Synthetic Lethality Triggered by Combining Olaparib with BRCA2-Rad51 Disruptors.

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.

Recombinant Adeno Associated Viral (AAV) vector type 9 delivery of Ex1-Q138-mutant huntingtin in the rat striatum as a short-time model for in vivo studies in drug discovery.

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.

A phenotypic screening assay for modulators of huntingtin-induced transcriptional dysregulation.

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.

VSTM2L is a novel secreted antagonist of the neuroprotective peptide Humanin.

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.

Immunolocalization of humanin in human sperm and testis.

We have discovered, by immunocytochemistry and immunoelectronmicroscopy, that humanin (HN) is expressed in human ejaculated sperm and testis. In sperm, the HN immunolabeling pattern depends on sperm morphology; in particular, HN is mainly localized in the midpiece of sperm in semen samples with normal morphology and in cytoplasmic residues and entire tail in those with abnormal morphology. We also found HN in the cytoplasm and nucleus of spermatocytes and spermatids and in experimentally uncoiled chromatin of mature ejaculated sperm. Because it has been established that HN has antiapoptotic properties, it is reasonably hypothesized that HN may play an important role in preventing apoptosis in human sperm and testis. Thus, the examination of the HN localization in normal and abnormal sperm could be proposed as an auxiliary test to better define sperm quality.

Humanin structural versatility and interaction with model cerebral cortex membranes.

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.