Cristiana Mollinari

Absence of caspase 8 and high expression of PED protect primitive neural cells from cell death

The mechanisms that control neural stem and progenitor cell survival are unknown. In several pathological conditions, death receptor (DR) ligands and inflammatory cytokines exert a deleterious effect on neurons, whereas primitive neural cells migrate and survive in the site of lesion. Here, we show that even in the presence of inflammatory cytokines, DRs are unable to generate death signals in primitive neural cells. Neural stem and progenitor cells did not express caspase 8, the presence of which is required for initiating the caspase cascade. However, exogenous or cytokine-mediated expression of caspase 8 was not sufficient to restore their DR sensitivity. Searching for molecules potentially able to block DR death-inducing signaling complex (DISC), we found that primitive neural cells expressed high levels of the death effector domain-containing protein PED (also known as PEA-15). PED localized in the DISC and prevented caspase 8 recruitment and activation. Moreover, lentiviral-mediated delivery of PED antisense DNA resulted in dramatic down-regulation of the endogenous gene expression and sensitization of primitive neural cells to apoptosis mediated by inflammatory cytokines and DRs. Thus, absence of caspase 8 and high expression of PED constitute two levels of protection from apoptosis induced by DRs and inflammatory cytokines in neural stem and progenitor cells.

Reduced GABAB receptor subunit expression and paired-pulse depression in a genetic model of absence seizures

Neocortical networks play a major role in the genesis of generalized spike-and-wave (SW) discharges associated with absence seizures in humans and in animal models, including genetically predisposed WAG/Rij rats. Here, we tested the hypothesis that alterations in GABA(B) receptors contribute to neocortical hyperexcitability in these animals. By using Real-Time PCR we found that mRNA levels for most GABA(B(1)) subunits are diminished in epileptic WAG/Rij neocortex as compared with age-matched non-epileptic controls (NEC), whereas GABA(B(2)) mRNA is unchanged. Next, we investigated the cellular distribution of GABA(B(1)) and GABA(B(2)) subunits by confocal microscopy and discovered that GABA(B(1)) subunits fail to localize in the distal dendrites of WAG/Rij neocortical pyramidal cells. Intracellular recordings from neocortical cells in an in vitro slice preparation demonstrated reduced paired-pulse depression of pharmacologically isolated excitatory and inhibitory responses in epileptic WAG/Rij rats as compared with NECs; moreover, paired-pulse depression in NEC slices was diminished by a GABA(B) receptor antagonist to a greater extent than in WAG/Rij rats further suggesting GABA(B) receptor dysfunction. In conclusion, our data identify changes in GABA(B) receptor subunit expression and distribution along with decreased paired-pulse depression in epileptic WAG/Rij rat neocortex. We propose that these alterations may contribute to neocortical hyperexcitability and thus to SW generation in absence epilepsy.

Neuroprotective Effects of Thymosin β4 in Experimental Models of Excitotoxicity

Abstract:  The aim of this study was to evaluate the possible neuroprotective effects of thymosin β4 in different models of excitotoxicity. The application of thymosin β4 significantly attenuated glutamate‐induced toxicity both in primary cultures of cortical neurons and in rat hippocampal slices. In in vivo experiments, the intracerebroventricular administration of thymosin β4 significantly reduced hippocampal neuronal loss induced by kainic acid. These results show that thymosin β4 induced a protective effect in models of excitotoxicity. The mechanisms underlying such an effect, as well as the real neuroprotective potential of thymosin β4, are worthy of further investigations.

Thymosin β4 targeting impairs tumorigenic activity of colon cancer stem cells

Thymosin β4(Tβ4) is an actin‐binding peptide overexpressed in several tumors, including colon carcinomas. The aim of this study was to investigate the role of Tβ4 in promoting the tumorigenic properties of colorectal cancer stem cells (CR‐CSCs), which are responsible for tumor initiation and growth. We first found that CR‐CSCs from different patients have higher Tβ4 levels than normal epithelial cells. Then, we used a lentiviral strategy to down‐regulate Tβ4 expression in CR‐CSCs and analyzed the effects of such modulation on proliferation, survival, and tumorigenic activity of CR‐CSCs. Empty vector‐transduced CR‐CSCs were used as a control. Targeting of the Tβ4 produced CR‐CSCs with a lower capacity to grow and migrate in culture and, interestingly, reduced tumor size and aggressiveness of CR‐CSC‐based xenografts in mice. Moreover, such loss in tumorigenic activity was accompanied by a significant increase of phosphatase and tensin homologue (PTEN) and a concomitant reduction of the integrin‐linked kinase (ILK) expression, which resulted in a decreased activation of protein kinase B (Akt). Accordingly, exogenous expression of an active form of Akt rescued all the protumoral features lost after Tβ4 targeting in CR‐CSCs. In conclusion, Tβ4 may have important implications for therapeutic intervention for treatment of human colon carcinoma.—Ricci‐Vitiani, L., Mollinari, C., di Martino, S., Biffoni, M., Pilozzi, E., Pagliuca, A., Chiara de Stefano, M., Circo, R., Merlo, D., De Maria, R., Garaci, E. Thymosin β4 targeting impairs tumorigenic activity of colon cancer stem cells. FASEB J. 24, 4291–4301 (2010). www.fasebj.org

GABAA receptors present higher affinity and modified subunit composition in spinal motor neurons from a genetic model of amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis is a neurodegenerative disease characterized by the selective degeneration of motor neurons in the spinal cord, brainstem and cerebral cortex. In this study we have analysed the electrophysiological properties of GABAA receptors and GABAA alpha1 and alpha2 subunits expression in spinal motor neurons in culture obtained from a genetic model of ALS (G93A) and compared with transgenic wild type SOD1 (SOD1) and their corresponding non transgenic litter mates (Control). Although excitotoxic motor neuron death has been extensively studied in relation to Ca2+‐dependent processes, strong evidence indicates that excitotoxic cell death is also remarkably dependent on Cl− ions and on GABAA receptor activation. In this study we have analysed the electrophysiological properties of GABAA receptors and the expression of GABAAα1 and α2 subunits in cultured motor neurons obtained from a genetic model of amyotrophic lateral sclerosis (G93A) and compared them with transgenic wild‐type Cu,Zn superoxide dismutase and their corresponding non‐transgenic littermates (Control). In all tested motor neurons, the application of γ‐aminobutyric acid (GABA) (0.5–100 μm) evoked an inward current that was reversibly blocked by bicuculline (100 μm), thus indicating that it was mediated by the activation of GABAA receptors. Our results indicate that the current density at high GABA concentrations is similar in control, Cu,Zn superoxide dismutase and G93A motor neurons. However, the dose‐response curve significantly shifted toward lower concentration values in G93A motor neurons and the extent of desensitization also increased in these neurons. Finally, multiplex single‐cell real‐time polymerase chain reaction and immunofluorescence revealed that the amount of GABAAα1 subunit was significantly increased in G93A motor neurons, whereas the levels of α2 subunit were unchanged. These data show that the functionality and expression of GABAA receptors are altered in G93A motor neurons inducing a higher Cl− influx into the cell with a possible consequent neuronal excitotoxicity acceleration.

Sublethal Doses of β-Amyloid Peptide Abrogate DNA-dependent Protein Kinase Activity

Background: Accumulation of DNA damage and deficiency in DNA repair may contribute to neuronal loss in Alzheimer disease. Results: Sublethal concentrations of aggregated β-amyloid peptides inhibit DNA-PK kinase activity in PC12 cells. Conclusion: DNA-PK inhibition may contribute to neurodegeneration by impairing DNA repair capability, inducing DNA damage accumulation. Significance: This represents a novel mechanism by which Aβ exerts its neurotoxic effects in Alzheimer disease. Accumulation of DNA damage and deficiency in DNA repair potentially contribute to the progressive neuronal loss in neurodegenerative disorders, including Alzheimer disease (AD). In multicellular eukaryotes, double strand breaks (DSBs), the most lethal form of DNA damage, are mainly repaired by the nonhomologous end joining pathway, which relies on DNA-PK complex activity. Both the presence of DSBs and a decreased end joining activity have been reported in AD brains, but the molecular player causing DNA repair dysfunction is still undetermined. β-Amyloid (Aβ), a potential proximate effector of neurotoxicity in AD, might exert cytotoxic effects by reactive oxygen species generation and oxidative stress induction, which may then cause DNA damage. Here, we show that in PC12 cells sublethal concentrations of aggregated Aβ(25–35) inhibit DNA-PK kinase activity, compromising DSB repair and sensitizing cells to nonlethal oxidative injury. The inhibition of DNA-PK activity is associated with down-regulation of the catalytic subunit DNA-PK (DNA-PKcs) protein levels, caused by oxidative stress and reversed by antioxidant treatment. Moreover, we show that sublethal doses of Aβ(1–42) oligomers enter the nucleus of PC12 cells, accumulate as insoluble oligomeric species, and reduce DNA-PK kinase activity, although in the absence of oxidative stress. Overall, these findings suggest that Aβ mediates inhibition of the DNA-PK-dependent nonhomologous end joining pathway contributing to the accumulation of DSBs that, if not efficiently repaired, may lead to the neuronal loss observed in AD.

Downregulation of thymosin β4 in neural progenitor grafts promotes spinal cord regeneration

Thymosin β4 (Tβ4) is an actin-binding peptide whose expression in developing brain correlates with migration and neurite extension of neurons. Here, we studied the effects of the downregulation of Tβ4 expression on growth and differentiation of murine neural progenitor cells (NPCs), using an antisense lentiviral vector. In differentiation-promoting medium, we found twice the number of neurons derived from the Tβ4-antisense-transduced NPCs, which showed enhanced neurite outgrowth accompanied by increased expression of the adhesion complex N-cadherin–β-catenin and increased ERK activation. Importantly, when the Tβ4-antisense-transduced NPCs were transplanted in vivo into a mouse model of spinal cord injury, they promoted a significantly greater functional recovery. Locomotory recovery correlated with increased expression of the regeneration-promoting cell adhesion molecule L1 by the grafted Tβ4-antisense-transduced NPCs. This resulted in an increased number of regenerating axons and in sprouting of serotonergic fibers surrounding and contacting the Tβ4-antisense-transduced NPCs grafted into the lesion site. In conclusion, our data identify a new role for Tβ4 in neuronal differentiation of NPCs by regulating fate determination and process outgrowth. Moreover, NPCs with reduced Tβ4 levels generate an L1-enriched environment in the lesioned spinal cord that favors growth and sprouting of spared host axons and enhances the endogenous tissue-repair processes.

DNA repair in post-mitotic neurons: a gene-trapping strategy

DNA repair is essential for maintaining the integrity of the genome. Although mutation often occurs during meiosis and mitosis, the DNA of post-mitotic neurons is also under risk of damage, for example, from free radicals. For humans, given that the lifespan of post-mitotic neurons is many decades, competent DNA repair could be a critical factor in slowing ageing and the progression of some pathologies. Double-strand breaks (DSBs) are considered the most lethal form of DNA damage which, if left unrepaired, can cause cell death. Mammalian cells repair DSBs by two pathways: homologous recombination (HR) and non-homologous end-joining (NHEJ). Although in vitro assays support the idea that the mature brain can repair DNA, repair of DSBs has not been directly demonstrated in living post-mitotic neurons. The aim of this study was to test directly the possibility of DSB repair in post-mitotic neurons by using a promoter-less gene-trapping (GT) vector. The promoter-less GT vector had a 6 kb GABAA receptor a6 subunit gene fragment containing part of exon 4 through to the middle of intron 8. The GT vector carried a splice acceptor site (SA), followed by stop codons in all three reading frames and an internal ribosome entry site (IRES) element linked to either b-galactosidase (lacZ) or green fluorescent protein (GFP). The IRES-lacZ or IRES-EGFP was inserted into exon 8 downstream of the SA of the a6 gene. Following a strand break in chromosomal DNA, the linear gene trap vector can be ligated into the gap. If this happens in the correct orientation and the gene is expressed in neurons, the result is a trapped gene which expresses GFP or b-galactosidase. By using the biolistic in-chamber technique, we transfected the GT vector, containing either GFP or lacZ, into cerebellar granule cell cultures and, after 48 h, we scored cells expressing the reporter gene. The expression of the marker present in the promoter-less GT vector would require the integration and re-ligation of its free ends into an actively transcribed genomic region. As expected, the frequency of integration was low. For the GFP reporter, just seven recombinant cells were observed on 29 coverslips (frequency of 7 10 7 – Figure 1b), whereas for lacZ, there were five cells in 27 coverslips (a frequency of 5 10 ; Figure 1d). On the contrary, transfection with both CMV-EGFP and RSVlacZ-positive control plasmids resulted in a large number of GFPand b-galactosidase-expressing cells (Figure 1a and c). We then looked if DNA ligation can happen in a wider range of neurons by transfecting organotypic cerebellar slices. Transfected organotypic cerebellar slices with the CMVEGFP-positive control plasmid produced GFP expression in numerous cells (Figure 1h). With the promoter-less gene trap constructs, some cells of various morphologies in the slices also expressed the reporter genes after transfection (Figure 1i). However, the frequency was low: in 35 transfected slices, there were only seven GFP-positive cells. Relative to the observed expression of the CMV-EGFP construct, this represents a relative frequency of 2.6 10 . The incorporation of the gene trap vector into the neuronal genome depends on the vector having free ends. In fact, for a number of transfections comparable to those with linearized plasmid (n1⁄4 30), cultures transfected with supercoiled (uncut) gene trap vectors had no positive cells (data not shown). If post-mitotic neurons have the ability to repair DNA, this repair activity should be enhanced following the induction of DSBs and so the frequency of GT events should increase. By switching the culture medium to minimal essential medium containing 5 mM Kþ /serum-free (K5/S ) and no insulin, cerebellar granule cells start apoptosis and undergo DNA fragmentation. To investigate the effect of induced DNA damage on GT frequency, cerebellar granule cells were switched to K5/S for 4 h and then transfected. The K5/S medium was then replaced with the previously saved cultureconditioned medium and the dishes were returned to the incubator for 48 h, when they were fixed. Cells remained viable and survived the crisis (Figure 1f and g). We found a strong increase in DNA recombination frequency, which increased from virtually undetectable to 4.7570.95 neurons/field (microscopic field of 800 500mm) with GFP construct (Figure 1e). The vector used in the current study could become trapped by enzymatic activities in the process of repairing DSBs arising from environmental DNA damage as an attempt to reverse it. To investigate if the increment in GT frequency observed by inducing DNA damage correlates with DNA repair machinery activation, we analysed changes in expression of genes involved in DSBs repair mechanism by real-time PCR, 4 and 8 h after apoptotic stimulus. After giving granule cells apoptotic stimulus, we first analysed RNA levels of the histone proteins (H1, H2A, H2B, H3 and H4) since chromatin modification has been documented during DSB repair as an early step in the cellular response, and they also indicate the occurrence of DNA damage. We indeed found, 4 h after the apoptotic stimulus, two-fold increase in H3, an approx. five-fold increase in H1 and H2A, 11-fold increase in H2B and 15-fold increase in H4 Cell Death and Differentiation (2005) 12, 307–309 & 2005 Nature Publishing Group All rights reserved 1350-9047/05

MiR-34a regulates cell proliferation, morphology and function of newborn neurons resulting in improved behavioural outcomes

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DNA Double Strand Breaks: A Common Theme in Neurodegenerative Diseases

miR-34a is involved in the regulation of the fate of different cell types. However, the mechanism by which it controls the differentiation programme of neural cells remains largely unknown. Here, we investigated the role of miR-34a in neurogenesis and maturation of developing neurons and identified Doublecortin as a new miR-34a target. We found that the overexpression of miR-34a in vitro significantly increases precursor proliferation and influences morphology and function of developing neurons. Indeed, miR-34a overexpressing neurons showed a decreased expression of several synaptic proteins and receptor subunits, a decrement of NMDA-evoked current density and, interestingly, a more efficient response to synaptic stimulus. In vivo, miR-34a overexpression showed stage-specific effects. In neural progenitors, miR-34a overexpression promoted cell proliferation, in migratory neuroblasts reduced the migration and in differentiating newborn neurons modulated process outgrowth and complexity. Importantly, we found that rats overexpressing miR-34a in the brain have better learning abilities and reduced emotionality.