ATM-deficient neural precursors develop senescence phenotype with disturbances in autophagy.

ATM is a kinase involved in DNA damage response (DDR), regulation of response to oxidative stress, autophagy and mitophagy. Mutations in the ATM gene in humans result in ataxi A-Telangiectasia disease (A-T) characterized by a variety of symptoms with neurodegeneration and premature ageing among them. Since brain is one of the most affected organs in A-T, we have focused on senescence of neural progenitor cells (NPCs) derived from A-T reprogrammed fibroblasts. Accordingly, A-T NPCs obtained through neural differentiation of iPSCs in 5% oxygen possessed some features of senescence including increased activity of SA-ß-gal and secretion of IL6 and IL8 in comparison to control NPCs. This phenotype of A-T NPC was accompanied by elevated oxidative stress. A-T NPCs exhibited symptoms of impaired autophagy and mitophagy with lack of response to chloroquine treatment. Additional sources of oxidative stress like increased oxygen concentration (20 %) and H2O2 respectively aggravated the phenotype of senescence and additionally disturbed the process of mitophagy. In both cases only A-T NPCs reacted to the treatment. We conclude that oxidative stress may be responsible for the phenotype of senescence and impairment of autophagy in A-T NPCs. Our results point to senescent A-T cells as a potential therapeutic target in this disease.

Comparative neuronal differentiation of self-renewing neural progenitor cell lines obtained from human induced pluripotent stem cells.

Most human neuronal disorders are associated with genetic alterations that cause defects in neuronal development and induce precocious neurodegeneration. In order to fully characterize the molecular mechanisms underlying the onset of these devastating diseases, it is important to establish in vitro models able to recapitulate the human pathology as closely as possible. Here we compared three different differentiation protocols for obtaining functional neurons from human induced pluripotent stem cells (hiPSCs): human neural progenitors (hNPs) obtained from hiPSCs were differentiated by co-culturing them with rat primary neurons, glial cells or simply by culturing them on matrigel in neuronal differentiation medium, and the differentiation level was compared using immunofluorescence, biochemical and electrophysiological methods. We show that the differentiated neurons displayed distinct maturation properties depending on the protocol used and the faster morphological and functional maturation was obtained when hNPs were co-cultured with rat primary neurons.

ATM-deficient human neural stem cells as an in vitro model system to study neurodegeneration.

Loss of ATM kinase, a transducer of the DNA damage response and redox sensor, causes the neurodegenerative disorder ataxia-telangiectasia (A-T). While a great deal of progress has been made in elucidating the ATM-dependent DNA damage response (DDR) network, a key challenge remains in understanding the selective susceptibility of the nervous system to faulty DDR. Several factors appear implicated in the neurodegenerative phenotype in A-T, but which of them plays a crucial role remains unclear, especially since mouse models of A-T do not fully mirror the respective human syndrome. Therefore, a number of human neural stem cell (hNSC) systems have been developed to get an insight into the molecular mechanisms of neurodegeneration as consequence of ATM inactivation. Here we review the hNSC systems developed by us an others to model A-T.

Brain and induced pluripotent stem cell-derived neural stem cells as an in vitro model of neurodegeneration in ataxia-telangiectasia.

The ataxia telangiectasia mutated (ATM) kinase is a key transducer of the cellular response to DNA double strand breaks and its deficiency causes ataxia-telangiectasia (A-T), a pleiotropic genetic disorder primarily characterized by cerebellar neuropathy, immunodeficiency and cancer predisposition. While enormous progress has been achieved in elucidating the biochemical and functional regulation of ATM in DNA damage response, and more recently in redox signalling and antioxidant defence, the factors that make neurons in A-T extremely vulnerable remain unclear. Given also that ATM knockout mice do not recapitulate the central nervous system phenotype, a number of human neural stem cell (hNSC) model systems have been developed to provide insights into the mechanisms of neurodegeneration associated with ATM dysfunction. Here we review the hNSC systems developed by us an others to model A-T.

Differential pathotropism of non-immortalized and immortalized human neural stem cell lines in a focal demyelination model.

Cell therapy is reaching the stage of phase I clinical trials for post-traumatic, post-ischemic, or neurodegenerative disorders, and the selection of the appropriate cell source is essential. In order to assess the capacity of different human neural stem cell lines (hNSC) to contribute to neural tissue regeneration and to reduce the local inflammation after an acute injury, we transplanted GMP-grade non-immortalized hNSCs and v-myc (v-IhNSC), c-myc T58A (T-IhNSC) immortalized cells into the corpus callosum of adult rats after 5 days from focal demyelination induced by lysophosphatidylcholine. At 15 days from transplantation, hNSC and T-IhNSC migrated to the lesioned area where they promoted endogenous remyelination and differentiated into mature oligodendrocytes, while the all three cell lines were able to integrate in the SVZ. Moreover, where demyelination was accompanied by an inflammatory reaction, a significant reduction of microglial cells' activation was observed. This effect correlated with a differential migratory pattern of transplanted hNSC and IhNSC, significantly enhanced in the former, thus suggesting a specific NSC-mediated immunomodulatory effect on the local inflammation. We provide evidence that, in the subacute phase of a demyelination injury, different human immortalized and non-immortalized NSC lines, all sharing homing to the stem niche, display a differential pathotropism, both through cell-autonomous and non-cell autonomous effects. Overall, these findings promote IhNSC as an inexhaustible cell source for large-scale preclinical studies and non-immortalized GMP grade hNSC lines as an efficacious, safe, and reliable therapeutic tool for future clinical applications.

A protein phosphatase feedback mechanism regulates the basal phosphorylation of Chk2 kinase in the absence of DNA damage.

The checkpoint kinase Chk2 is an effector component of the ATM-dependent DNA damage response (DDR) pathway. The activation of Chk2 by genotoxic stress involves its phosphorylation on T68 by ATM and additional auto/transphosphorylations. Here we demonstrate that in unperturbed cells, chemical inhibition of Chk2 by VRX0466617 (VRX) enhances the phosphorylation of Chk2-T68 throughout the cell cycle phases. This event, dependent on the presence of ATM and catalytically functional Chk2, is not consequential to DNA damage, as neither gamma-H2AX nuclear foci nor increased ATM activation is detected in VRX-treated cells, suggesting the involvement of other regulatory proteins. As serine/threonine protein phosphatases (PPs) regulate the phosphorylation and deactivation of proteins of the DDR pathway, we analyzed their role in phospho-T68-Chk2 regulation. We found that intracellular inhibition of PP1 and PP2A-like activities by okadaic acid markedly raised the accumulation of Chk2-pT68 without DNA damage induction, and this phenomenon was also seen when PP1-C, PP2A-C, and Wip1/PPM1D were simultaneously knockdown by siRNA. Altogether, these data indicate a novel mechanism in undamaged cells where PPs function to maintain the balance between ATM and its direct substrate Chk2 through a regulatory circuit.

Mild hypoxia enhances proliferation and multipotency of human neural stem cells.

BACKGROUND: Neural stem cells (NSCs) represent an optimal tool for studies and therapy of neurodegenerative diseases. We recently established a v-myc immortalized human NSC (IhNSC) line, which retains stem properties comparable to parental cells. Oxygen concentration is one of the most crucial environmental conditions for cell proliferation and differentiation both in vitro and in vivo. In the central nervous system, physiological concentrations of oxygen range from 0.55 to 8% oxygen. In particular, in the in the subventricular zone niche area, it's estimated to be 2.5 to 3%. METHODOLOGY/PRINCIPAL FINDINGS: We investigated in vitro the effects of 1, 2.5, 5, and 20% oxygen concentrations on IhNSCs both during proliferation and differentiation. The highest proliferation rate, evaluated through neurosphere formation assay, was obtained at 2.5 and 5% oxygen, while 1% oxygen was most noxious for cell survival. The differentiation assays showed that the percentages of beta-tubIII+ or MAP2+ neuronal cells and of GalC+ oligodendrocytes were significantly higher at 2.5% compared with 1, 5, or 20% oxygen at 17 days in vitro. Mild hypoxia (2.5 to 5% oxygen) promoted differentiation into neuro-oligodendroglial progenitors as revealed by the higher percentage of MAP2+/Ki67+ and GalC+/Ki67+ residual proliferating progenitors, and enhanced the yield of GABAergic and slightly of glutamatergic neurons compared to 1% and 20% oxygen where a significant percentage of GFAP+/nestin+ cells were still present at 17 days of differentiation. CONCLUSIONS/SIGNIFICANCE: These findings raise the possibility that reduced oxygen levels occurring in neuronal disorders like cerebral ischemia transiently lead to NSC remaining in a state of quiescence. Conversely, mild hypoxia favors NSC proliferation and neuronal and oligodendroglial differentiation, thus providing an important advance and a useful tool for NSC-mediated therapy of ischemic stroke and neurodegenerative diseases like Parkinson's disease, multiple sclerosis, and Alzheimer's disease.

REGgamma proteasome activator is involved in the maintenance of chromosomal stability.

REGgamma is a member of the 11S regulatory particle that activates the 20S proteasome. Studies in REGgamma deficient mice indicated an additional role for this protein in cell cycle regulation and proliferation control. In this paper we demonstrate that REGgamma protein is equally expressed throughout the cell cycle, but undergoes a distinctive subcellular localization at mitosis. Thus, while in interphase cells REGgamma is nuclear, in telophase cells it localizes on chromosomes, suggesting a role in mitotic progression. Furthermore, we found that REGgamma overexpression weakens the mitotic arrest induced by spindle damage, allowing premature exit from mitosis, whereas REGgamma depletion has the opposite effect, thus reflecting a new REGgamma function, unrelated to its role as proteasome activator. Additionally, we found that primary cells from REGgamma-/- mice and human fibroblasts with depleted expression of REGgamma or overexpressing a dominant negative mutant unable to activate the 20S proteasome, demonstrated a marked aneuploidy (chromosomal gains and losses), supernumerary centrosomes and multipolar spindles. These findings thus underscore a previously uncharacterized function of REGgamma in centrosome and chromosomal stability maintenance.

Biochemical and cellular characterization of VRX0466617, a novel and selective inhibitor for the checkpoint kinase Chk2.

VRX0466617 is a novel selective small-molecule inhibitor for Chk2 discovered through a protein kinase screening program. In this study, we provide a detailed biochemical and cellular characterization of VRX0466617. We show that VRX0466617 blocks the enzymatic activity of recombinant Chk2, as well as the ionizing radiation (IR)-induced activation of Chk2 from cells pretreated with the compound, at doses between 5 and 10 micromol/L. These doses of VRX0466617 inhibited, to some extent, the phosphorylation of Chk2 Ser(19) and Ser(33-35), but not of Chk2 Thr(68), which is phosphorylated by the upstream ataxia-telangiectasia mutated (ATM) kinase. Interestingly, VRX0466617 induced the phosphorylation of Chk2 Thr(68) even in the absence of DNA damage, arising from the block of its enzymatic activity. VRX0466617 prevented the IR-induced Chk2-dependent degradation of Hdmx, concordant with the in vivo inhibition of Chk2. Analysis of ATM/ATM and Rad3-related substrates Smc1, p53, and Chk1 excluded a cross-inhibition of these kinases. VRX0466617 did not modify the cell cycle phase distribution, although it caused an increase in multinucleated cells. Whereas VRX0466617 attenuated IR-induced apoptosis, in short-term assays it did not affect the cytotoxicity by the anticancer drugs doxorubicin, Taxol, and cisplatin. These results underscore the specificity of VRX0466617 for Chk2, both in vitro and in vivo, and support the use of this compound as a biological probe to study the Chk2-dependent pathways.

DNA damage-induced cell cycle regulation and function of novel Chk2 phosphoresidues.

Chk2 kinase is activated by DNA damage to regulate cell cycle arrest, DNA repair, and apoptosis. Phosphorylation of Chk2 in vivo by ataxia telangiectasia-mutated (ATM) on threonine 68 (T68) initiates a phosphorylation cascade that promotes the full activity of Chk2. We identified three serine residues (S19, S33, and S35) on Chk2 that became phosphorylated in vivo rapidly and exclusively in response to ionizing radiation (IR)-induced DNA double-strand breaks in an ATM- and Nbs1-dependent but ataxia telangiectasia- and Rad3-related-independent manner. Phosphorylation of these residues, restricted to the G(1) phase of the cell cycle, was induced by a higher dose of IR (>1 Gy) than that required for phosphorylation of T68 (0.25 Gy) and declined by 45 to 90 min, concomitant with a rise in Chk2 autophosphorylation. Compared to the wild-type form, Chk2 with alanine substitutions at S19, S33, and S35 (Chk2(S3A)) showed impaired dimerization, defective auto- and trans-phosphorylation activities, and reduced ability to promote degradation of Hdmx, a phosphorylation target of Chk2 and regulator of p53 activity. Besides, Chk2(S3A) failed to inhibit cell growth and, in response to IR, to arrest G(1)/S progression. These findings underscore the critical roles of S19, S33, and S35 and argue that these phosphoresidues may serve to fine-tune the ATM-dependent response of Chk2 to increasing amounts of DNA damage.

MRE11 mutations and impaired ATM-dependent responses in an Italian family with ataxia-telangiectasia-like disorder.

Hypomorphic mutations of the MRE11 gene are the hallmark of the radiosensitive ataxia-telangiectasia-like disorder (ATLD). Here, we describe a new family with two affected siblings, ATLD5 and ATLD6, now aged 37 and 36, respectively. They presented with late onset cerebellar degeneration slowly progressing until puberty and absence of telangiectasias, and were cancer-free. Both patients were wild-type for ATM and NBS1, but compound heterozygotes for MRE11 gene mutations [1422C-->A, T481K; 1714C-->T, R571X]. The 1422C-->A allele was inherited from the mother, whereas the 1714C-->T, allele paternally inherited, was apparently null as a result of nonsense-mediated mRNA decay (NMD). Interestingly, the 1714C-->T mutation is the same as previously identified in an unrelated English ATLD family (probands ATLD3 and ATLD4), suggesting an important role for NMD in saving potentially lethal mutations. Lymphoblastoid cell lines (LCLs) derived from ATLD5 and ATLD6 were normal for ATM, but defective for Mre11, Rad50 and Nbs1 (the MRN complex) protein expression. Their response to gamma-radiation was abnormal, as evidenced by the enhanced radiosensitivity, attenuated autophosphorylation of ATM-S1981 and phosphorylation of the ATM targets p53-S15 and Smc1-S966, failure to form Mre11 nuclear foci and defective G1 checkpoint arrest. The fibroblasts, but not LCLs, from ATLD5 and ATLD6 showed an impaired ATM-dependent Chk2 phosphorylation. These findings further underscore the interconnection between ATM activity and MRN function, which rationalizes the clinical similarity between ataxia-telangiectasia (A-T) and ATLD.