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

The orphan nuclear receptor steroidogenic factor 1 (SF-1, also called Ad4BP and officially designated NR5A1) has emerged as an essential regulator of endocrine development and function. Initially identified as a tissue-specific transcriptional regulator of the cytochrome P450 steroid hydroxylases, SF-1 has considerably broader roles, as evidenced from studies in knockout mice lacking SF-1. The SF-1-knockout mice lacked adrenal glands and gonads and therefore died from adrenal insufficiency within the first week after birth. In addition, SF-1 knockout mice exhibited male-to-female sex reversal of their internal and external genitalia, impaired expression of multiple markers of pituitary gonadotropes, and agenesis of the ventromedial hypothalamic nucleus (VMH). These studies delineated essential roles of SF-1 in regulating endocrine differentiation and function at multiple levels, particularly with respect to reproduction. This chapter will review the experiments that established SF-1 as a pivotal, global determinant of endocrine differentiation and function. We next discuss recent insights into the mechanisms controlling the expression and function of SF-1 as well as the current status of research aimed at delineating its roles in specific tissues. Finally, we highlight areas where additional studies are needed to expand our understanding of SF-1 action. I. Initial Isolation of Steroidogenic Factor 1Steroid hormones are essential for fluid and electrolyte balance, intermediary metabolism, sexual differentiation, and reproductive function. Once the pathways of steroid hormone biosynthesis were defined and shown to involve the concerted actions of several cytochrome P450 mixed-function oxidases, attention turned to elucidating the mechanisms that regulate the expression of these enzymes. With the isolation of the bovine 21-hydroxylase cDNA (White et al., 1984b), followed shortly thereafter by the cloning of cDNAs encoding the side-chain cleavage enzyme (Matteson et al., 1984;Morohashi et al., 1984) and 11␤-hydroxylase (John et al., 1984), these questions could be addressed at a molecular level.

Alzheimer’s disease (AD) is characterized by the accumulation of intraneuronal tau and extracellular amyloid-β (Aβ) peptide. A triple transgenic (Tg) mouse (3xTg-AD) was reported to develop Aβ plaques and tau inclusions as well as remarkable accumulations of intracellular Aβ that were suggested to be the initiators of AD pathogenesis. However, it was unclear whether the anti-Aβ antibodies were able to distinguish Aβ peptide from the same Aβ epitopes within the amyloid precursor protein (APP). To further elucidate the identity of the immunoreactive intraneuronal material in 3xTg-AD mice, we conducted immunohistochemical, biochemical and ultrastructural studies using a well characterized panel of antibodies that distinguish Aβ within APP from cleaved Aβ peptides. We found that the intraneuronal material shared epitopes with full-length APP but not free Aβ. To demonstrate unequivocally that this intraneuronal material was not free Aβ peptide, we generated 3xTg-AD mice deficient for β-secretase (BACE), the protease required for Aβ generation from APP. In the absence of Aβ production, robust intraneuronal APP immunostaining was detected in the 3xTg-AD/BACE(−/−) mice. Finally, we found that the formation of tau lesions was not different between 3xTg-AD versus 3xTg-AD/BACE(−/−) mice, thereby demonstrating that tau pathology forms independently from Aβ peptide generation in this mouse model. Although we cannot corroborate the presence of intraneuronal Aβ peptide in 3xTg-AD mice, our findings warrant further study as to the role of aberrant APP accumulation in this unique model of AD.

The orphan nuclear receptor steroidogenic factor 1 (SF-1) plays key roles in endocrine development and function. Initially identified as a positive regulator of the cytochrome P450 steroid hydroxylases, analyses of knockout mice deficient in SF-1 revealed that SF-1 is essential for adrenal and gonadal development, pituitary gonadotropin expression and formation of the ventromedial hypothalamic nucleus. Although more limited in scope, analyses of SF-1 in humans similarly have suggested that SF-1 is important for differentiated function in adrenocortical and gonadotrope adenomas. In the hope of extending our understanding of SF-1 function by identifying possible roles of SF-1 in clinical endocrine disorders, we isolated the FTZ-F1 gene encoding human SF-1 and mapped it to chromosome 9q33. In this report, we characterize the sequence and structural organization of the human cDNA and gene encoding SF-1, providing new insights into comparative aspects of SF-1 structure that will facilitate efforts to study the role of this transcription factor in human endocrine disorders.

Yeast aspartic protease 3 (YAP3p), a basic-residue specific proprotein processing enzyme, was shown to be a membrane-associated protease. The membrane association of YAP3p was demonstrated to be through a glycophosphatidylinositol anchor situated in the carboxy terminus of the enzyme. Carboxy-terminal truncation of YAP3p by 37 amino acids resulted in secretion of YAP3p into the growth medium. Western blot analysis after sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed two secreted forms of YAP3p with apparent molecular masses of approximately 180 and approximately 90 kDa. YAP3p has an isoelectric point of approximately 4.5 as determined by isoelectric focusing gel electrophoresis. Treatment of YAP3p with endoglycosidase H reduced the size of both forms of the protein to approximately 65 kDa, consistent with the presence of 10 potential N-linked glycosylation sites in the deduced amino acid sequence of this protein. Removal of the N-linked sugars did not affect the enzymatic activity of YAP3p. Analysis of the effect of temperature on the stability and the rate of enzymatic activity of YAP3p showed that the enzyme retained 100% of its activity when incubated for 1 h at 37 degrees C, while incubation at 50 degrees C for 1 h resulted in approximately 80% loss of activity. The dependence of activity on temperature demonstrated a calculated Q10 of 1.95.