PolyQ repeat expansions in ATXN2 associated with ALS are CAA interrupted repeats

Amyotrophic lateral sclerosis (ALS) is a devastating, rapidly progressive disease leading to paralysis and death. Recently, intermediate length polyglutamine (polyQ) repeats of 27-33 in ATAXIN-2 (ATXN2), encoding the ATXN2 protein, were found to increase risk for ALS. In ATXN2, polyQ expansions of ≥ 34, which are pure CAG repeat expansions, cause spinocerebellar ataxia type 2. However, similar length expansions that are interrupted with other codons, can present atypically with parkinsonism, suggesting that configuration of the repeat sequence plays an important role in disease manifestation in ATXN2 polyQ expansion diseases. Here we determined whether the expansions in ATXN2 associated with ALS were pure or interrupted CAG repeats, and defined single nucleotide polymorphisms (SNPs) rs695871 and rs695872 in exon 1 of the gene, to assess haplotype association. We found that the expanded repeat alleles of 40 ALS patients and 9 long-repeat length controls were all interrupted, bearing 1-3 CAA codons within the CAG repeat. 21/21 expanded ALS chromosomes with 3CAA interruptions arose from one haplotype (GT), while 18/19 expanded ALS chromosomes with <3CAA interruptions arose from a different haplotype (CC). Moreover, age of disease onset was significantly earlier in patients bearing 3 interruptions vs fewer, and was distinct between haplotypes. These results indicate that CAG repeat expansions in ATXN2 associated with ALS are uniformly interrupted repeats and that the nature of the repeat sequence and haplotype, as well as length of polyQ repeat, may play a role in the neurological effect conferred by expansions in ATXN2.     

Drosophila DJ-1 mutants are selectively sensitive to environmental toxins associated with Parkinson's disease

Parkinson's disease (PD) is a common neurodegenerative disorder that displays both sporadic and inherited forms. Exposure to several common environmental toxins acting through oxidative stress has been shown to be associated with PD. One recently identified inherited PD gene, DJ-1, may have a role in protection from oxidative stress, thus potentially linking a genetic cause with critical environmental risk factors. To develop an animal model that would allow integrative study of genetic and environmental influences, we have generated Drosophila lacking DJ-1 function. Fly DJ-1 homologs exhibit differential expression: DJ-1beta is ubiquitous, while DJ-1alpha is predominantly expressed in the male germline. DJ-1alpha and DJ-1beta double knockout flies are viable, fertile, and have a normal lifespan; however, they display a striking selective sensitivity to those environmental agents, including paraquat and rotenone, linked to PD in humans. This sensitivity results primarily from loss of DJ-1beta protein, which also becomes modified upon oxidative stress. These studies demonstrate that fly DJ-1 activity is selectively involved in protection from environmental oxidative insult in vivo and that the DJ-1beta protein is biochemically responsive to oxidative stress. Study of these flies will provide insight into the critical interplay of genetics and environment in PD.     

Nitrated lipids decompose to nitric oxide and lipid radicals and cause vasorelaxation

Nitric oxide-derived oxidants such as nitrogen dioxide and peroxynitrite have been receiving increasing attention as mediators of nitric oxide toxicity. Indeed, nitrated and nitrosated compounds have been detected in biological fluids and tissues of healthy subjects and in higher yields in patients under inflammatory or infectious conditions as a consequence of nitric oxide overproduction. Among them, nitrated lipids have been detected in vivo. Here, we confirmed and extended previous studies by demonstrating that nitrolinoleate, chlolesteryl nitrolinoleate, and nitrohydroxylinoleate induce vasorelaxation in a concentration-dependent manner while releasing nitric oxide that was characterized by chemiluminescence-and EPR-based methodologies. As we first show here, diffusible nitric oxide production is likely to occur by isomerization of the nitrated lipids to the corresponding nitrite derivatives that decay through homolysis and/or metal ion/ascorbate-assisted reduction. The homolytic mechanism was supported by EPR spin-trapping studies with 3,5-dibromo-4-nitrosobenzenesulfonic acid that trapped a lipid-derived radical during nitrolinoleate decomposition. In addition to provide a mechanism to explain nitric oxide production from nitrated lipids, the results support their role as endogenous sources of nitric oxide that may play a role in endothelium-independent vasorelaxation.     

Modulation of GvHD by suicide-gene transduced donor T lymphocytes: clinical applications in mismatched transplantation

In allogeneic hematopoietic cell transplantation (allo-HCT), donor lymphocytes play a central therapeutic role in both GvL and immune reconstitution. However, the full exploitation of these therapeutic properties is limited by the occurrence of GvHD. Different strategies have been investigated to obtain all the benefits derived from donor lymphocytes while avoiding the risk of GvHD. The genetic engineering of donor lymphocytes with the herpes simplex virus-thymidine kinase (HSV-TK) suicide gene confers the ability to modulate GvHD by invivo ganciclovir-induced elimination of the transduced cells. The suicide-gene strategy has applications in both donor lymphocyte infusion (DLI) for disease relapse and in add-back infusions after T-cell depleted allo-HCT. TK cell DLI resulted in anti-tumor activity in a relevant proportion of treated patients. Haplo-identical stem cell transplantation (haplo-HCT) is a promising therapeutic option for patients with high risk hematologic malignancies lacking an HLA-matched donor. However, the profound T-cell depletion required to overcome the risk of lethal GvHD has been associated with a marked delayed T-cell recovery with a prolonged risk of post-transplant viral, fungal and other opportunistic infections. TK cell add-backs efficiently promote early immune reconstitution after haplo-HCT and prevent disease relapse, providing a unique tool for the control of GvHD. The genetic manipulation of donor lymphocytes with a suicide gene is a promising strategy to increase feasibility and safety of allo-HCT.     

Mechanisms of Suppression of {alpha}-Synuclein Neurotoxicity by Geldanamycin in Drosophila

Parkinson's disease is a common neurodegenerative disease characterized by the loss of dopaminergic neurons in the substantia nigra pars compacta and the accumulation of the protein alpha-synuclein into aggregates called Lewy bodies and Lewy neurites. Parkinson's disease can be modeled in Drosophila where directed expression of alpha-synuclein induces compromise of dopaminergic neurons and the formation of Lewy body-like aggregates. The molecular chaperone Hsp70 protects cells from the deleterious effects of alpha-synuclein, indicating a potential therapeutic approach to enhance neuron survival in Parkinson's disease. We have now investigated the molecular mechanisms by which the drug geldanamycin protects neurons against alpha-synuclein toxicity. Our studies show that geldanamycin sensitizes the stress response within normal physiological parameters to enhance chaperone activation, offering protection against alpha-synuclein neurotoxicity. Further, geldanamycin uncouples neuronal toxicity from Lewy body and Lewy neurite formation such that dopaminergic neurons are protected from the effects of alpha-synuclein expression despite the continued presence of (and even increase in) inclusion pathology. These studies indicate that compounds that modulate the stress response are a promising approach to treat Parkinson's disease.     

N-terminal deletion affects catalytic activity of saporin toxin

Single-chain ribosome inactivating proteins (RIPs) are cytotoxic components of macromolecular pharmaceutics for immunotherapy of cancer and other human diseases. Saporin belongs to a family of single-chain RIPs sharing sequence and structure homology. In a preliminary attempt to define an active saporin polypeptide of minimum size we have generated proteins with deletions at the N-terminus and at the C-terminus. An N-terminal (sapDelta1-20) deletion mutant of saporin displayed defective catalytic activity, drastically reduced cytotoxicity but increased ability to interact with liposomes inducing their permeabilization at low pH. A C-terminal (sapDelta239-253) deletion mutant showed instead a moderate reduction in cytotoxic activity. A substantial alteration of secondary structure was evidenced by Fourier transformed infrared spectroscopy (FTIR) in the sapDelta1-20 mutant. It can be hypothesized that the defective functions of sapDelta1-20 are due to alterations of its spatial configuration.     

Nitroglycerin drives endothelial nitric oxide synthase activation via the phosphatidylinositol 3-kinase/protein kinase B pathway

Nitroglycerin (GTN) has been clinically used to treat angina pectoris and acute heart episodes for over 100 years. The effects of GTN have long been recognized and active research has contributed to the unraveling of numerous metabolic routes capable of converting GTN to the potent vasoactive messenger nitric oxide. Recently, the mechanism by which minute doses of GTN elicit robust pharmacological responses was revisited and eNOS activation was implicated as an important route mediating vasodilation induced by low GTN doses (1-50nM). Here, we demonstrate that at such concentrations the pharmacologic effects of nitroglycerin are largely dependent on the phosphatidylinositol 3-kinase, Akt/PKB, and phosphatase and tensin homolog deleted on chromosome 10 (PTEN) signal transduction axis. Furthermore, we demonstrate that nitroglycerin-dependent accumulation of 3,4,5-InsP(3), probably because of inhibition of PTEN, is important for eNOS activation, conferring a mechanistic basis for GTN pharmacological action at pharmacologically relevant doses.     

Detection of Ras GTPase protein radicals through immuno-spin trapping

Over the past decade immuno-spin trapping (IST) has been used to detect and identify protein radical sites in numerous heme and metalloproteins. To date, however, the technique has had little application toward nonmetalloproteins. In this study, we demonstrate the successful application of IST in a system free of transition metals and present the first conclusive evidence of (?)NO-mediated protein radical formation in the HRas GTPase. HRas is a nonmetalloprotein that plays a critical role in regulating cell-growth control. Protein radical formation in Ras GTPases has long been suspected of initiating premature release of bound guanine nucleotide. This action results in altered Ras activity both in vitro and in vivo. As described herein, successful application of IST may provide a means for detecting and identifying radical-mediated Ras activation in many different cancers and disease states in which Ras GTPases play an important role.     

Chronic Nerve Growth Factor Exposure Increases Apoptosis in a Model of In Vitro Induced Conjunctival Myofibroblasts

In the conjunctiva, repeated or prolonged exposure to injury leads to tissue remodeling and fibrosis associated with dryness, lost of corneal transparency and defect of ocular function. At the site of injury, fibroblasts (FB) migrate and differentiate into myofibroblasts (myoFB), contributing to the healing process together with other cell types, cytokines and growth factors. While the physiological deletion of MyoFB is necessary to successfully end the healing process, myoFB prolonged survival characterizes the pathological process of fibrosis. The reason for myoFB persistence is poorly understood. Nerve Growth Factor (NGF), often increased in inflamed stromal conjunctiva, may represent an important molecule both in many inflammatory processes characterized by tissue remodeling and in promoting wound-healing and well-balanced repair in humans. NGF effects are mediated by the specific expression of the NGF neurotrophic tyrosine kinase receptor type 1 (trkA^NGFR) and/or the pan-neurotrophin glycoprotein receptor (p75^NTR). Therefore, a conjunctival myoFB model (TGFβ1-induced myoFB) was developed and characterized for cell viability/proliferation as well as αSMA, p75^NTR and trkA^NGFR expression. MyoFB were exposed to acute and chronic NGF treatment and examined for their p75^NTR/trkA^NGFR, αSMA/TGFβ1 expression, and apoptosis. Both NGF treatments significantly increased the expression of p75^NTR, associated with a deregulation of both αSMA/TGFβ1 genes. Acute and chronic NGF exposures induced apoptosis in p75^NTR expressing myoFB, an effect counteracted by the specific trkA^NGFR and/or p75^NTR inhibitors. Focused single p75^NTR and double trkA^NGFR/p75^NTR knocking-down experiments highlighted the role of p75^NTR in NGF-induced apoptosis. Our current data indicate that NGF is able to trigger in vitro myoFB apoptosis, mainly via p75^NTR. The trkA^NGFR/p75^NTR ratio in favor of p75^NTR characterizes this process. Due to the lack of effective pharmacological agents for balanced tissue repairs, these new findings suggest that NGF might be a suitable therapeutic tool in conditions with impaired tissue healing.     

Cryopreservation of umbilical cord mesenchymal cells in xenofree conditions

Background aimsMesenchymal stromal cells (MSC) are being used to treat and prevent a variety of clinical conditions. To be readily available, MSC must be cryopreserved until infusion. However, the optimal cryopreservation methods, cryoprotector solutions and MSC sensitivity to dimethyl sulfoxide (DMSO) exposure are unknown. This study investigated these issues.MethodsMSC samples were obtained from human umbilical cord (n = 15), expanded with Minimal Essential Medium-alpha (α-MEM) 10% human serum (HS), resuspended in 25 mL solution (HS, 10% DMSO, 20% hydroxyethyl starch) and cryopreserved using the BioArchive^® system. After a mean of 18 ± 7 days, cell suspensions were thawed and diluted until a DMSO concentration of 2.5% was reached. Samples were tested for cell quantification and viability, immunophenotype and functional assays.ResultsPost-thaw cell recovery: 114 ± 2.90% (mean ± SEM). Recovery of viable cells: 93.46 ± 4.41%, 90.17 ± 4.55% and 81.03 ± 4.30% at 30 min, 120 min and 24 h post-thaw, respectively. Cell viability: 89.26 ± 1.56%, 72.71 ± 2.12%, 70.20 ± 2.39% and 63.02 ± 2.33% (P < 0.0001) pre-cryopreservation and 30 min, 120 min and 24 h post-thaw, respectively. All post-thaw samples had cells that adhered to culture bottles. Post-thaw cell expansion was 4.18 ± 0.17 ×, with a doubling time of 38 ± 1.69 h, and their capacity to inhibit peripheral blood mononuclear cells (PBMC) proliferation was similar to that observed before cryopreservation. Differentiation capacity, cell-surface marker profile and cytogenetics were not changed by the cryopreservation procedure.ConclusionsA method for cryopreservation of MSC in bags, in xenofree conditions, is described that facilitates their clinical use. The MSC functional and cytogenetic status and morphologic characteristics were not changed by cryopreservation. It was also demonstrated that MSC are relatively resistant to exposure to DMSO, but we recommend cell infusion as soon as possible.