Prevention of the degeneration of human dopaminergic neurons in an astrocyte co-culture system allowing endogenous drug metabolism
2015-08, Efremova, Liudmila, Schildknecht, Stefan, Adam, Martina, Pape, Regina, Gutbier, Simon, Hanf, Benjamin, Bürkle, Alexander, Leist, Marcel
Background and purpose
Few neuropharmacological model systems use human neurons. Moreover, available test systems rarely reflect functional roles of co-cultured glial cells. There is no human in vitro counterpart of the widely used 1-methyl-4-phenyl-tetrahydropyridine (MPTP) mouse model of Parkinson's disease.
We generated such a model by growing an intricate network of human dopaminergic neurons on a dense layer of astrocytes. In these co-cultures, MPTP was metabolized to 1-methyl-4-phenyl-pyridinium (MPP+) by the glial cells, and the toxic metabolite was taken up through the dopamine transporter into neurons. Cell viability was measured biochemically and by quantitative neurite imaging, siRNA techniques were also used.
We initially characterized the activation of PARP. As in mouse models, MPTP exposure induced (poly-ADP-ribose) synthesis and neurodegeneration was blocked by PARP inhibitors. Several different putative neuroprotectants were then compared in mono-cultures and co-cultures. Rho kinase inhibitors worked in both models; CEP1347, ascorbic acid or a caspase inhibitor protected mono-cultures from MPP+ toxicity, but did not protect co-cultures, when used alone or in combination. Application of GSSG prevented degeneration in co-cultures, but not in mono-cultures. The surprisingly different pharmacological profiles of the models suggest that the presence of glial cells, and the in situ generation of the toxic metabolite MPP+ within the layered cultures played an important role in neuroprotection.
Conclusions and Implications
Our new model system is a closer model of human brain tissue than conventional cultures. Its use for screening of candidate neuroprotectants may increase the predictiveness of a test battery.
Assessment of Unique and Combined Functions of Poly(ADP-Ribose) Polymerases by Using RNA Interference
2014, Hanf, Benjamin
Poly(ADP-ribose) polymerases (PARP) catalyze the synthesis of poly (ADP-ribose) (PAR), a reversible modification of proteins, using NAD+ as a substrate. Poly(ADP-ribosyl)ation produced by PARP-1 and PARP-2 is involved in cytoplasmic and nuclear processes, such as chromatin remodeling, DNA damage signaling and repair, RNA processing, and regulation of cell death. Genetic knockout mouse models of PARP-1 and PARP-2 have revealed a degree of redundancy in cellular PARP functions. However, advancements in elucidating this redundancy have been hindered by the embryonic lethality of the combined PARP-1 and PARP-2 genetic knockout in mice. To date there are several in vitro studies on the cellular depletion of PARP-1 and PARP-2, but these reports did neither aim to investigate this degree of redundancy nor try to provide detailed understanding of the consequences of a combined knockdown of PARP-1 and PARP-2.
In the present work, a first systematic study on the unique and combined functions of PARP-1 and PARP-2 was provided by RNA interference of both proteins in two different cellular approaches. The first approach using a doxycycline inducible microRNA-adapted shRNA (shRNAmir) system had revealed design difficulties in the expression of a polycistronic head to tail configuration to achieve concurrent expression of two shRNAmir sequences, a design formerly reported to be successful but also problematic in some instances. Here, expression of only the second shRNAmir sequence (i.e. PARP-1) was successful, whereas PARP-2 shRNAmir expression could not be demonstrated in stable PARP-1 and PARP-2 shRNAmir expressing HeLa S3 clonal cell populations. Thus, this first approach using concurrent expression of two shRNAmir sequences was not successful in generating a combined knockdown of PARP-1 and PARP-2 in a cellular in vitro system. Moreover, the study design also cautioned before use of older published target siRNA sequences (likely to express off-target effects), although being successful in generating a stable PARP-1 shRNAmir HeLa S3 clonal cell populations. Therefore, in an alternative approach, previously observed difficulties were addressed and concurrent knockdown of PARP-1 and PARP-2 was performed in transient siRNA transfections in two different human cancer cell lines.
To deplete PARP-1 and PARP-2 protein expression in the alternative approach, new and effective PARP-1 and PARP-2 siRNA were generated for use in transient siRNA transfections. Here, single and combined transfections of PARP-1 and PARP-2 siRNA demonstrated a strong knockdown of PARP-1 and/or PARP-2 protein expression in western blot analysis and quantifications of relative mRNA levels in HeLa S3 and U2OS cells. Furthermore, both PARP-1 and PARP 2 siRNA were able to strikingly reduce poly(ADP ribose) formation after oxidative stress, demonstrating a functional loss of poly(ADP-ribosyl)ation capacities in cells.
In following analyses of population doubling, cell proliferation after genotoxic stress, clonogenic survival, cell death, and finally cell cycle phase distributions in HeLa S3 and U2OS cells, no functional redundancies between PARP-1 and PARP-2 could be observed. In contrast, a novel function of PARP-2 during cellular proliferation in HeLa S3 and U2OS cell lines was demonstrated. Depletion of PARP-2, but not PARP-1, significantly reduced cellular proliferation dependent processes as examined by population doubling, cell proliferation after genotoxic stress and clonogenic survival. Moreover, this new PARP-2 function during cellular proliferation was also independent of oxidative or genotoxic stress and could not be attributed to alterations in cell death. Changes in cell cycle have been found instead to mediate this new PARP-2 function, demonstrating a cell-type and p53 independent G1 phase cell cycle arrest. Finally, this G1 phase cell cycle arrest was shown to be independent of PARP catalytic activity, which might be due the reported function of PARP-2 as a transcriptional repressor of cell cycle related promoters, such as c-MYC, which regulate the G1 phase cell cycle checkpoint.
In summary, this first systematic study on unique and combined functions of PARP-1 and PARP-2 demonstrated no functional redundancies of PARP-1 and PARP-2 in the endpoints analyzed. In contrast, a novel catalytic- and PARP-1-independent function of PARP-2 during cellular proliferation was demonstrated within the present work, which might advance understanding of targeting PARP-2 in cancer therapy to suppress tumor growth.
Inflammatory and age-related pathologies in mice with ectopic expression of human PARP-1
2010, Mangerich, Aswin, Herbach, Nadja, Hanf, Benjamin, Fischbach, Arthur, Popp, Oliver, Moreno-Villanueva, Maria, Bruns, Oliver T., Bürkle, Alexander
Poly(ADP-ribose) polymerase-1 (PARP-1) is a sensor for DNA strand breaks and some unusual DNA structures and catalyzes poly(ADP-ribosyl)ation of nuclear proteins with NAD+ serving as substrate. PARP-1 is involved in the regulation of genomic integrity, transcription, inflammation, and cell death. Due to its versatile role, PARP-1 is discussed both as a longevity factor and as an aging-promoting factor. Recently, we generated a mouse model with ectopic integration of full-length hPARP-1 [Mangerich, A., Scherthan, H., Diefenbach, J., Kloz, U., van der Hoeven, F., Beneke, S. and Bürkle, A., 2009. A caveat in mouse genetic engineering: ectopic gene targeting in ES cells by bidirectional extension of the homology arms of a gene replacement vector carrying human PARP-1. Transgenic Res. 18, 261 279]. Here, we show that hPARP-1 mice exhibit impaired survival rates accompanied by reduced hair growth and premature development of several inflammation and age-associated pathologies, such as adiposity, kyphosis, nephropathy, dermatitis, pneumonitis, cardiomyopathy, hepatitis, and anemia. Moreover, mutant male mice showed impaired glucose tolerance, yet without developing manifest diabetes. Overall tumor burden was comparable in wild-type and hPARP-1 mice, but tumor spectrum was shifted in mutant mice, showing lower incidence of sarcomas, but increased incidence of carcinomas. Furthermore, DNA repair was delayed in splenocytes of hPARP-1 mice, and gene expression of pro-inflammatory cytokines was dysregulated. Our results suggest that in hPARP-1 mice impaired DNA repair, accompanied by a continuous low-level increase in pro-inflammatory stimuli, causes development of chronic diseases leading to impaired survival.