Stav Kemeny

Neuron death during development and in Alzheimer’s disease (AD) is associated with aberrant regulation/induction of cell cycle proteins. However, the proximal events in this process are unknown. Cell cycle initiation requires dephosphorylation of cyclin-dependent kinases by cell division cycle 25A (Cdc25A). Here, we show that Cdc25A is essential for neuronal death in response to NGF deprivation or β-amyloid (Aβ) treatment and describe the mechanisms by which it is regulated in these paradigms. Cdc25A mRNA, protein and Cdc25A phosphatase activity were induced by NGF deprivation and Aβ treatment. Enhanced Cdc25A expression was also observed in rat brains infused with Aβ and in Aβ-overexpressing AβPPswe-PS1dE9 mice. In cultured neurons Cdc25A inhibition by chemical inhibitors or shRNA prevented cell death and neurite degeneration caused by NGF deprivation or Aβ. Additionally, Cdc25A inhibition diminished distal signaling events including Cdk-dependent elevation of phospho-pRb and subsequent caspase-3 activation. Mechanism studies revealed that Cdc25A induction by NGF deprivation and Aβ is mediated by activation of Forkhead transcription factors that in turn suppress miR-21, a negative regulator of Cdc25A. Our studies thus identify Cdc25A as a required upstream element of the apoptotic cell cycle pathway that is required for neuron death in response to trophic factor deprivation and to Aβ exposure and therefore as a potential target to suppress pathologic neuron death.

Parkinson’s disease (PD) is associated with excessive cell death causing selective loss of dopaminergic neurons. Dysfunction of the Ubiquitin Proteasome System (UPS) is associated with the pathophysiology of PD. Mutations in Parkin which impair its E3-ligase activity play a major role in the pathogenesis of inherited PD. ARTS (Sept4_i2) is a mitochondrial protein, which initiates caspase activation upstream of cytochrome c release in the mitochondrial apoptotic pathway. Here we show that Parkin serves as an E3-ubiquitin ligase to restrict the levels of ARTS through UPS-mediated degradation. Though Parkin binds equally to ARTS and Sept4_i1 (H5/PNUTL2), the non-apoptotic splice variant of Sept4, Parkin ubiquitinates and degrades only ARTS. Thus, the effect of Parkin on ARTS is specific and probably related to its pro-apoptotic function. High levels of ARTS are sufficient to promote apoptosis in cultured neuronal cells, and rat brains treated with 6-OHDA reveal high levels of ARTS. However, over-expression of Parkin can protect cells from ARTS-induced apoptosis. Furthermore, Parkin loss-of-function experiments reveal that reduction of Parkin causes increased levels of ARTS and apoptosis. We propose that in brain cells in which the E3-ligase activity of Parkin is compromised, ARTS levels increase and facilitate apoptosis. Thus, ARTS is a novel substrate of Parkin. These observations link Parkin directly to a pro-apoptotic protein and reveal a novel connection between Parkin, apoptosis, and PD.

ARTS (Sept4_i2) is a pro-apoptotic tumor suppressor protein that functions as an antagonist of X-linked IAP (XIAP) to promote apoptosis. It is generally thought that mitochondrial outer membrane permeabilization (MOMP) occurs before activation of caspases and is required for it. Here, we show that ARTS initiates caspase activation upstream of MOMP. In living cells, ARTS is localized to the mitochondrial outer membrane. In response to apoptotic signals, ARTS translocates rapidly to the cytosol in a caspase-independent manner, where it binds XIAP and promotes caspase activation. This translocation precedes the release of cytochrome C and SMAC/Diablo, and ARTS function is required for the normal timing of MOMP. We also show that ARTS-induced caspase activation leads to cleavage of the pro-apoptotic Bcl-2 family protein Bid, known to promote MOMP. We propose that translocation of ARTS initiates a first wave of caspase activation that can promote MOMP. This leads to the subsequent release of additional mitochondrial factors, including cytochrome C and SMAC/Diablo, which then amplifies the caspase cascade and causes apoptosis.

The yeast S. cerevisiae is a central model organism in eukaryotic cell studies and a major component in many food and biotechnological industrial processes. However, the wide knowledge regarding genetics and molecular biology of S. cerevisiae is based on an extremely narrow range of strains. Studies of natural populations of S. cerevisiae, not associated with human activities or industrial fermentation environments, are very few. We isolated a panel of S. cerevisiae strains from a natural microsite, “Evolution Canyon” at Mount Carmel, Israel, and studied their genomic biodiversity. Analysis of 19 microsatellite loci revealed high allelic diversity and variation in ploidy level across the panel, from diploids to tetraploids, confirmed by flow cytometry. No significant differences were found in the level of microsatellite variation between strains derived from the major localities or microniches, whereas strains of different ploidy showed low similarity in allele content. Maximum genetic diversity was observed among diploids and minimum among triploids. Phylogenetic analysis revealed clonal, rather than sexual, structure of the triploid and tetraploid subpopulations. Viability tests in tetrad analysis also suggest that clonal reproduction may predominate in the polyploid subpopulations.