Gila Maor

Green tea polyphenol (-)-epigallocatechin-3-gallate prevents N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced dopaminergic neurodegeneration

In the present study we demonstrate neuroprotective property of green tea extract and (–)‐epigallocatechin‐3‐gallate in N‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine mice model of Parkinsons disease. N‐Methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine neurotoxin caused dopamine neuron loss in substantia nigra concomitant with a depletion in striatal dopamine and tyrosine hydroxylase protein levels. Pretreatment of mice with either green tea extract (0.5 and 1 mg/kg) or (–)‐epigallocatechin‐3‐gallate (2 and 10 mg/kg) prevented these effects. In addition, the neurotoxin caused an elevation in striatal antioxidant enzymes superoxide dismutase (240%) and catalase (165%) activities, both effects being prevented by (–)‐epigallocatechin‐3‐gallate. (–)‐Epigallocatechin‐3‐gallate itself also increased the activities of both enzymes in the brain. The neuroprotective effects are not likely to be caused by inhibition of N‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine conversion to its active metabolite 1‐methyl‐4‐phenylpyridinium by monoamine oxidase‐B, as both green tea and (–)‐epigallocatechin‐3‐gallate are very poor inhibitors of this enzyme in vitro (770 µg/mL and 660 µM, respectively). Brain penetrating property of polyphenols, as well as their antioxidant and iron‐chelating properties may make such compounds an important class of drugs to be developed for treatment of neurodegenerative diseases where oxidative stress has been implicated.

Attenuation of 6-hydroxydopamine (6-OHDA)-induced nuclear factor-kappaB (NF-kappaB) activation and cell death by tea extracts in neuronal cultures

Antioxidant and anti-inflammatory therapy approaches have been in the focus of attention in the treatment of neurodegenerative Parkinsons and Alzheimers diseases where oxidative stress has been implicated. Tea extracts have been previously reported to possess radical scavenger, iron chelating and anti-inflammatory properties in a variety of tissues. The purpose of this study was to investigate potential neuroprotective effects of tea extracts and possible signal pathway involved in a neuronal cell model of Parkinsons disease. We demonstrated highly potent antioxidant-radical scavenging activities of green tea (GT) and black tea (BT) extracts on brain mitochondrial membrane fraction, against iron (2.5 microM)-induced lipid peroxidation. Both extracts (0.6-3 microM total polyphenols) were shown to attenuate the neurotoxic action of 6-hydroxydopamine (6-OHDA)-induced neuronal death. 6-OHDA (350 and 50 microM) activated the iron dependent inflammatory redox sensitive nuclear factor-kappaB (NF-kappaB) in rat pheochromocytoma (PC12) and human neuroblastoma (NB) SH-SY5Y cells, respectively. Immunofluorescence and electromobility shift assays showed increased nuclear translocation and binding activity of NF-kappaB after exposure to 6-OHDA in NB SH-SY5Y cells, with a concomitant disappearance from the cytoplasm. Introduction of GT extract (0.6, 3 microM total polyphenols) before 6-OHDA inhibited both NF-kappaB nuclear translocation and binding activity induced by this toxin in NB SH-SY5Y cells. Neuroprotection was attributed to the potent antioxidant and iron chelating actions of the polyphenolic constituents of tea extracts, preventing nuclear translocation and activation of cell death promoting NF-kappaB. Brain penetrating property of polyphenols may make such compounds an important class of drugs for treatment of neurodegenerative diseases.

Gene expression analysis in N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine mice model of Parkinson's disease using cDNA microarray: effect of R-apomorphine.

To establish the possible roles of oxidative stress, inflammatory processes and other unknown mechanisms in neurodegeneration, we investigated brain gene alterations in N‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP) mice model of Parkinsons disease using Atlas mouse cDNA expression array membrane. The expression of 51 different genes involved in oxidative stress, inflammation, glutamate and neurotrophic factors pathways as well as in still undefined processes, such as cell cycle regulators and signal transduction molecules, was differentially affected by the treatment. The present study indicates the involvement of an additional cascade of events that might act in parallel to oxidative stress and inflammation to converge eventually into a common pathway leading to neurodegeneration. The attenuation of these gene changes by R‐apomorphine, an iron chelator‐radical scavenger drug, supports our previous findings in vivo where R‐apomorphine was neuroprotective.

Iron and α-synuclein in the substantia nigra of MPTP-treated mice

One of the prominent pathological features of Parkinson’s disease (PD) is the abnormal accumulation of iron in the substantia nigra pars compacta (SNpc), in the reactive microglia, and in association with neuromelanin, within the melanin-containing dopamine (DA) neurons. Lewy body, the morphological hallmark of PD, is composed of lipids, redox-active iron, and aggregated α-synuclein, concentrating in its peripheral halo and ubiquitinated, hyperphosphorylated, neurofilament proteins. The capacity of free iron to enhance and promote the generation of toxic reactive oxygen radicals has been discussed numerous times. Recent observations, that iron induces aggregation of inert α-synuclein to toxic aggregates, have reinforced the critical role of iron in oxidative stress-induced pathogenesis of DA neuron degeneration and protein degradation via ubiquitination. N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)- and 6-hydroxydopamine-induced neurodegeneration in rodents and nonhuman primates is associated with increased presence of iron and α-synuclein in the SNpc. The accumulation of iron in MPTP-induced neurodegeneration has been linked to nitric oxide-dependent mechanism, resulting in degradation of prominent iron regulatory proteins by ubiquitination. Radical scavengers such as R-apomorphine and green tea catechin polyphenol (−)-epigallocatechin-3-gallate, as well as the recently developed brain-permeable VK-28 series derivative iron chelators, which are neuroprotective against these neurotoxins in mice and rats, prevent the accumulation of iron and α-synuclein in SNpc. This study supports the notion that a combination of iron chelation and antioxidant therapy, as emphasized on several occasions, might be a significant approach to neuroprotection in PD and other neurodegenerative diseases.

Effects of R‐ and S‐apomorphine on MPTP‐induced nigro‐striatal dopamine neuronal loss

In order to establish whether the antioxidant and iron‐chelating activities of R‐apomorphine (R‐APO), a D1‐D2 receptor agonist, may contribute to its neuroprotective property, its S‐isomer, which is not a dopamine agonist, was studied. The neuroprotective property of R‐ and S‐APO has been studied in the MPTP model of Parkinsons disease (PD). Both S‐APO (0.5–1 mg/kg, subcutaneous) and R‐APO (10 mg/kg) pretreatment of C57‐BL mice, protected against MPTP (24 mg/kg, intraperitoneally) induced dopamine (DA) depletion and reduction in tyrosine hydroxylase (TH) activity. However, only R‐APO prevented nigro‐striatal neuronal cell degeneration, as indicated by the immunohistochemistry of TH positive neurones in substantia nigra and by western analysis of striatal TH content. R‐APO prevented the reduction of striatal‐GSH and the increase in the ratio of GSSG over total glutathione, caused by MPTP treatment. In vitro both R‐APO and S‐APO inhibited monoamine oxidase A and B activity at relatively high concentrations (100 and 300 µmol/L, respectively). The elevated activity of TH induced by the two enantiomers may contribute to the maintenance of normal DA levels, suggesting that one of the targets of these molecules may involve upregulation of TH activity. It is suggested that the antioxidant and iron‐chelating properties, possible monoamine oxidase inhibitory actions, together with activation of DA receptors, may participate in the mechanism of neuroprotection by APO enantiomers against MPTP.

Fas (CD95/Apo-1)–Mediated Damage to Ventricular Myocytes Induced by Cytotoxic T Lymphocytes From Perforin-Deficient Mice: A Major Role for Inositol 1,4,5-Trisphosphate

Cytotoxic T lymphocytes (CTLs) that infiltrate the heart are important immune effectors implicated in heart transplant rejection, myocarditis, and other cardiomyopathies. To investigate the mechanism(s) underlying CTL damage to the myocardium through activation of the Fas receptor (Fas/CD95/Apo-1) by the Fas ligand, we explored the interaction between peritoneal exudate CTLs (PELs), derived from perforin gene-knockout (P-/-) mice, and murine ventricular myocytes. Fas expression on isolated ventricular myocytes was demonstrated immunohistochemically. Action potentials, [Ca2+]i transients, and contractions of myocytes conjugated to P-/- PELs or treated with the apoptosis-inducing anti-Fas monoclonal antibody Jo2 were recorded. Action potential characteristics of nonconjugated myocytes and myocytes conjugated with P-/- PELs were, respectively, as follows: Vm, -73.2+/-1.5 and -53.6+/-6.4 mV (mean+/-SEM); action potential amplitude, 117.9+/-3.9 and 74.3+/-21.2 mV; and action potential duration at 80% repolarization, 17+/-6 and 42+/-13 milliseconds (all P<.05). P-/- PELs also induced early and delayed afterdepolarizations as well as arrhythmogenic activity. Diastolic [Ca2+]i increased during the cytocidal interaction with P-/- PELs, from a fluorescence ratio of 0.82+/-0.05 (n=7) to 1.98+/-0.09 (n=13) (P<.05). All of the effects caused by P-/- PELs were reproduced by incubating the myocytes with Jo2. Heparin (50 microg/mL), an antagonist of inositol trisphosphate (IP3)-operated sarcoplasmic reticulum Ca2+ channels, or U-73122 (2 micromol/L), a phospholipase C inhibitor, but not the inactive agonist U-73343, prevented Fas-mediated myocyte dysfunction. Additionally, intracellular application (through the patch pipette) of the active IP3 analogue, inositol 1,4,5-trisphosphate, but not the inactive analogue, inositol 1,3,4-trisphosphate, caused electrophysiological changes resembling those resulting from P-/- PELs and Jo2, suggesting that CTL-induced Fas-based myocyte dysfunction is mediated by IP3. We conclude that a Fas-based perforin-independent mechanism of CTL action can account for the immunopathology seen in the allotransplanted heart, myocarditis, and dilated cardiomyopathy.

Testosterone Stimulates Insulin-Like Growth Factor-I and Insulin-Like Growth Factor-I-Receptor Gene Expression in the Mandibular Condyle—A Model of Endochondral Ossification

Puberty is associated with an increase in the plasma concentration of sex steroids, GH, and insulin-like growth factor-I (IGF-I). Gonadal steroid hormones are important for the normal pubertal growth spurt and skeletal growth. The mechanism by which gonadal steroids induce skeletal growth is still not fully understood. To better understand the direct effect sex steroids have on bone growth, we studied an isolated organ culture system of the mandibular condyle, derived from 3.5–5.5-week-old male and female mice. We found that testosterone 10−6 m, but not estradiol, stimulated thymidine incorporation into the DNA of male-derived condyle. Three days of testosterone treatment doubled the condyle size and increased the chondroprogenitor zone, while maintaining the normal gradient of the developing chondrocytes. Immunohistochemistry and in situ hybridization techniques showed that testosterone stimulated IGF-I and IGF-I-R and their messenger RNAs (mRNAs) mainly in the mature chondrocyte layer. Immunoneutralizat...

Early and Late Gene Changes in MPTP Mice Model of Parkinson's Disease Employing cDNA Microarray

Recently, we reported specific brain gene expression changes in the chronic MPTP model in the late stage of degeneration, employing cDNA expression array, which indicate a “domino” cascade of events involved in neuronal cell death. In an attempt to elucidate early gene expression profile in the region of the substantia nigra (SN) and the striatum of acute MPTP-treated mice (3–24 h), we elected a restricted number of genes affected by the long-term MPTP treatment, and their expression was examined. Specifically, we detected alterations in the expression of genes implicated in oxidative-stress, inflammatory processes, signal transduction and glutamate toxicity. These pro-toxic genes appear to be compensated by the elevated expression in trophic factors and antioxidant defenses, which are also activated by short exposure to MPTP. The time course of these gene expression changes indicates the importance of investigating the early gene cascade of events occurring prior to late nigrostriatal dopamine neuronal cell death.

Early and late molecular events in neurodegeneration and neuroprotection in Parkinson’s disease MPTP model as assessed by cDNA microarray; the role of iron

Possible cell death mechanisms for pars compacta nigro-striatal dopamine neurons in Parkinson’s disease include oxidative stress, inflammatory processes, nitric oxide, iron accumulation, glutamate toxicity and diminished neurotrophic factor responses. There is a notion that Parkinson’s disease is not a single disorder but a syndrome that can be initiated by several factors. Because of limitations of biochemical methods in the global analysis of neuronal death, a full picture of events has not been established. However, recently developed cDNA microarray or microchips, in which the global expression of thousands of genes can be assessed simultaneously, is changing the prospect for understanding the disease process, its progression, response to drugs, etc. The neurotoxin N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) is considered the most valid model of Parkinson’s disease. We employed the technique of cDNA microarray gene expression to determine the mechanism of action of MPTP in mouse substantia nigra. Also, we studied neuroprotective processes induced by several compounds, including R-apomorphine and the green tea polyphenol epigallo-catechin-3-gallate (EGCG). This was done in two ways: (1) the time-dependent acute effect of MPTP, for determining which of the initial genes might lead to dopamine neuron death and (2) gene expression at the time of MPTP-induced dopamine neuron death. We observed that early (acute MPTP) gene expression differs from effects seen at the time of death (chronic MPTP), and that early gene changes are crucial for setting into action genes that eventually cause dopamine neuron death. Furthermore, this process is a cascade of “domino” effects, some of which were previously established by biochemical means. However, our findings show an additional large number of events previously unknown. The neuroprotective drugs reversed some but not all of the gene expression, suggesting involvement of these genes in the neurodegenerative process. Because of the profound complexity of “domino” effect it is now reasonable to understand why a single neuroprotective drug has not shown clinical neuroprotective efficacy. Future multi neuroprotective drugs may be necessary for treatment of not only Parkinson’s disease, but other neurodegenerative diseases (e.g. Alzheimer’s disease) and detrimental states (e.g. ischaemia).

Insulin enhances the growth of cartilage in organ and tissue cultures of mouse neonatal mandibular condyle.

SummaryCondylar cartilages were cultured in the form of organ cultures on top of collagen sponges in medium containing 2% fetal calf serum and were treated with 3.5–350 nM insulin for 6 days. Doses of 175 nM of insulin caused a marked increase (+96%) in DNA synthesis and in proteoglycan production (+74%), features that manifested themselves structurally by a 60% increase in overall size of the cultured explants. Using a tissue culture system comprised of cartilage progenitor cells, insulin was found to enhance the differentiation of the progenitor cells so that by 6 days in culture and appreciable nodule of differentiated chondrocytes developed. The latter was surrounded by perichondrial cells whereas the extracellular matrix within the newly formed, insulin-induced, nodule reacted positively for cartilagespecific antigens (type II collagen and bone sialoprotein). It is suggested that insulin induces a direct stimulatory effect on progenitor cell proliferation, cartilage differentiation, and extracellular matrix deposition.