Alessio Cardinale

Oxidative Stress Induces p53-Mediated Apoptosis in Glia: p53 Transcription-Independent Way to Die

Oxidative stress has been implicated in the pathogenesis of stroke, traumatic brain injuries, and neurodegenerative diseases affecting both neuronal and glial cells in the central nervous system (CNS). The tumor suppressor protein p53 plays a pivotal function in neuronal apoptosis triggered by oxidative stress. We investigated the role of p53 and related molecular mechanisms that support oxidative stress‐induced apoptosis in glia. For this purpose, we exposed C6 glioma cells and primary cultures of rat cortical astrocytes to an H2O2‐induced oxidative stress protocol followed by a recovery period. We evaluated the effects of pifithrin‐α (PF‐α), which has been reported to protect neurons from ischemic insult by specifically inhibiting p53 DNA‐binding activity. Strikingly, PF‐α was unable to prevent oxidative stress‐induced astrocyte apoptosis. We demonstrate that p53 is able to mediate an apoptotic response by direct signaling at mitochondria, despite its transcriptional activity. The z‐VAD‐fmk‐sensitive apoptotic response requires a caspase‐dependent MDM‐2 degradation, leading to p53 mitochondrial targeting accompanied by cytochrome c release and nucleosomal fragmentation.

The potential of intracellular antibodies for therapeutic targeting of protein-misfolding diseases.

Misfolding diseases are a wide group of devastating disorders characterized by the accumulation of pathological protein aggregates. Although these disorders still lack an effective treatment, new antibody-based strategies are emerging and entering clinical trials. The intrabody approach is a gene-based technology developed to neutralize or modify the function of intracellular and extracellular target antigens. Because intrabodies can potentially target all the different isoforms of a misfolding-prone protein, including pathological conformations, they are emerging as therapeutic molecules for the treatment of misfolding diseases, including Alzheimers, Parkinsons, Huntingtons and prion diseases. This review will provide a description of the intrabody approach, an update of preclinical studies on misfolding diseases and an outlook on the intrabody delivery issue for therapeutic purposes.

The mode of action of Y13-259 scFv fragment intracellularly expressed in mammalian cells

The anti‐p21ras Y13‐259 single‐chain Fv fragment (scFv) neutralizes the activity of p21‐ras when intracellularly expressed in different systems. We have studied the mode of action of this inhibition in 3T3 K‐ras fibroblasts and demonstrated that (i) this antibody fragment is highly aggregating when cytoplasmically expressed and (ii) the p21‐ras antigen is sequestered in these aggregates in an antibody‐dependent manner. This co‐segregation leads to an efficient inhibition of DNA synthesis. These results suggest that an antigen can be diverted from its normal location inside the cells in an antibody mediated way, prospecting a new mode of action for intracellular antibodies in vivo.

Selective re-routing of prion protein to proteasomes and alteration of its vesicular secretion prevent PrPSc formation

Conversion of the cellular prion protein (PrPC) into the abnormal scrapie isoform (PrPSc) is the hallmark of prion diseases, which are fatal and transmissible neurodegenerative disorders. ER‐retained anti‐prion recombinant single‐chain Fv fragments have been proved to be an effective tool for inhibition of PrPC trafficking to the cell surface and antagonize PrPSc formation and infectivity. In the present study, we have generated the secreted version of 8H4 intrabody (Sec‐8H4) in order to compel PrPC outside the cells. The stable expression of the Sec‐8H4 intrabodies induces proteasome degradation of endogenous prion protein but does not influence its glycosylation profile and maturation. Moreover, we found a dramatic diverting of PrPC traffic from its vesicular secretion and, most importantly, a total inhibition of PrPSc accumulation in NGF‐differentiated Sec‐8H4 PC12 cells. These results confirm that perturbing the intracellular traffic of endogenous PrPC is an effective strategy to inhibit PrPSc accumulation and provide convincing evidences for application of intracellular antibodies in prion diseases.

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.

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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Combating protein misfolding and aggregation by intracellular antibodies.

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.

The Response to Oxidative DNA Damage in Neurons: Mechanisms and Disease

Conformational or misfolding diseases are a large class of devastating human disorders associated with protein misfolding and aggregation. Most conformational diseases are caused by a combination of genetic and environmental factors, suggesting that spontaneous events can destabilize the protein involved in the pathology or impair the clearance mechanisms of misfolded aggregates. Aging is one of the risk factors associa-ted to these events, and the clinical relevance of conformational disorders is growing dramatically, as they begin to reach epidemic proportions due to increases in mean lifespan. Currently, there are no effective strategies to slow or prevent these diseases. Intrabodies are promising therapeutic agents for the treatment of misfolding diseases, because of their virtually infinite ability to specifically recognize the different conformations of a protein, including pathological isoforms, and because they can be targeted to the potential sites of aggregation (both intra- and extracellular sites). These molecules can work as neutralizing agents against amylo-idogenic proteins by preventing their aggregation, and/or as molecular shunters of intracellular traffic by re-routing the protein from its potential aggregation site. The fast-developing field of recombinant antibody technology provides intrabodies with enhanced binding specificity and stability, together with lower immunogenicity, for use in a clinical setting. This review provides an update on the applications of intrabodies in misfolding diseases, with particular emphasis on an evaluation of their multiple and feasible modes of action.

DNA Double Strand Breaks: A Common Theme in Neurodegenerative Diseases

There is a growing body of evidence indicating that the mechanisms that control genome stability are of key importance in the development and function of the nervous system. The major threat for neurons is oxidative DNA damage, which is repaired by the base excision repair (BER) pathway. Functional mutations of enzymes that are involved in the processing of single-strand breaks (SSB) that are generated during BER have been causally associated with syndromes that present important neurological alterations and cognitive decline. In this review, the plasticity of BER during neurogenesis and the importance of an efficient BER for correct brain function will be specifically addressed paying particular attention to the brain region and neuron-selectivity in SSB repair-associated neurological syndromes and age-related neurodegenerative diseases.