Biomedical subjects

W Michael Zawada

Publications and source records attributed to W Michael Zawada.

8 recordsLinked to original sources

PubMed2007

Angiotensin type 1 receptor antagonist losartan, reduces MPTP-induced degeneration of dopaminergic neurons in substantia nigra.

BACKGROUND: Recent attention has focused on understanding the role of the brain-renin-angiotensin-system (RAS) in stroke and neurodegenerative diseases. Direct evidence of a role for the brain-RAS in Parkinson's disease (PD) comes from studies demonstrating the neuroprotective effect of RAS inhibitors in several neurotoxin based PD models. In this study, we show that an antagonist of the angiotensin II (Ang II) type 1 (AT1) receptor, losartan, protects dopaminergic (DA) neurons against 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) toxicity both in primary ventral mesencephalic (VM) cultures as well as in the substantia nigra pars compacta (SNpc) of C57BL/6 mice (Fig. 1). RESULTS: In the presence of exogenous Ang II, losartan reduced MPP+ (5 muM) induced DA neuronal loss by 72% in vitro. Mice challenged with MPTP showed a 62% reduction in the number of DA neurons in the SNpc and a 71% decrease in tyrosine hydroxylase (TH) immunostaining of the striatum, whereas daily treatment with losartan lessened MPTP-induced loss of DA neurons to 25% and reduced the decrease in striatal TH+ immunostaining to 34% of control. CONCLUSION: Our study demonstrates that the brain-RAS plays an important neuroprotective role in the MPTP model of PD and points to AT1 receptor as a potential novel target for neuroprotection.

Journal Article↗

PubMed2005

Angiotensin II protects cultured midbrain dopaminergic neurons against rotenone-induced cell death.

In this study, we demonstrate that angiotensin II (Ang II) protects dopamine (DA) neurons from rotenone toxicity in vitro. Primary ventral mesencephalic (VM) cultures from E15 rats were grown for 5 days and then cultured in the presence of the mitochondrial complex I inhibitor, rotenone. Acute exposure (20 h) to 20 nM rotenone reduced the number of tyrosine hydroxylase-positive (TH+) neurons by 50 +/- 6% when compared to untreated cultures. Pre-treatment of VM cultures with 100 nM Ang II decreased TH+ neuronal loss to 25 +/- 10% at the 20-nM rotenone concentration. Ang II in the presence of the angiotensin type 1 receptor (AT1R) antagonist, losartan, was even more effective in protecting DA neurons showing a loss of only 13 +/- 4% at 20 nM rotenone. Conversely, the AT2R antagonist, PD123319, abolished the protective effects of Ang II. Furthermore, both the NMDA receptor antagonist, MK801, and the antioxidant, alpha-tocopheryl succinate (vitamin E analogue), prevented rotenone-induced toxicity. Here, we show that acute exposure of VM cultures to the pesticide rotenone leads to dopaminergic neuronal cell death and that angiotensin acting through the AT2 receptor protects dopamine neurons from rotenone toxicity.

Angiotensin II↗

PubMed2004

Multiple molecular determinants in the carboxyl terminus regulate dopamine transporter export from endoplasmic reticulum.

The plasma membrane dopamine transporter (DAT) has an essential role in terminating dopaminergic neurotransmission by reuptake of dopamine into the presynaptic neurons. Therefore, the amount of DAT at the cell surface is a critical determinant of DAT function. In this study, we examined the role of the carboxyl terminus of DAT in trafficking of the transporter through the biosynthetic pathway to the plasma membrane. Live cell fluorescence microscopy and cell surface biotinylation were used to study the effects of systematic deletions and alanine substitutions in the carboxyl terminus on DAT localization. It was found that alanine substitutions of Lys-590 and Asp-600 significantly delayed the delivery of DAT to the plasma membrane because of retention of DAT in the endoplasmic reticulum (ER). Most surprising, mutation of Gly-585 to alanine completely blocked the exit of DAT from the ER and surface expression of the transporter. The effect of these three mutations on ER export of DAT was demonstrated in porcine aortic endothelial cells and the immortalized neuronal cell line 1RB3AN27. In primary cultures of rat embryonic midbrain neurons, DAT G585A, K590A, and D600A mutants were restricted to the cell soma and did not traffic to the dendrites or axonal processes. These data are consistent with the model whereby the local conformation and/or intramolecular interactions of the sequences of the DAT carboxyl terminus proximal to the last transmembrane domain are essential for the ER export of the transporter.

Alanine↗

PubMed2003

Hypoxia induces differentiation of pulmonary artery adventitial fibroblasts into myofibroblasts.

Activation of the alpha-smooth muscle actin (alpha-SMA) gene during the conversion of fibroblasts into myofibroblasts is an essential feature of various fibrotic conditions. Microvascular compromise and thus local environmental hypoxia are important components of the fibrotic response. The present study was thus undertaken to test the hypothesis that hypoxia can induce transdifferentiation of vascular fibroblasts into myofibroblasts and also to evaluate potential signaling mechanisms governing this process. We found that hypoxia significantly upregulates alpha-SMA protein levels in bovine pulmonary artery adventitial fibroblasts. Increased alpha-SMA expression is controlled at the transcriptional level because the alpha-SMA gene promoter activity, assayed via a luciferase reporter, was markedly increased in transfected fibroblasts exposed to hypoxia. Hypoxic induction of the alpha-SMA gene was mimicked by overexpression of constitutively active Galphai2 (alphai2Q205L) but not Galpha16 (alpha-16Q212L). Blockade of hypoxia-induced alpha-SMA expression with pertussis toxin, a Galphai antagonist, confirmed a role for Galphai in the hypoxia-induced transdifferentiation process. c-Jun NH2-terminal kinase (JNK) inhibitor II and SB202190, but not U0126, also attenuated alpha-SMA expression in hypoxic fibroblasts, suggesting the importance of JNK in the differentiation process. Hypoxia-induced increase in bromodeoxyuridine incorporation, which occurred concomitantly with hypoxia-induced differentiation, was blocked by U0126, suggesting that DNA synthesis and alpha-SMA expression take place through simultaneously activated parallel signaling pathways. Neutralizing antibody against transforming growth factor-beta1 blocked only 30% of the hypoxia-induced alpha-SMA promoter activity. Taken together, our results suggest that hypoxia induces differentiation of vascular fibroblasts into myofibroblasts by upregulating the expression of alpha-SMA, and this increase in alpha-SMA level occurs through Galphai- and JNK-dependent signaling pathways.

Actins↗

PubMed2003

The pesticide rotenone induces caspase-3-mediated apoptosis in ventral mesencephalic dopaminergic neurons.

In vivo, the pesticide rotenone induces degeneration of dopamine neurons and parkinsonian-like pathology in adult rats. In the current study, we utilized primary ventral mesencephalic (VM) cultures from E15 rats as an in vitro model to examine the mechanism underlying rotenone-induced death of dopamine neurons. After 11 h of exposure to 30 nm rotenone, the number of dopamine neurons identified by tyrosine hydroxylase (TH) immunostaining declined rapidly with only 23% of the neurons surviving. By contrast, 73% of total cells survived rotenone treatment, indicating that TH+ neurons are more sensitive to rotenone. Examination of the role of apoptosis in TH+ neuron death, revealed that 10 and 30 nm rotenone significantly increased the number of apoptotic TH+ neurons from 7% under control conditions to 38 and 55%, respectively. The increase in apoptotic TH+ neurons correlated with an increase in immunoreactivity for active caspase-3 in TH+ neurons. The caspase-3 inhibitor, DEVD, rescued a significant number of TH+ neurons from rotenone-induced death. Furthermore, this protective effect lasted for at least 32 h post-rotenone and DEVD exposure, indicating lasting neuroprotection achieved with an intervention prior to the death commitment point. Our results show for the first time in primary dopamine neurons that, at low nanomolar concentrations, rotenone induces caspase-3-mediated apoptosis. Understanding the mechanism of rotenone-induced apoptosis in dopamine neurons may contribute to the development of new neuroprotective strategies against Parkinson's disease.

Animals↗

PubMed2003

Neural stem cells and alcohol.

This article summarizes the proceedings of a symposium held at the 2002 Research Society on Alcoholism Meeting in San Francisco, California. The aim of this symposium was to review research on the effects of ethanol on neural stems cells and neurogenesis. Ethanol is known to alter neurogenesis during development; however, recent studies indicate that the brain forms new neurons from stem cells throughout life. Furthermore, stem cells can be transplanted into the brain, creating exciting new possibilities to study brain function. The symposium covered these research areas. Dr. Michael W. Miller reviewed knowledge on the effects of ethanol on stem cell proliferation and differentiation during development. Dr. Wu Ma described studies in culture indicating that (1) neural stem cells express functional muscarinic acetylcholine receptors (mAchR), (2) mAchR-mediated proliferation involves Ca signaling and mitogen-activated protein kinase phosphorylation, and (3) phosphoinositol-3 kinase is a downstream effector for mAchR-mediated cell proliferation via activation of Akt. Drs. Kim Nixon and Fulton T. Crews followed with in vivo studies on ethanol's effects on adult neural stem cell proliferation and differentiation. Dr. W. Michael Zawada described studies directed at dopamine neuron cell transplants into mammalian central nervous system. These studies clearly establish that ethanol has significant effects on stem cells.

Adult↗

PubMed2002

Overexpression of human alpha-synuclein causes dopamine neuron death in primary human mesencephalic culture.

Mutations in the alpha-synuclein gene have been linked to rare cases of familial Parkinson's disease (PD). Alpha-synuclein is a major component of Lewy bodies (LB), a pathological hallmark of PD. Transgenic mice and Drosophila expressing either wild-type or mutant human alpha-synuclein develop motor deficits, LB-like inclusions in some neurons, and neuronal degeneration. However, the relationship between abnormal aggregates of alpha-synuclein and human dopamine (DA) neuron degeneration remains unclear. In this report, we have investigated the influence of alpha-synuclein expression on DA neurons in primary culture of embryonic human mesencephalon. Two days after culture, human DA cells were transduced with wild-type or mutant human (Ala(53)Thr) alpha-synuclein adenoviruses and maintained for 5 days. Overexpression of mutant and wild-type human alpha-synuclein resulted in 49% (P<0.01) and 27% (P<0.05) loss of DA neurons, respectively, while not affecting viability of other cells in the culture. Overexpression of rat alpha-synuclein or GFP (green fluorescent protein) had no effect on DA neuron survival. Cytoplasmic inclusions of alpha-synuclein were detected immunohistochemically in DA cells transduced with mutant human alpha-synuclein, but not wild-type alpha-synuclein. These results show that overexpression of human alpha-synuclein, particularly the mutant form, can cause human DA neuron death, suggesting that alpha-synuclein may have a primary role in the pathogenesis of PD.

Adenoviridae↗

PubMed2002

Xenografts of MHC-deficient mouse embryonic mesencephalon improve behavioral recovery in hemiparkinsonian rats.

The limited availability of human embryonic tissue for dopamine cell transplants in Parkinson's patients has led to an increased interest in using xenogeneic donor tissue. Unfortunately, without aggressive immunosuppression, such brain xenografts are rejected by the host immune system. Chronic brain xenograft rejection is largely mediated by helper T cells, which require presentation of xenoantigens by major histocompatability complex (MHC) class II for their activation. We examined survival and function of xenografts of E13 mouse mesencephalon deficient in either MHC class I, class II, or both after transplantation into adult hemiparkinsonian rats without immunosuppression. Recipients received grafts from C57BL/6 mice that were either: 1) wild-type (wt), 2) MHC class I knockout (KO), 3) MHC class II KO, 4) MHC class I and II double KO, or 5) saline sham transplants. At 6 weeks after transplantation, recipients of MHC class I KO, class II KO, and double KO xenografts significantly reduced methamphetamine-induced circling rate while rats with wt xenografts and sham-operated rats showed no improvement. MHC class II KO grafts had the greatest number of surviving dopamine neurons. All transplants, including saline sham controls, contained infiltrating host MHC class II-positive cells. Saline sham grafts and MHC class II KO xenografts contained significantly fewer infiltrating host MHC class II-positive cells than did wt grafts. Our results show that MHC class II-deficient xenografts survive transplantation for at least 6 weeks in the absence of immunosuppression, reduce rotational asymmetry, and provoke lesser immune reaction than wt grafts.

Animals↗