In BRCA2-defective cells, poly(adenosine diphosphate [ADP]-ribose) polymerase inhibitors can trigger synthetic lethality, as two independent DNA-repairing mechanisms are simultaneously impaired. Here, we have pharmacologically induced synthetic lethality, which was triggered by combining two different small organic molecules. When administered with a BRCA2-Rad51 disruptor in nonmutant cells, Olaparib showed anticancer activity comparable to that shown when administered alone in BRCA2-defective cells. This strategy could represent an innovative approach to anticancer drug discovery and could be extended to other synthetic lethality pathways.

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Humanin (HN) is a 24-residue peptide displaying a protective activity in vitro against a range of cytotoxic and neurotoxic insults, as well as mediating in vivo amelioration of Alzheimer disease (AD)-related memory impairment in experimental models. Published evidence suggests that the mechanisms through which HN exerts its cyto- and neuroprotective activity may include its secretion and binding to membrane-associated receptors. Here, we describe the identification of a new modulator of HN neuroprotective activity, V-set and transmembrane domain containing 2 like (VSTM2L), previously known as C20orf102. VSTM2L interacts with HN in both yeast and mammalian cells, is secreted in cultured cells, is present in serum, and is selectively expressed in the central nervous system. VSTM2L colocalizes with HN in distinct brain areas as well as in primary cultured neurons, where it plays a role in the modulation of neuronal viability. When tested in HN neuroprotection bioassays, VSTM2L acts as a strong antagonist of HN neuroprotective activity. In summary, VSTM2L is the first example of a secreted antagonist of HN and may play a role in the modulation of HN biological functions.

Humanin (HN) is a recently identified neuroprotective peptide able to inhibit neurotoxicity induced by various insults which can be related to Alzheimer disease (AD) as well as to cell death induced by other stimuli. Previous CD and NMR studies demonstrated that HN adopts an unordered conformation in water, a alpha-helix conformation in 30% TFE, and a beta-sheet structure in PBS. Furthermore, other studies clearly indicated HN as a secreted peptide, able to prevent neuronal cell death caused by amyloid beta (Abeta) derivatives. Although Abeta was found to interact with neuronal membranes, currently there is not experimental evidence unveiling HN interaction with membranes. In this paper a spin labeling technique coupled with electron paramagnetic resonance (EPR) and circular dichroism (CD) has been used to study the structure and dynamics of HN in solution and for the first time in the presence of model cerebral cortex membranes (CCM). We have demonstrated that HN has a great tendency to aggregate even at low concentrations in water solutions at different ionic strengths and monomerizes in the TFE apolar environment. We also showed that HN slightly perturbs model CCM at the surface assuming a clear beta-sheet conformation. In addition, HN increases the fluidity of the bilayer core without penetrating into the membrane.

Huntington's Disease is a rare neurodegenerative disease caused by an abnormal expansion of CAG repeats encoding polyglutamine in the first exon of the huntingtin gene. N-terminal fragments containing polyglutamine (polyQ) sequences aggregate and can bind to cellular proteins, resulting in several pathophysiological consequences for affected neurons such as changes in gene transcription. One transcriptional pathway that has been implicated in HD pathogenesis is the CREB binding protein (CBP)/cAMP responsive element binding (CREB) pathway. We developed a phenotypic assay to screen for compounds that can reverse the transcriptional dysregulation of the pathway caused by induced mutated huntingtin protein (µHtt). 293/T-REx cells were stably co-transfected with an inducible full-length mutated huntingtin gene containing 138 glutamine repeats and with a reporter gene under control of the cAMP responsive element (CRE). One clone, which showed reversible inhibition of µHtt-induced reporter activity upon treatment with the neuroprotective Rho kinase inhibitor Y27632, was used for the development of a high-throughput phenotypic assay suitable for a primary screening campaign, which was performed on a library of 24,000 compounds. Several hit compounds were identified and validated further in a cell viability adenosine triphosphate assay. The assay has the potential for finding new drug candidates for the treatment of HD.

Huntington's disease (HD) is an inherited neurodegenerative disorder characterized by dyskinesia, cognitive impairment and emotional disturbances, presenting progressive neurodegeneration in the striatum and intracellular mutant Huntingtin (mHTT) aggregates in various areas of the brain. Recombinant Adeno Associated Viral (rAAV) vectors have been successfully used to transfer foreign genes to the brain of adult animals. In the present study we report a novel in vivo rat HD model obtained by stereotaxic injection of rAAV serotype2/9 containing Exon1-Q138 mHTT (Q138) and Exon1-Q17 wild type HTT (Q17; control), respectively in the right and in the left striatum, and expressed as C-terminal GFP fusions to facilitate detection of infected cells and aggregate production. Immunohistochemical analysis of brain slices from animals sacrificed twenty-one days after viral infection showed that Q138 injection resulted in robust formation of GFP-positive aggregates in the striatum, increased GFAP and microglial activation and neurodegeneration, with little evidence of any of these events in contralateral tissue infected with wild type (Q17) expressing construct. Differences in the relative metabolite concentrations (N-Acetyl Aspartate/Creatine and Myo-Inositol/Creatine) were observed by H1 MR Spectroscopy. By quantitative RT-PCR we also demonstrated that mHTT induced changes in the expression of genes previously shown to be altered in other rodent HD models. Importantly, administration of reference compounds previously shown to ameliorate the aggregation and neurodegeneration phenotypes in preclinical HD models was demonstrated to revert the mutant HTT-dependent effects in our model. In conclusion, the AAV2/9-Q138/Q17 exon 1 HTT stereotaxic injection represents a useful first-line in vivo preclinical model for studying the biology of mutant HTT exon 1 in the striatum and to provide early evidence of efficacy of therapeutic approaches.

Humanin (HN) is a 24-amino acid peptide showing potent neuroprotective properties against damage associated with Alzheimer's disease (1). In neuronal cells, HN acts by inhibiting apoptosis via multiple mechanisms: it can suppress mitochondrially-mediated apoptosis by interfering with Bax activation (2), additionally it can bind to the Insulin Growth Factor-Binding Protein 3 (IGFBP-3) antagonizing its apoptotic actions (3). Ying et al. (4) demonstrated that HN inhibited beta-amyloid (Aβ) 42-induced neurotoxicity by binding to G-protein-coupled receptor FPRL-1. Moreover, neuronal protection by HN involves activation of tyrosine kinases, STAT-3 phosphorylation (5) and the binding to a complex or complexes involving CNTFR/WSX-1/gp180 (6). The homologues of HN in the rat is called Rattin (HNr) (7) and share, like HN, neuroprotective properties. HN and HNr have a broader spectrum of protective activity also in non-neuronal cells. Regarding the male reproductive system, studies reported the presence of HN both in human and rat, proposing that this peptide may play a role as a testicular anti-apoptotic factor (8-10). This study aimed to investigate the presence and to localize HN, mainly by immunocytochemistry (ICC), directly in human, mouse and rabbit sperm. MATERIAL AND METHODS Immunocytochemistry (ICC)Human semen samples were evaluated following World Health Organization guidelines (11) and processed for the ICC analyses. Ejaculated spermatozoa, washed in phosphate-buffered saline (PBS), were smeared on glass slides, air dried, rinsed in PBS and saturated for 20 min with PBS-bovine serum albumin (BSA) 1% containing 5% normal goat serum (NGS). The specimens were then incubated overnight at 4°C using two different rabbit polyclonal anti-HN antibodies (Sigma-Aldrich, USA and a self made antibody P04, kindly gift of Prof. Matzuoka) both diluted 1:50 in PBS/0.1% BSA/1% NGS. For the detection a goat antirabbit FITC conjugate antibody (Sigma-Aldrich), diluted 1:300, was used. The same ICC analysis was carried out also in sperm from rabbit and mouse. The fluorescence was observed with a Leitz Aristoplan light microscope equipped with fluorescence apparatus. Incubation in primary antibodies was omitted in control samples. Test for antibody specificityPreadsorption: An aliquot of rabbit polyclonal anti-HN antibody (Sigma-Aldrich) was pre-incubated 64

Humanin (HN) is a peptide showing neuroprotective properties against damage associated with Alzheimer’s disease. In male reproductive system HN is expressed in human and rat testis. This preliminary study aimed to localize HN by immunocytochemistry (ICC) directly in human, mouse and rabbit sperm. Semen samples were processed for the ICC analyses using two different rabbit polyclonal anti-HN antibodies. Western blotting analyses were performed using fresh human semen samples. Normal human, rabbit and mouse sperm, showed HN labeling in sub-acrosomal and mid-piece regions, whereas human abnormally shaped sperm were stained also at acrosomal level and particularly in the flagellum. Western blotting analysis revealed the presence of HN in analyzed human semen samples. HN was localized directly on human, rabbit and mouse spermatozoa. In human samples, we demonstrated a different localization pattern in normally shaped sperm compared to abnormal sperm probably due to a protective effect of HN in pathological sperm