Laurie H. Sanders

Oxidative damage to macromolecules in human Parkinson disease and the rotenone model

Parkinson disease (PD), the most common neurodegenerative movement disorder, is associated with selective degeneration of nigrostriatal dopamine neurons. Although the underlying mechanisms contributing to neurodegeneration in PD seem to be multifactorial, mitochondrial impairment and oxidative stress are widely considered to be central to many forms of the disease. Whether oxidative stress is a cause or a consequence of dopaminergic death, there is substantial evidence for oxidative stress both in human PD patients and in animal models of PD, especially using rotenone, a complex I inhibitor. There are many indices of oxidative stress, but this review covers the recent evidence for oxidative damage to nucleic acids, lipids, and proteins in both the brain and the peripheral tissues in human PD and in the rotenone model. Limitations of the existing literature and future perspectives are discussed. Understanding how each particular macromolecule is damaged by oxidative stress and the interplay of secondary damage to other biomolecules may help us design better targets for the treatment of PD.

LRRK2 mutations cause mitochondrial DNA damage in iPSC-derived neural cells from Parkinson's disease patients: reversal by gene correction.

Parkinsons disease associated mutations in leucine rich repeat kinase 2 (LRRK2) impair mitochondrial function and increase the vulnerability of induced pluripotent stem cell (iPSC)-derived neural cells from patients to oxidative stress. Since mitochondrial DNA (mtDNA) damage can compromise mitochondrial function, we examined whether LRRK2 mutations can induce damage to the mitochondrial genome. We found greater levels of mtDNA damage in iPSC-derived neural cells from patients carrying homozygous or heterozygous LRRK2 G2019S mutations, or at-risk individuals carrying the heterozygous LRRK2 R1441C mutation, than in cells from unrelated healthy subjects who do not carry LRRK2 mutations. After zinc finger nuclease-mediated repair of the LRRK2 G2019S mutation in iPSCs, mtDNA damage was no longer detected in differentiated neuroprogenitor and neural cells. Our results unambiguously link LRRK2 mutations to mtDNA damage and validate a new cellular phenotype that can be used for examining pathogenic mechanisms and screening therapeutic strategies.

Mitochondrial DNA damage: Molecular marker of vulnerable nigral neurons in Parkinson's disease

DNA damage can cause (and result from) oxidative stress and mitochondrial impairment, both of which are implicated in the pathogenesis of Parkinsons disease (PD). We therefore examined the role of mitochondrial DNA (mtDNA) damage in human postmortem brain tissue and in in vivo and in vitro models of PD, using a newly adapted histochemical assay for abasic sites and a quantitative polymerase chain reaction (QPCR)-based assay. We identified the molecular identity of mtDNA damage to be apurinic/apyrimidinic (abasic) sites in substantia nigra dopamine neurons, but not in cortical neurons from postmortem PD specimens. To model the systemic mitochondrial impairment of PD, rats were exposed to the pesticide rotenone. After rotenone treatment that does not cause neurodegeneration, abasic sites were visualized in nigral neurons, but not in cortex. Using a QPCR-based assay, a single rotenone dose induced mtDNA damage in midbrain neurons, but not in cortical neurons; similar results were obtained in vitro in cultured neurons. Importantly, these results indicate that mtDNA damage is detectable prior to any signs of degeneration - and is produced selectively in midbrain neurons under conditions of mitochondrial impairment. The selective vulnerability of midbrain neurons to mtDNA damage was not due to differential effects of rotenone on complex I since rotenone suppressed respiration equally in midbrain and cortical neurons. However, in response to complex I inhibition, midbrain neurons produced more mitochondrial H2O2 than cortical neurons. We report selective mtDNA damage as a molecular marker of vulnerable nigral neurons in PD and suggest that this may result from intrinsic differences in how these neurons respond to complex I defects. Further, the persistence of abasic sites suggests an ineffective base excision repair response in PD.

PCR Based Determination of Mitochondrial DNA Copy Number in Multiple Species

Mitochondrial DNA (mtDNA) copy number is a critical component of overall mitochondrial health. In this chapter, we describe methods for isolation of both mtDNA and nuclear DNA (nucDNA) and measurement of their respective copy numbers using quantitative PCR. Methods differ depending on the species and cell type of the starting material and availability of specific PCR reagents.

Role of Pseudomonas aeruginosa dinB-Encoded DNA Polymerase IV in Mutagenesis

Pseudomonas aeruginosa is a human opportunistic pathogen that chronically infects the lungs of cystic fibrosis patients and is the leading cause of morbidity and mortality of people afflicted with this disease. A striking correlation between mutagenesis and the persistence of P. aeruginosa has been reported. In other well-studied organisms, error-prone replication by Y family DNA polymerases contributes significantly to mutagenesis. Based on an analysis of the PAO1 genome sequence, P. aeruginosa contains a single Y family DNA polymerase encoded by the dinB gene. As part of an effort to understand the mechanisms of mutagenesis in P. aeruginosa, we have cloned the dinB gene of P. aeruginosa and utilized a combination of genetic and biochemical approaches to characterize the activity and regulation of the P. aeruginosa DinB protein (DinB(Pa)). Our results indicate that DinB(Pa) is a distributive DNA polymerase that lacks intrinsic proofreading activity in vitro. Modest overexpression of DinB(Pa) from a plasmid conferred a mutator phenotype in both Escherichia coli and P. aeruginosa. An examination of this mutator phenotype indicated that DinB(Pa) has a propensity to promote C-->A transversions and -1 frameshift mutations within poly(dGMP) and poly(dAMP) runs. The characterization of lexA+ and DeltalexA::aacC1 P. aeruginosa strains, together with in vitro DNA binding assays utilizing cell extracts or purified P. aeruginosa LexA protein (LexA(Pa)), indicated that the transcription of the dinB gene is regulated as part of an SOS-like response. The deletion of the dinB(Pa) gene sensitized P. aeruginosa to nitrofurazone and 4-nitroquinoline-1-oxide, consistent with a role for DinB(Pa) in translesion DNA synthesis over N2-dG adducts. Finally, P. aeruginosa exhibited a UV-inducible mutator phenotype that was independent of dinB(Pa) function and instead required polA and polC, which encode DNA polymerase I and the second DNA polymerase III enzyme, respectively. Possible roles of the P. aeruginosa dinB, polA, and polC gene products in mutagenesis are discussed.

Autophagy Protects Against Aminochrome-Induced Cell Death in Substantia Nigra-Derived Cell Line

Aminochrome, the precursor of neuromelanin, has been proposed to be involved in the neurodegeneration neuromelanin-containing dopaminergic neurons in Parkinsons disease. We aimed to study the mechanism of aminochrome-dependent cell death in a cell line derived from rat substantia nigra. We found that aminochrome (50μM), in the presence of NAD(P)H-quinone oxidoreductase, EC 1.6.99.2 (DT)-diaphorase inhibitor dicoumarol (DIC) (100μM), induces significant cell death (62 ± 3%; p < 0.01), increase in caspase-3 activation (p < 0.001), release of cytochrome C, disruption of mitochondrial membrane potential (p < 0.01), damage of mitochondrial DNA, damage of mitochondria determined with transmission electron microscopy, a dramatic morphological change characterized as cell shrinkage, and significant increase in number of autophagic vacuoles. To determine the role of autophagy on aminochrome-induced cell death, we incubated the cells in the presence of vinblastine and rapamycin. Interestingly, 10μM vinblastine induces a 5.9-fold (p < 0.001) and twofold (p < 0.01) significant increase in cell death when the cells were incubated with 30μM aminochrome in the absence and presence of DIC, respectively, whereas 10μM rapamycin preincubated 24 h before addition of 50μM aminochrome in the absence and the presence of 100μM DIC induces a significant decrease (p < 0.001) in cell death. In conclusion, autophagy seems to be an important protective mechanism against two different aminochrome-induced cell deaths that initially showed apoptotic features. The cell death induced by aminochrome when DT-diaphorase is inhibited requires activation of mitochondrial pathway, whereas the cell death induced by aminochrome alone requires inhibition of autophagy-dependent degrading of damaged organelles and recycling through lysosomes.

Extensive uptake of α-synuclein oligomers in astrocytes results in sustained intracellular deposits and mitochondrial damage

Abstract The presence of Lewy bodies, mainly consisting of aggregated &agr;‐synuclein, is a pathological hallmark of Parkinsons disease (PD) and dementia with Lewy bodies (DLB). The &agr;‐synuclein inclusions are predominantly found in neurons, but also appear frequently in astrocytes. However, the pathological significance of &agr;‐synuclein inclusions in astrocytes and the capacity of glial cells to clear toxic &agr;‐synuclein species remain unknown. In the present study we investigated uptake, degradation and toxic effects of oligomeric &agr;‐synuclein in a co‐culture system of primary neurons, astrocytes and oligodendrocytes. Alpha‐synuclein oligomers were found to co‐localize with the glial cells and the astrocytes were found to internalize particularly large amounts of the protein. Following ingestion, the astrocytes started to degrade the oligomers via the lysosomal pathway but, due to incomplete digestion, large intracellular deposits remained. Moreover, the astrocytes displayed mitochondrial abnormalities. Taken together, our data indicate that astrocytes play an important role in the clearance of toxic &agr;‐synuclein species from the extracellular space. However, when their degrading capacity is overburdened, &agr;‐synuclein deposits can persist and result in detrimental cellular processes. HighlightsAstrocytes rapidly ingest large amounts of oligomeric &agr;‐synuclein.Due to incomplete lysosomal degradation, the &agr;‐synuclein is intracellularly stored.The accumulation of &agr;‐synuclein induces astrocytic mitochondrial impairments.Our results emphasize an important role of astrocytes in &agr;‐synucleinopathies.

SLIDING CLAMP-DNA INTERACTIONS ARE REQUIRED FOR VIABILITY AND CONTRIBUTE TO DNA POLYMERASE MANAGEMENT IN Escherichia coli

Sliding clamp proteins topologically encircle DNA and play vital roles in coordinating the actions of various DNA replication, repair, and damage tolerance proteins. At least three distinct surfaces of the Escherichia coli beta clamp interact physically with the DNA that it topologically encircles. We utilized mutant beta clamp proteins bearing G66E and G174A substitutions (beta159), affecting the single-stranded DNA-binding region, or poly-Ala substitutions in place of residues 148-HQDVR-152 (beta(148-152)), affecting the double-stranded DNA binding region, to determine the biological relevance of clamp-DNA interactions. As part of this work, we solved the X-ray crystal structure of beta(148-152), which verified that the poly-Ala substitutions failed to significantly alter the tertiary structure of the clamp. Based on functional assays, both beta159 and beta(148-152) were impaired for loading and retention on a linear primed DNA in vitro. In the case of beta(148-152), this defect was not due to altered interactions with the DnaX clamp loader, but rather was the result of impaired beta(148-152)-DNA interactions. Once loaded, beta(148-152) was proficient for DNA polymerase III (Pol III) replication in vitro. In contrast, beta(148-152) was severely impaired for Pol II and Pol IV replication and was similarly impaired for direct physical interactions with these Pols. Despite its ability to support Pol III replication in vitro, beta(148-152) was unable to support viability of E. coli. Nevertheless, physiological levels of beta(148-152) expressed from a plasmid efficiently complemented the temperature-sensitive growth phenotype of a strain expressing beta159 (dnaN159), provided that Pol II and Pol IV were inactivated. Although this strain was impaired for Pol V-dependent mutagenesis, inactivation of Pol II and Pol IV restored the Pol V mutator phenotype. Taken together, these results support a model in which a sophisticated combination of competitive clamp-DNA, clamp-partner, and partner-DNA interactions serve to manage the actions of the different E. coli Pols in vivo.

The GO system prevents ROS-induced mutagenesis and killing in Pseudomonas aeruginosa

Inactivation of the Pseudomonas aeruginosa mutM, mutY, or mutT gene conferred a 2.4-, 17.2-, or 38.1-fold increase in spontaneous mutation frequency, respectively. Importantly, the mutY and mutT strains each displayed a robust H(2)O(2)-induced mutation frequency. In addition, the mutM, mutY, and mutT mutations severely sensitized P. aeruginosa to killing by H(2)O(2), suggesting that these gene products act to repair one or more cytotoxic lesions in P. aeruginosa. Nucleotide sequence analysis of a fragment of the rpoB gene from rifampicin resistant mutM-, mutY-, and, mutT-deficient strains was consistent with this conclusion. These findings are discussed in terms of possible roles for mutM, mutY, and mutT in contributing to survival and mutagenesis of P. aeruginosa colonizing the airways of cystic fibrosis patients.

Phenotype of the zebrafish masterblind (mbl) mutant is dependent on genetic background

The zebrafish masterblind (mbl) mutant is characterized by the lack of olfactory placodes and optic vesicles, reduced telencephalon, an expanded epiphysis (Heisenberg et al. [ 1996 ] Development 123:191–203), and enlarged jaw. To understand the cellular events giving rise to the olfactory placode defect of this mutant, we examined the expression pattern of the distal‐less‐3 (dlx3) gene in mbl. In the mutant, dlx3, which is normally expressed in the developing nose and ear, showed reduced expression in the olfactory placode field, but normal expression in the developing ear. To determine whether the loss of dlx3 expression was due to cell loss, we assayed cell death by using TUNEL labeling. Although cell death in the mutant was not concentrated in the region of dlx3 expression, there was increased cell death in the forebrain, epiphysis, and jaw region, as compared with that in wild‐type controls. This cell death phenotype was cyclical in nature, showing an increase and decrease in cell death on a roughly 24‐hr cycle. Further analysis showed that this cyclical phenotype was specific to the genetic background. The severity of the mbl phenotype, including cell death, expanded epiphysis, and enlarged jaw, decreased when the mutation was moved from the original “TL” background to the “AB” background. Thus, the severity of developmental defects in the mbl mutant is strongly dependent on genetic background. We examined the contribution of cell death to the morphologic defects of mbl by blocking cell death by using zVADfmk, a known caspase inhibitor. We found that this treatment partially rescued the expanded jaw defect and that this rescue was dependent on the genetic background. Therefore, the mbl mutant phenotypes result, in part, from genetic background effects that alter the pattern of programmed cell death early in development. Developmental Dynamics 227:291–300, 2003.