No abstract

The transcription factors Aft1 and Aft2 from Saccharomyces cerevisiae regulate the expression of genes involved in iron homeostasis. These factors induce the expression of iron regulon genes in iron-deficient yeast but are inactivated in iron-replete cells. Iron inhibition of Aft1/Aft2 was previously shown to be dependent on mitochondrial components required for cytosolic iron sulfur protein biogenesis. We presently show that the nuclear monothiol glutaredoxins Grx3 and Grx4 are critical for iron inhibition of Aft1 in yeast cells. Cells lacking both glutaredoxins show constitutive expression of iron regulon genes. Overexpression of Grx4 attenuates wild type Aft1 activity. The thioredoxin-like domain in Grx3 and Grx4 is dispensable in mediating iron inhibition of Aft1 activity, whereas the conserved cysteine that is part of the conserved CGFS motif in monothiol glutaredoxins is essential for this function. Grx3 and Grx4 interact with Aft1 as shown by two-hybrid interactions and co-immunoprecipitation assays. The interaction between glutaredoxins and Aft1 is not modulated by the iron status of cells but is dependent on the conserved glutaredoxin domain Cys residue. Thus, Grx3 and Grx4 are novel components required for Aft1 iron regulation that most likely occurs in the nucleus.

The yeast mitochondrion is shown to contain a pool of copper that is distinct from that associated with the two known mitochondrial cuproenzymes, superoxide dismutase (Sod1) and cytochrome c oxidase (CcO) and the copper-binding CcO assembly proteins Cox11, Cox17, and Sco1. Only a small fraction of mitochondrial copper is associated with these cuproproteins. The bulk of the remainder is localized within the matrix as a soluble, anionic, low molecular weight complex. The identity of the matrix copper ligand is unknown, but the bulk of the matrix copper fraction is not protein-bound. The mitochondrial copper pool is dynamic, responding to changes in the cytosolic copper level. The addition of copper salts to the growth medium leads to an increase in mitochondrial copper, yet the expansion of this matrix pool does not induce any respiration defects. The matrix copper pool is accessible to a heterologous cuproenzyme. Co-localization of human Sod1 and the metallochaperone CCS within the mitochondrial matrix results in suppression of growth defects of sod2⌬ cells. However, in the absence of CCS within the matrix, the activation of human Sod1 can be achieved by the addition of copper salts to the growth medium.Copper is an essential cell nutrient acting as a cofactor in nearly 20 enzymes (1). However, excess accumulation of copper ions results in toxicity. Evidence for the effectiveness of copper ions as a toxin comes from its historic use as a fungicide, molluscide, and algicide. Homeostatic mechanisms exist in cells to regulate the cellular concentration of copper ions, thus maintaining copper balance and minimizing deleterious effects. Cells appear to maintain a quota for essential metal ions; this quota is primarily the quantity necessary to metallate the various copper proteins (2, 3). Copper ions are required for at least three key enzymes in Saccharomyces cerevisiae. The cuproenzymes include the cytosolic superoxide dismutase Sod1, 1 the plasma membrane ferroxidase Fet3, and the mitochondrial inner membrane enzyme cytochrome c oxidase (CcO). The copper quota of the yeast S. cerevisiae is about 5 ϫ 10 5 atoms per cell (3, 4).It is unclear what fraction of the 5 ϫ 10 5 copper atoms per cell is from copper in Sod1, Fet3, and CcO. Expression of these copper-binding proteins varies with growth conditions, suggesting that the copper may be distributed differently depending on growth conditions. Clearly, a significant fraction of the cellular copper is associated with Sod1; however, not all Sod1 molecules are metallated (4, 5). Fet3 requires four copper ions for activity, but levels of this protein are dependent on iron status of the medium (6). CcO levels vary depending on whether the cells are grown by fermentation or respiration. In addition, a varying quantity of cellular copper exists bound to two metallothioneins, Cup1 and Crs5 (7,8). Expression of CUP1 and CRS5 is regulated by copper levels through the copper-responsive transcription factor Ace1 (9, 10). An increase in the free Cu(I) ion pool activates Ace1 throu...

Fibroblast growth factor (FGF) and epidermal growth factor (EGF) are critical for the development of the nervous system. We previously discovered that FGF2 and EGF had opposite effects on motor neuron differentiation from human fetal neural stem cells (hNSCs), but the underlying mechanisms remain unclear. Here, we show that FGF2 and EGF differentially affect the temporal patterns of Akt and glycogen synthase kinase 3 beta (GSK3β) activation. High levels of phosphatidylinositol 3-kinase (PI3K)/Akt activation accompanied with GSK3β inactivation result in reduction of the motor neuron transcription factor HB9. Inhibition of PI3K/Akt by chemical inhibitors or RNA interference or overexpression of a constitutively active form of GSK3β enhances HB9 expression. Consequently, PI3K inhibition increases hNSCs differentiation into HB9+/microtubule-associated protein 2 (MAP2)+ motor neurons in vitro. More importantly, blocking PI3K not only enhances motor neuron differentiation from hNSCs grafted into the ventral horn of adult rat spinal cords, but also permits ectopic generation of motor neurons in the dorsal horn by overriding environmental influences. Our data suggest that FGF2 and EGF affect the motor neuron fate decision in hNSCs differently through a fine tuning of the PI3K/AKT/GSK3β pathway, and that manipulation of this pathway can enhance motor neuron generation.

Neural stem cells (NSCs) have some specified properties, but are generally uncommitted and so can change their fate after exposure to environmental cues. It is unclear to what extent this NSC plasticity can be modulated by extrinsic cues and what are the molecular mechanisms underlying neuronal fate determination. Basic fibroblast growth factor (bFGF) is a well known mitogen for proliferating NSCs. However, its role in guiding stem cells for neuronal subtype specification is undefined. Here we report that in vitro expanded human fetal forebrain-derived neural stem cells can generate cholinergic neurons with spinal motor neuron properties when treated with bFGF within a specific time window. bFGF induces NSCs to express the motor neuron marker Hb9, which is blocked by specific FGF receptor inhibitors and bFGF neutralizing antibodies. This development of spinal motor neuron properties is independent of selective proliferation or survival and does not require high levels of MAPK activation. Thus our study indicates that bFGF can play an important role in modulating plasticity and neuronal fate of human NSCs and presumably has implications for exploring the full potential of brain NSCs for clinical applications, particularly spinal motor neuron regeneration.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease caused by the selective loss of both spinal and upper motor neurons. One strategy in treating ALS is to use stem cells to replace lost spinal motor neurons. However, transplanted stem cell-derived motor neurons may not survive when exposed to the harsh microenvironment in the spinal cord of ALS. In particular, dysfunctional astrocytes and overactivated microglia in ALS may limit the survival of motor neurons generated from cell replacement therapy. On the other hand, stem cells may provide large quantities of motor neurons that can be used for studying glia-mediated toxic mechanisms and potential therapies in ALS. Here we will review methods and molecular factors for directed differentiation of stem cells into spinal motor neurons, the potential uses of these models for dissecting the mechanisms underlying glia-induced motor neuron degeneration and screening for new therapeutics aimed at protecting motor neurons in ALS, as well as discuss challenges facing the development of motor neuron replacement-based cell therapies for recovery in ALS.