specify the excitatory lineage. We uncover the mechanism by which Ptf1a represses excitatory cell fate in the inhibitory lineage. We identify Prdm13 as a direct target of Ptf1a and reveal that Prdm13 actively represses excitatory cell fate by binding to regulatory sequences near the Tlx1 and Tlx3 genes to silence their expression. Prdm13 acts through multiple mechanisms including interactions with the bHLH factor Ascl1 to repress Ascl1 activation of Tlx3. Thus, Prdm13 is a key component of a highly coordinated transcriptional network that determines the balance of inhibitory versus excitatory neurons in the dorsal spinal cord.

Nicotinamide mononucleotide (NMN) adenylyltransferase 2 (Nmnat2) catalyzes the synthesis of NAD from NMN and ATP. The Nmnat2 transcript is expressed predominately in the brain; we report here that Nmnat2 is a low abundance protein expressed in neurons. Previous studies indicate that Nmnat2 localizes to Golgi. As Nmnat2 is not predicted to contain a signal sequence, lipid-binding domain, or transmembrane domain, we investigated the nature of this interaction. These experiments reveal that Nmnat2 is palmitoylated in vitro, and this modification is required for membrane association. Surprisingly, exogenous Nmnat2 is toxic to neurons, indicating that protein levels must be tightly regulated. To analyze Nmnat2 localization in neurons (previous experiments relied on exogenous expression in HeLa cells), mouse brains were fractionated, showing that Nmnat2 is enriched in numerous membrane compartments including synaptic terminals. In HeLa cells, in addition to Golgi, Nmnat2 localizes to Rab7-containing late endosomes. These studies show that Nmnat2 is a neuronal protein peripherally attached to membranes via palmitoylation and suggest that Nmnat2 is transported to synaptic terminals via an endosomal pathway. Nicotinamide mononucleotide (NMN)2 adenylyltransferases (Nmnat) catalyze the synthesis of NAD from NMN and ATP (1-3). Humans and mice express three isoforms, each from a separate gene: Nmnat1, -2, and -3; whereas invertebrates such as Drosophila melanogaster have only one (4). Nmnat2 is unique in that its transcript is expressed predominately in the brain (5-7). In contrast, Nmnat1 and -3 transcripts are widely expressed, although they do not necessarily overlap (2). Furthermore, Nmnat2 localizes to Golgi (8), whereas Nmnat1 is nuclear (8, 9), and Nmnat3 associates with mitochondria (8, 10). This suggests that 1) Nmnat1, -2, and -3 have evolved specialized roles in vertebrate NAD metabolism, and 2) the subcellular location of NAD synthesis is cell type-specific.In addition to its role as a cofactor, NAD is also a substrate for numerous enzymes including sirtuins, poly(ADP-ribose) polymerases, and ADP-ribosyl cyclases (e.g. CD38) (11). These enzymes regulate diverse cellular processes including transcription, apoptosis, and calcium signaling (12)(13)(14). In eukaryotes, NAD is synthesized either de novo from tryptophan or recycled from nicotinic acid, nicotinamide, or nicotinamide riboside (1, 11). Because neurons require an enormous amount of energy to propagate action potentials, it is not surprising that insufficient dietary intake of NAD precursors results in severe neurological dysfunction (15,16).It is unclear what specific role Nmnat2 has in the brain that cannot be met by Nmnat1 or -3. To address this question, we have first sought to understand how Nmnat2 interacts with the Golgi, its cellular and developmental protein expression, and its localization in brain cells. We report here that Nmnat2 is a developmentally regulated, low abundance neuronal protein that localizes not only to Golgi but also to vesicles ...

A mutation was recovered in the slr0721 gene, which encodes the decarboxylating NADP ؉ -dependent malic enzyme in the cyanobacterium Synechocystis sp. strain PCC 6803, yielding the mutant 3WEZ. Under continuous light, 3WEZ exhibits poor photoautotrophic growth while growing photoheterotrophically on glucose at rates nearly indistinguishable from wild-type rates. Interestingly, under diurnal light conditions (12 h of light and 12 h of dark), normal photoautotrophic growth of the mutant is completely restored.Cyanobacteria are photoautotrophic gram-negative eubacteria that are capable of performing oxygenic photosynthesis. Synechocystis sp. strain PCC 6803 is a naturally transformable (7) unicellular cyanobacterium and has proven to be one of the best model organisms for studying the mechanism and regulation of oxygenic photosynthesis (14); it has also been used in a variety of global gene expression (6,8,13) and metabolomic (15, 16) studies. In addition to autotrophic growth, the presence of an unidentified mutation in the Williams strain of Synechocystis (14) confers glucose tolerance to this organism. With glucose as a carbon source, this strain can be grown under mixotrophic, photoheterotrophic (continuous photosynthetic illumination at 20 to 40 mol of photons m Ϫ2 s Ϫ1 in the presence of the photosystem II inhibitor dichloromethylurea [DCMU]), and heterotrophic (nonphotosynthetic continuous illumination at Ͻ1 mol of photons m Ϫ2 s Ϫ1