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Over the last three decades, neurodegenerative diseases have received increasing attention due to their frequency in the aging population and the social and economic burdens they are posing. In parallel, an era's worth of research in neuroscience has shaped our current appreciation of the complex relationship between nutrition and the central nervous system. Particular branches of nutrition continue to galvanize neuroscientists, in particular the diverse roles that bioactive food derivatives play on health and disease. Bioactive food derivatives are nowadays recognized to directly impact brain homeostasis, specifically with respect to their actions on cellular mechanisms of oxidative stress, neuroinflammation, mitochondrial dysfunction, apoptosis and autophagy. However, ambiguities still exist regarding the significance of the influence of bioactive food derivatives on human health. In turn, gut microbiota dysbiosis is emerging as a novel player in the pathogenesis of neurodegenerative diseases. Currently, several routes of communication exist between the gut and the brain, where molecules are either released in the bloodstream or directly transported to the CNS. As such, bioactive food derivatives can modulate the complex ecosystem of the gut-brain axis, thus, targeting this communication network holds promises as a neuroprotective tool. This review aims at addressing one of the emerging aspects of neuroscience, particularly the interplay between food bioactive derivatives and neurodegeneration. We will specifically address the role that polyphenols and omega-3 fatty acids play in preventing neurodegenerative diseases and how dietary intervention complements available pharmacological approaches., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

3,4-Methylenedioxymethamphetamine (MDMA, "ecstasy") is an amphetamine-related drug that may damage the dopaminergic nigrostriatal system. To investigate the mechanisms that sustain this toxic effect and ascertain their sex-dependence, we evaluated in the nigrostriatal system of MDMA-treated (4 × 20 mg/kg, 2 h apart) male and female mice the activity of superoxide dismutase (SOD), the gene expression of SOD type 1 and 2, together with SOD1/2 co-localization with tyrosine hydroxylase (TH)-positive neurons. In the same mice and brain areas, activity of glutathione peroxidase (GPx) and of β2/β5 subunits of the ubiquitin-proteasome system (UPS) were also evaluated. After MDMA, SOD1 increased in striatal TH-positive terminals, but not nigral neurons, of males and females, while SOD2 increased in striatal TH-positive terminals and nigral neurons of males only. Moreover, after MDMA, SOD1 gene expression increased in the midbrain of males and females, whereas SOD2 increased only in males. Finally, MDMA increased the SOD activity in the midbrain of females, without affecting GPx activity, decreased the β2/β5 activities in the striatum of males and the β2 activity in the midbrain of females. These results suggest that the mechanisms of MDMA-induced neurotoxic effects are sex-dependent and dopaminergic neurons of males could be more sensitive to SOD2- and UPS-mediated toxic effects., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Costa, Caputi, Serra, Simola, Rullo, Stamatakos, Sanna, Germain, Martinoli, Candeletti, Morelli and Romualdi.)

Neuroinflammation has been implicated in the pathogenesis of neurodegeneration and is now accepted as a common molecular feature underpinning neuronal damage and death. Palmitic acid (PA) may represent one of the links between diet and neuroinflammation. The aims of this study were to assess whether PA induced toxicity in neuronal cells by modulating microglial inflammatory responses and/or by directly targeting neurons. We also determined the potential of oleic acid (OA), a monounsaturated fatty acid, to counteract inflammation and promote neuroprotection. We measured the ability of PA to induce the secretion of interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α), the induction of the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signalling pathways, as well as the phosphorylation of c-Jun, and the expression of inducible nitric oxide synthase (iNOS). Finally, to determine whether PA exerted an indirect neurotoxic effect on neuronal cells, we employed a microglia-neuron co-culture paradigm where microglial cells communicate with neuronal cells in a paracrine fashion. Herein, we demonstrate that PA induces the activation of the NF-κB signalling pathway and c-Jun phosphorylation in N9 microglia cells, in the absence of increased cytokine secretion. Moreover, our data illustrate that PA exerts an indirect as well as a direct neurotoxic role on neuronal PC12 cells and these effects are partially prevented by OA. These results are important to establish that PA interferes with neuronal homeostasis and suggest that dietary PA, when consumed in excess, may induce neuroinflammation and possibly concurs in the development of neurodegeneration.

The polyphenol trans-ε-viniferin (viniferin) is a dimer of resveratrol, reported to hold antioxidant and anti-inflammatory properties. The aims of our study were to evaluate the neuroprotective potential of viniferin in the nerve growth factor (NGF)-differentiated PC12 cells, a dopaminergic cellular model of Parkinson's disease (PD) and assess its anti-inflammatory properties in a N9 microglia-neuronal PC12 cell co-culture system. The neuronal cells were pre-treated with viniferin, resveratrol or their mixture before the administration of 6-hydroxydopamine (6-OHDA), recognized to induce parkinsonism in rats. Furthermore, N9 microglia cells, in a co-culture system with neuronal PC12, were pre-treated with viniferin, resveratrol or their mixture to investigate whether these polyphenols could reduce lipopolysaccharide (LPS)-induced inflammation. Our results show that viniferin as well as a mixture of viniferin and resveratrol protects neuronal dopaminergic cells from 6-OHDA-induced cytotoxicity and apoptosis. Furthermore, when viniferin, resveratrol or their mixture was used to pre-treat microglia cells in our co-culture system, they reduced neuronal cytotoxicity induced by glial activation. Altogether, our data highlight a novel role for viniferin as a neuroprotective and anti-inflammatory molecule in a dopaminergic cellular model, paving the way for nutraceutical therapeutic avenues in the complementary treatments of PD.

A high-fat diet induces hypothalamic inflammation in rodents which, in turn, contributes to the development of obesity by eliciting both insulin and leptin resistance. However, the mechanism by which long-chain saturated fatty acids trigger inflammation is still contentious. To elucidate this mechanism, the effect of fatty acids on the expression of the pro-inflammatory cytokines IL-6 and TNFα was investigated in the mHypoE-N42 hypothalamic cell line (N42). N42 cells were treated with lauric acid (LA) and palmitic acid (PA). PA challenge was carried out in the presence of either a TLR4 inhibitor, a ceramide synthesis inhibitor (L-cycloserine), oleic acid (OA) or eicosapentaenoic acid (EPA). Intracellular ceramide accumulation was quantified using LC-ESI-MS/MS. PA but not LA upregulated IL-6 and TNFα . L-cycloserine, OA and EPA all counteracted PA-induced intracellular ceramide accumulation leading to a downregulation of IL-6 and TNFα . However, a TLR4 inhibitor failed to inhibit PA-induced upregulation of pro-inflammatory cytokines.In conclusion, PA induced the expression of IL-6 and TNFα in N42 neuronal cells independently of TLR4 but, partially, via ceramide synthesis with OA and EPA being anti-inflammatory by decreasing PA-induced intracellular ceramide build-up. Thus, ceramide accumulation represents one on the mechanisms by which PA induces inflammation in neurons.

Diabetes mellitus (DM), a group of diseases characterized by defective glucose metabolism, is the most widespread metabolic disorder affecting over 400 million adults worldwide. This pathological condition has been implicated in the pathogenesis of a number of central encephalopathies and peripheral neuropathies. In further support of this notion, recent epidemiological evidence suggests a link between DM and Parkinson's disease (PD), with hyperglycemia emerging as one of the culprits in neurodegeneration involving the nigrostriatal pathway, the neuroanatomical substrate of the motor symptoms affecting parkinsonian patients. Indeed, dopaminergic neurons located in the mesencephalic substantia nigra appear to be particularly vulnerable to oxidative stress and degeneration, likely because of their intrinsic susceptibility to mitochondrial dysfunction, which may represent a direct consequence of hyperglycemia and hyperglycemia-induced oxidative stress. Other pathological pathways induced by increased intracellular glucose levels, including the polyol and the hexosamine pathway as well as the formation of advanced glycation end-products, may all play a pivotal role in mediating the detrimental effects of hyperglycemia on nigral dopaminergic neurons. In this review article, we will examine the epidemiological as well as the molecular and cellular clues supporting the potential susceptibility of nigrostriatal dopaminergic neurons to hyperglycemia., (Copyright © 2019 Sergi, Renaud, Simola and Martinoli.)

Over the last two decades, the increase in the incidence of neurodegenerative diseases due to the increasingly ageing population has resulted in a major social and economic burden. At present, a large body of literature supports the potential use of functional nutrients, which exhibit potential neuroprotective properties to mitigate these diseases. Among the most studied dietary molecules, polyphenols stand out because of their multiple and often overlapping reported modes of action. However, ambiguity still exists as to the significance of their influence on human health. This review discusses the characteristics and functions of polyphenols that shape their potential therapeutic actions in neurodegenerative diseases while the less-explored gaps in knowledge of these nutrients will also be highlighted.

Epidemiological evidence suggests a correlation between diabetes and age-related neurodegenerative disorders, including Alzheimer's and Parkinson's diseases. Hyperglycemia causes oxidative stress in vulnerable tissues such as the brain. We recently demonstrated that elevated levels of glucose lead to the death of dopaminergic neurons in culture through oxidative mechanisms. Considering the lack of literature addressing dopaminergic alterations in diabetes with age, the goal of this study was to characterize the state of 2 critical dopaminergic pathways in the nicotinamide-streptozotocin rat model of long-term hyperglycemia, specifically the nigrostriatal motor pathway and the reward-associated mesocorticolimbic pathway. Neuronal and glial alterations were evaluated 3 and 6 months after hyperglycemia induction, demonstrating preferential degeneration of the nigrostriatal pathway complemented by a noticeable astrogliosis and loss of microglial cells throughout aging. Behavioral tests confirmed the existence of motor impairments in hyperglycemic rats that resemble early parkinsonian symptomatology in rats, pensuing from nigrostriatal alterations. These results solidify the relation between hyperglycemia and nigrostriatal dopaminergic neurodegeneration, providing new insight on the higher occurrence of Parkinson's disease in diabetic patients., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Chronic pain is associated with autonomic disturbance. However, specific effects of chronic back pain on sympathetic regulation remain unknown. Chronic pain is also associated with structural changes in the anterior cingulate cortex (ACC), which may be linked to sympathetic dysregulation. The aim of this study was to determine whether sympathetic regulation and ACC surface and volume are affected in a rat model of chronic back pain, in which complete Freund Adjuvant (CFA) is injected in back muscles. Sympathetic regulation was assessed with renal blood flow (RBF) changes induced by electrical stimulation of a hind paw, while ACC structure was examined by measuring cortical surface and volume. RBF changes and ACC volume were compared between control rats and rats injected with CFA in back muscles segmental (T10) to renal sympathetic innervation or not (T2). In rats with CFA, chronic inflammation was observed in the affected muscles in addition to increased nuclear factor-kappa B (NF-kB) protein expression in corresponding spinal cord segments (p=0.01) as well as decreased ACC volume (p<0.05). In addition, intensity-dependent decreases in RBF during hind paw stimulation were attenuated by chronic pain at T2 (p's<0.05) and T10 (p's<0.05), but less so at T10 compared with T2 (p's<0.05). These results indicate that chronic back pain alters sympathetic functions through non-segmental mechanisms, possibly by altering descending regulatory pathways from ACC. Yet, segmental somato-sympathetic reflexes may compete with non-segmental processes depending on the back region affected by pain and according to the segmental organization of the sympathetic nervous system., (Copyright © 2017 IBRO. Published by Elsevier Ltd. All rights reserved.)

Parkinson's disease (PD) is a progressive neurodegenerative disorder, primarily affecting dopaminergic neurons in the substantia nigra. There is currently no cure for PD and present medications aim to alleviate clinical symptoms, thus prevention remains the ideal strategy to reduce the prevalence of this disease. The goal of this study was to investigate whether oleuropein (OLE), the major phenolic compound in olive derivatives, may prevent neuronal degeneration in a cellular dopaminergic model of PD, differentiated PC12 cells exposed to the potent parkinsonian toxin 6-hydroxydopamine (6-OHDA). We also investigated OLE's ability to mitigate mitochondrial oxidative stress and modulate the autophagic flux. Our results obtained by measuring cytotoxicity and apoptotic events demonstrate that OLE significantly decreases neuronal death. OLE could also reduce mitochondrial production of reactive oxygen species resulting from blocking superoxide dismutase activity. Moreover, quantification of autophagic and acidic vesicles in the cytoplasm alongside expression of specific autophagic markers uncovered a regulatory role for OLE against autophagic flux impairment induced by bafilomycin A1. Altogether, our results define OLE as a neuroprotective, anti-oxidative and autophagy-regulating molecule, in a neuronal dopaminergic cellular model.

The role of secreted soluble factors in the modification of cellular responses is a recurrent theme in the study of all tissues and systems. In an attempt to make straightforward the very complex relationships between the several cellular subtypes that compose multicellular organisms, in vitro techniques have been developed to help researchers acquire a detailed understanding of single cell populations. One of these techniques uses inserts with a permeable membrane allowing secreted soluble factors to diffuse. Thus, a population of cells grown in inserts can be co-cultured in a well or dish containing a different cell type for evaluating cellular changes following paracrine signaling in the absence of cell-cell contact. Such insert co-culture systems offer various advantages over other co-culture techniques, namely bidirectional signaling, conserved cell polarity and population-specific detection of cellular changes. In addition to being utilized in the field of inflammation, cancer, angiogenesis and differentiation, these co-culture systems are of prime importance in the study of the intricate relationships that exist between the different cellular subtypes present in the central nervous system, particularly in the context of neuroinflammation. This article offers general methodological guidelines in order to set up an experiment in order to evaluating cellular changes mediated by secreted soluble factors using an insert co-culture system. Moreover, a specific protocol to measure the neuroinflammatory effects of cytokines secreted by lipopolysaccharide-activated N9 microglia on neuronal PC12 cells will be detailed, offering a concrete understanding of insert co-culture methodology.

Sheltered in a bony cage, populated by cells with little regenerative potential, the central nervous system (CNS) could likely not withstand classic inflammation without risking major sequelae. As a consequence, it had to develop an original way to provide surveillance, defence and reparation, which relies on both the complex architecture of the periphery-nervous parenchyma exchange zones, and the tightly regulated collaboration between all the cell populations that reside in or pass through the CNS. Despite its tight regulation, neuroinflammation is sometimes the cause of irreversible loss but it is also where the solution stands. The specific immune crosstalk that takes place in the CNS needs to be decoded in order to identify the best therapeutic strategies aimed at helping the CNS to restore homeostasis in problematic situations, such as in the case of neurodegenerative disorders. This review deals with this double-edged sword nature of neuroinflammation., (© 2015 médecine/sciences – Inserm.)

Parkinson's disease (PD) is the second most common neurodegenerative disease, and it is characterized by the loss of the neurotransmitter dopamine and neuronal degeneration in the substantia nigra pars compacta. Thus far, current therapeutic strategies have failed to address neuronal degeneration. It has been reported that overproduction of reactive oxygen species, resulting in oxidative stress, and neuroinflammation play an important role in neurodegenerative diseases through the induction of macromolecular oxidative damage and modulation of intracellular signaling pathways concurring to neuronal cell death. Indeed, anti-oxidant and anti-inflammatory drugs have been the subject of recommendation as a complementary therapy alongside an effective symptomatic treatment to hamper the progression of PD. Today, much attention is paid to polyphenols in light of their potent capacity to reduce oxidative stress and inflammation, while having much fewer side effects than most other drugs. Camellia sinensis L. is the most common ancient herbal tea prepared as a beverage worldwide and it possesses numerous beneficial effects on human health. Epigallocatechin-3-gallate is the best-known bioactive component of C. sinensis and is recognized to exert potent neuroprotective effects against oxidative stress, neuroinflammation, protein aggregation, autophagy, and neuronal cell death in vitro as well as in vivo. The present review appraises the available literature on the beneficial role of epigallocatechin-3-gallate pertaining to dopaminergic degeneration characteristic of PD with particular emphasis on its possible mechanisms of action.

A growing number of studies have revealed that natural molecules own interesting antioxidant and anti-apoptotic properties in cell culture as well as in animal models of human diseases such as cancer, inflammatory and neurodegenerative diseases. During the past sixty years, several cucurbitacins have been isolated from a number of cucurbitaceous species, amongst others. Cucurbitacins are triterpenoid compounds originally identify as the bitter components of the Cucurbit family that demonstrated several pro-survival activities in various model of cellular decay. Specifically, Cucurbitacin E (CuE), an oxygenated tetracyclic triterpenoid, has been investigated in a wider array of bioactivities, mainly immunomodulatory. Recently, CuE has been reported to possess anti-inflammatory and anti-tumorigenic properties mediated by its action on the cellular cytoskeleton, on mitotic pathways as well as on cellular autophagy. Few studies also pinpoint the role of CuE in the nervous system as cytostatic for gliomas and neuroprotective in a model of Parkinson's diseases. This review deals with the use of CuE in various experimental models as one of the most promising therapeutic natural molecules against cancer proliferation, as an immunomudulator and for the prevention of neurodegeneration.

Natural molecules are under intensive study for their potential as preventive and/or adjuvant therapies for neurodegenerative disorders such as Parkinson's disease (PD). We evaluated the neuroprotective potential of cucurbitacin E (CuE), a tetracyclic triterpenoid phytosterol extracted from the Ecballium elaterium (Cucurbitaceae), using a known cellular model of PD, NGF-differentiated PC12. In our postmitotic experimental paradigm, neuronal cells were treated with the parkinsonian toxin 1-methyl-4-phenylpyridinium (MPP(+)) to provoke significant cellular damage and apoptosis or with the potent N,N-diethyldithiocarbamate (DDC) to induce superoxide (O2(•-)) production, and CuE was administered prior to and during the neurotoxic treatment. We measured cellular death and reactive oxygen species to evaluate the antioxidant and antiapoptotic properties of CuE. In addition, we analyzed cellular macroautophagy, a bulk degradation process involving the lysosomal pathway. CuE showed neuroprotective effects on MPP(+)-induced cell death. However, CuE failed to rescue neuronal cells from oxidative stress induced by MPP(+) or DDC. Microscopy and western blot data show an intriguing involvement of CuE in maintaining lysosomal distribution and decreasing autophagy flux. Altogether, these data indicate that CuE decreases neuronal death and autophagic flux in a postmitotic cellular model of PD.

Nuclear receptors (Nurs) represent a large family of gene expression regulating proteins. Gathering evidence indicates an important role for Nurs as transcription factors in dopamine neurotransmission. Nur77, a member of the Nur superfamily, plays a role in mediating the effects of antiparkinsonian and neuroleptic drugs. Besides, Nur77 survival and apoptotic roles depend largely on its subcellular localization. Estrogens are known for their neuroprotective properties, as demonstrated in animal and clinical studies. However, their action on Nur77 translocation pertaining to neuroprotection has not been investigated yet. The aim of our study was to perform a kinetic study on the effect of neurotoxic 6-hydroxydopamine (6-OHDA) and 17β-estradiol (E2) on the subcellular localization of Nur77 with reference to the modulation of apoptosis in PC12 cells. Our results demonstrate that E2 administration alone does not affect Nur77 cytoplasmic/nuclear ratio, mRNA levels, or apoptosis in PC12 cells. The neurotoxin 6-OHDA significantly enhances cytoplasmic localization of Nur77 after merely 3 h, while precipitating apoptosis. 6-OHDA also increases Nur77 transcription, which could partly explain the rise in cytoplasmic localization of the protein. Finally, treatment with both E2 and 6-OHDA delays Nur77 accumulation in the cytoplasm and delays cell death for a few hours in our cellular paradigm. Pre-treatment with E2 does not alter the increase in levels of Nur77 mRNA produced by 6-OHDA, suggesting that a raise in nuclear translocation is likely responsible for the stabilization of the cytoplasmic/nuclear ratio until 6 h. These results suggest an intriguing cooperation between E2 and Nur77 toward cellular fate guidance.

Resveratrol (RESV), a polyphenolic natural compound, has long been acknowledged to have cardioprotective and antiinflammatory actions. Evidence suggests that RESV has antioxidant properties that reduce the formation of reactive oxygen species leading to oxidative stress and apoptotic death of dopaminergic (DAergic) neurons in Parkinson's disease (PD). Recent literature has recognized hyperglycemia as a cause of oxidative stress reported to be harmful for the nervous system. In this context, our study aimed (a) to evaluate the effect of RESV against high glucose (HG)-induced oxidative stress in DAergic neurons, (b) to study the antiapoptotic properties of RESV in HG condition, and c) to analyze RESV's ability to modulate p53 and GRP75, a p53 inactivator found to be under expressed in postmortem PD brains. Our results suggest that RESV protects DAergic neurons against HG-induced oxidative stress by diminishing cellular levels of superoxide anion. Moreover, RESV significantly reduces HG-induced apoptosis in DAergic cells by modulating DNA fragmentation and the expression of several genes implicated in the apoptotic cascade, such as Bax, Bcl-2, cleaved caspase-3, and cleaved PARP-1. RESV also prevents the pro-apoptotic increase of p53 in the nucleus induced by HG. Such data strengthens the correlation between hyperglycemia and neurodegeneration, while providing new insight on the high occurrence of PD in patients with diabetes. This study enlightens potent neuroprotective roles for RESV that should be considered as a nutritional recommendation for preventive and/or complementary therapies in controlling neurodegenerative complications in diabetes.

Under normal conditions, most of the central nervous system (CNS) is protected by the blood brain barrier (BBB) from systemic inflammation progression and from the infiltration of immune cells. As a consequence, the CNS developed an original way to provide surveillance, defense and repair, which relies on the complex process of neuroinflammation. Despite tight regulation, neuroinflammation is frequently the cause of irreversible nerve cell loss but it is also where the solution lies. Specific immune crosstalk taking place in the CNS needs to be decoded in order to identify the best therapeutic strategies aimed at helping the CNS restore homeostasis in difficult conditions such as in neurodegenerative disorders. This review deals with the double-edged sword nature of neuroinflammation and the use of resveratrol in various models as one of the most promising therapeutic molecules for preventing the consequences of nerve cell autodestruction.

A growing body of evidence indicates that the majority of Parkinson's disease (PD) cases are associated with microglia activation with resultant elevation of various inflammatory mediators and neuroinflammation. In this study, we investigated the effects of 2 natural molecules, quercetin and sesamin, on neuroinflammation induced by the Parkinsonian toxin 1-methyl-4-phenylpyridinium (MPP(+)) in a glial-neuronal system. We first established that quercetin and sesamin defend microglial cells against MPP(+)-induced increases in the mRNA or protein levels of 3 pro-inflammatory cytokines (interleukin-6, IL-1β and tumor necrosis factor-alpha), as revealed by real time-quantitative polymerase chain reaction and enzyme-linked immunoabsorbent assay, respectively. Quercetin and sesamin also decrease MPP(+)-induced oxidative stress in microglial cells by reducing inducible nitric oxide synthase protein expression as well as mitochondrial superoxide radicals. We then measured neuronal cell death and apoptosis after MPP(+) activation of microglia, in a microglial (N9)-neuronal (PC12) coculture system. Our results revealed that quercetin and sesamin rescued neuronal PC12 cells from apoptotic death induced by MPP(+) activation of microglial cells. Altogether, our data demonstrate that the phytoestrogen quercetin and the lignan sesamin diminish MPP(+)-evoked microglial activation and suggest that both these molecules may be regarded as potent, natural, anti-inflammatory compounds.

We have demonstrated previously that the brassinosteroid (BR) 24-epibrassinolide exerts neuroprotective effects deriving from its antioxidative properties. In this study, we synthesized 2 natural BRs and 5 synthetic analogs and analyzed their neuroprotective actions in neuronal PC12 cells, against 1-methyl-4-phenylpyridinium (MPP(+)), a neurotoxin known to induce oxidative stress and degenerescence of dopaminergic neurons characteristic of Parkinsonian brains. We also tested the neuroprotective potential of 2 commercially available BRs. Our results disclosed that 6 of the 9 BRs and analogs tested protected neuronal PC12 cells against MPP(+) toxicity. In addition, our structure-activity study suggests that the steroid B-ring and lateral chain play an important role for their neuroprotective action., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Oxidative stress and apoptosis are frequently cited to explain neuronal cell damage in various neurodegenerative disorders, such as Parkinson' s disease. Brassinosteroids (BRs) are phytosterols recognized to promote stress tolerance of vegetables via modulation of the antioxidative enzyme cascade. However, their antioxidative effects on mammalian neuronal cells have never been examined so far. We analyzed the ability of 24-epibrassinolide (24-Epi), a natural BR, to protect neuronal PC12 cells from 1-methyl-4-phenylpyridinium- (MPP(+)-) induced oxidative stress and consequent apoptosis in dopaminergic neurons. Our results demonstrate that 24-Epi reduces the levels of intracellular reactive oxygen species and modulates superoxide dismutase, catalase, and glutathione peroxidase activities. Finally, we determined that the antioxidative properties of 24-Epi lead to the inhibition of MPP(+)-induced apoptosis by reducing DNA fragmentation as well as the Bax/Bcl-2 protein ratio and cleaved caspase-3. This is the first time that the potent antioxidant and neuroprotective role of 24-Epi has been shown in a mammalian neuronal cell line.

Reactive oxygen species produced by oxidative stress may participate in the apoptotic death of dopamine neurons distinctive of Parkinson's disease. Resveratrol, a red wine extract, and quercetin, found mainly in green tea, are two natural polyphenols, presenting antioxidant properties in a variety of cellular paradigms. The aim of this study was to evaluate the effect of resveratrol and quercetin on the apoptotic cascade induced by the administration of 1-methyl-4-phenylpyridinium ion (MPP(+)), a Parkinsonian toxin, provoking the selective degeneration of dopaminergic neurons. Our results show that a pre-treatment for 3 h with resveratrol or quercetin before MPP(+) administration could greatly reduce apoptotic neuronal PC12 death induced by MPP(+). We also demonstrated that resveratrol or quercetin modulates mRNA levels and protein expression of Bax, a pro-apoptotic gene, and Bcl-2, an anti-apoptotic gene. We then evaluated the release of cytochrome c and the nuclear translocation of the apoptosis-inducing factor (AIF). Altogether, our results indicate that resveratrol and quercetin diminish apoptotic neuronal cell death by acting on the expression of pro- and anti-apoptotic genes. These findings support the role of these natural polyphenols in preventive and/or complementary therapies for several human neurodegenerative diseases caused by oxidative stress and apoptosis.

Oxidative stress is regarded as a mediator of nerve cell death in several neurodegenerative disorders, such as Parkinson's disease. Sesamin, a lignan mainly found in sesame oil, is currently under study for its anti-oxidative and possible neuroprotective properties. We used 1-methyl-4-phenyl-pyridine (MPP(+)) ion, the active metabolite of the potent parkinsonism-causing toxin 1-methyl-4-phenyl-1,2,5,6-tetrahydropyridine, to produce oxidative stress and neurodegeneration in neuronal PC12 cells, which express dopamine, as well as neurofilaments. Our results show that picomolar doses of sesamin protected neuronal PC12 cells from MPP(+)-induced cellular death, as revealed by colorimetric measurements and production of reactive oxygen species. We also demonstrated that sesamin acted by rescuing tyrosine hydroxylase levels from MPP(+)-induced depletion. Sesamin, however, did not modulate dopamine transporter levels, and estrogen receptor-alpha and -beta protein expression. By examining several parameters of cell distress, we found that sesamin also elicited a strong increase in superoxide dismutase activity as well as protein expression and decreased catalase activity and the MPP(+) stimulated inducible nitric oxide synthase protein expression, in neuronal PC12 cells. Finally, sesamin possessed significant anti-inflammatory properties, as disclosed by its potential to reduce MPP(+)-induced interleukin-6 mRNA levels in microglia. From these studies, we determined the importance of the lignan sesamin as a neuroprotective molecule and its possible role in complementary and/or preventive therapies of neurodegenerative diseases.

Phytoestrogens, and particularly resveratrol, a red wine polyphenol, are currently under study for their therapeutic antioxidant properties. Administration of the dopaminergic neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) to C57BL/6 mice targets nigrostriatal dopaminergic neurons, leading to cell death and striatal dopamine (DA) depletion. The aim of the present study was to analyze the protective effect of a diet rich in resveratrol against MPTP-induced neuronal death. Male mice were kept on a phytoestrogen-free diet, supplemented or not with 50 or 100 mg/kg/day of resveratrol for 1 or 2 weeks, after which MPTP was injected intraperitoneally. We observed that daily administration of resveratrol prevented MPTP-induced depletion of striatal DA, and maintained striatal tyrosine hydroxylase (TH) protein levels. Our results also demonstrated that mice treated with resveratrol prior to MPTP administration showed more abundant TH-immunopositive neurons than mice given only MPTP, indicating that resveratrol protects nigral neurons from MPTP insults. Altogether, these data revealed that resveratrol can counteract the toxic effects of the neurotoxin MPTP and, as such, it may be regarded as a powerful molecule for complementary neuroprotective therapy.

Parkinson's disease (PD) is a movement disorder characterized by a progressive loss of nigrostriatal dopaminergic neurons. Microglia activation and neuroinflammation have been associated with the pathogenesis of PD. Indeed, cytokines have been proposed as candidates that mediate the apoptotic cell death of dopaminergic neurons seen in PD. In this study, we investigated the effect of two natural polyphenols, resveratrol and quercetin, on neuroinflammation. For glial cells, we observed that lipopolysaccharide (LPS)-induced mRNA levels of two proinflammatory genes, interleukin 1-alpha and tumor necrosis factor-alpha, are strongly decreased by treatments with resveratrol or quercetin. We also undertook microglial-neuronal coculture to examine the influence of resveratrol and quercetin on dopaminergic neuronal cell death evoked by LPS-activated microglia. Cytotoxicity assays were performed to evaluate the percentage of cell death, with apoptotic cells identified by both the TdT-mediated dUTP nick end labeling technique and the detection of cleaved caspase-3. We report that treatment of N9 microglial cells with resveratrol or quercetin successfully reduced the inflammation-mediated apoptotic death of neuronal cells in our coculture system. Altogether our results demonstrate that resveratrol and quercetin diminished apoptotic neuronal cell death induced by microglial activation and suggest that these two phytoestrogens may be potent antiinflammatory compounds., ((c) 2007 Wiley-Liss, Inc.)

Dopaminergic cell loss in the mesencephalic substantia nigra is the hallmark of Parkinson's disease and may be associated with abnormal oxidative metabolic activity. However, the delicate balance underlying dopamine decline and oxidative stress is still a matter of debate. The aim of this study was to analyze the possible modulation of D2 agonists and antagonists on MPP+ (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridinium ion) -induced cellular death in differentiated and undifferentiated PC12 cells. Using colorimetric assays, western blots and reverse transcriptase-PCR, we demonstrated that two D2 agonists, bromocriptine and quinpirole, consistently increased MPP+ -induced cytotoxicity in both differentiated and undifferentiated PC12 cells, whereas D2 antagonists do not modulate cell death. However, this increase in cellular death was reversed when bromocriptine or quinpirole were used in presence of D2 antagonists. On the other hand, 1-{2-[bis-(4-fluorophenyl)methoxy]ethyl}-4-(3-phenylpropyl)piperazine (GBR 12909), a potent inhibitor of the dopamine transporter, partially reversed MPP+ -induced cellular death and completely abolished the increase of cellular death induced by bromocriptine. Dopamine agonists and antagonists also modulate the expression of the dopamine transporter in PC12 cells; in particular, bromocriptine may alter MPP+ uptake by increasing DAT expression We also show that, in our cellular paradigm, D2 receptor mRNA levels are more abundant that D3 mRNA levels and MPP+ and /or bromocriptine could not modulate D2 gene expression while D3 gene expression clearly decrease after MPP+ and /or bromocriptine treatment.

Oxidative stress plays an important role in the pathogenesis of neurodegenerative diseases such as Parkinson's disease (PD). Neuronal death in the substantia nigra of PD patients is partly caused by exacerbated oxidative damage. Our previous studies demonstrated that oxidative stress can alter the structure and stability of neurofilament (NF) proteins and that 17-alpha- and 17-beta-estradiol are potent neuroprotective agents. The aim of this study was to investigate the cytoskeletal target of neuroprotection by estrogens in neuronal PC12 cells. We induced oxidative stress by MPP+ administration for 24 h, and 17-alpha- and 17-beta-estradiol were used as neuroprotective drugs. We measured gene expression and protein expression of each NF subunit, NFL, NFM, and NFH, by semiquantitative RT-PCR, Western blot, and immunofluorescence. Our results demonstrate that NFL mRNA and protein levels are not modulated by MPP+ or estradiol isomers, whereas NFM gene expression, as well as protein expression, are strongly influenced by MPP+, 17-alpha-, and 17-beta-estradiol after a 24-h treatment. Finally, mRNA levels of the most phosphorylated subunits, NFH, are not changed by MPP+ or treatment with both estradiol isomers, whereas NFH protein expression is decreased by the same treatments. These results suggest that oxidative stress affects neuronal cytoskeleton, maybe though proteolysis and/or abnormal structural changes in NFs. Then, 17-alpha- and 17-beta-estradiol might help the neuronal cell in recovering after oxidative stress by inducing protein expression of NFM and NFH subunits.

Several studies have demonstrated that C57 and DBA mice exhibit behavioural differences in diverse learning tasks as well as variations in the expression of long-term potentiation (LTP) in the hippocampus. In the present investigation, we tested the possibility that these differences between the two strains might be attributable to differential regulation of hippocampal alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptors by calcium-dependent mechanisms. Using in vitro receptor autoradiography, we found that calcium treatment of C57 mice sections resulted in a marked increase of 3H-AMPA binding in areas CA3 and CA1 of the hippocampus and in the dentate gyrus. However, we discovered that the ability of calcium to upregulate 3H-AMPA binding in the DBA strain was much lower than in corresponding regions from the C57 strain. Western blot and immunohistochemical experiments indicated that truncation of AMPA receptor subunits by calcium-dependent mechanisms was possibly not responsible for the binding differences, as no significant variations in glutamate receptor subunit 1 (GluR1) and GluR2/3 immunoreactivity were observed between the two strains after calcium treatment. Interestingly, we found that strain-related variations in the regulation of 3H-AMPA binding by calcium were totally eliminated when brain sections were preincubated with preferential inhibitors of lipoxygenase (LO) pathways of arachidonic acid (AA) metabolism. Taken together, these results suggest that calcium-induced regulation of AMPA receptors varies between the two strains and that this variation might be linked to the production of specific AA metabolites., (Copyright 2004 Elsevier B.V.)

We investigated the dopaminergic (DA) neuronal degeneration in animals subjected to systemic treatment of rotenone via subcutaneous delivery. Behavioral observations revealed a hypokinetic period in rats sacrificed at 3 and 5 days, and dystonic episodes in animals sacrificed at 8 days. Less than 20% of the total number of animals given rotenone depicted brain lesions after 8 days of treatment, as demonstrated by a significant loss of DA fibers in the striatum, but not of DA nigral neurons. Tyrosine hydroxylase-negative striatal territories were characterized by post-synaptic toxicity as demonstrated by a decreased number of interneurons labeled for choline acetyltransferase, NADPH-diaphorase, parvalbumin, and projection neurons labeled for calbindin and nerve growth factor inducible-B (NGFI-B). Post-synaptic neurodegeneration was demonstrated further by abundant striatal staining for Fluoro-Jade. Decrease in the nuclear orphan receptor Nurr1 expression was the only significant change observed at the level of the substantia nigra. Autopsy reports confirmed that animals suffered from severe digestion problems. These data suggest that hypokinesia observed between 3 and 5 days is the result of general health problems rather than a specific motor deficit associated to Parkinson's disease (PD) symptoms. Overall, the effects of rotenone toxicity are widespread, and subcutaneous administration of this toxin does not provide the neuropathological and behavioral basis for a relevant and reliable PD model.

Oxidative stress is currently considered a mediator of cell death in several neurodegenerative diseases. Notably, it may play an important role in the degeneration of dopamine neurons of the substantia nigra in Parkinson's disease. We examined the effect of a strong oxidant, the herbicide paraquat, on cell distress using native and neuronal pheochromocytoma PC12 cells. Paraquat administration for 8 hours induced a significant cellular death in both native and in neuronal PC12 cells. Since the anti-oxidant properties of estrogens may promote neuroprotection in vitro and in vivo, we then investigated the ability of estradiol stereoisomers, 17alpha-estradiol and 17- beta-estradiol, to rescue PC12 cells submitted to paraquat-induced oxidative stress. Our results show a protective effect of both estradiol stereoisomers in neuronal PC12 cells treated with paraquat, whereas this effect could not be observed in native PC12 cells. We also demonstrate that estrogen receptor beta protein expression is modulated by paraquat administration in native PC12 cells, while paraquat does not change estrogen receptor beta ?expression in neuronal PC12 cells. Paraquat also decreases estrogen receptor alpha in neuronal PC12 cells, thus suggesting new routes for paraquat to collapse cellular metabolism. Besides, the oxidation of dihydrodhodamine-123 into fluorescent rhodamine in the presence of paraquat but not in presence of paraquat and 17 alpha-estradiol or 17 beta-estradiol, sustain a possible direct scavenging role of both estradiol stereoisomers.

It is now well recognized that many environmental contaminants are capable of disrupting endocrine processes in a variety of species, including birds, mammals, reptiles, and fish. Among these contaminants are toxaphene and polychlorinated biphenyls (PCBs), two of the most prevalent contaminants present in aquatic food chains of the Great Lakes and the Canadian Arctic region. We set out to investigate the possible endocrine-modulating activities of toxaphene, the PCB congener 3,3',4,4'-tetrachlorobiphenyl (TeCB), an equimolar mixture of both compounds (toxaphene/TeCB), and estradiol (E(2)) (E(2)/toxaphene, E(2)/TeCB) on prolactin (PRL) mRNA expression. Concentrations ranging from 10(-7) to 10(-11)M for both toxaphene and TeCB were assayed but only toxaphene modulated PRL mRNA levels, as determined by relative quantitative reverse transcriptase-polymerase chain reaction. Maximal induction by toxaphene was seen at 10(-7)M, resulting in a fourfold increase in PRL mRNA levels. No interactions were observed for combinations of the test substances. Our study demonstrates that toxaphene may exhibit estrogen-like activity by modulating PRL mRNA levels in GH(3) cells.

Oxidative stress has been recently considered as a mediator of nerve cell death in several neurodegenerative diseases. We studied the effect of the parkinsonism-inducing toxine 1-methyl-4-phenyl-pyridine (MPP+) on several parameters of cell distress using native and neuronal PC12 cells. Then, since estrogens have been reported to prevent neuronal degeneration caused by oxidative damage, we investigated the ability of 17beta- estradiol (E2); two Ginkgo biloba extracts, EGb 761 and Cp 202; as well as two flavonoids, quercetin and kaempferol, to rescue PC12 cells submitted to MPP+- induced oxidative stress. Our results consistently show that both Ginkgo biloba extracts could prevent cell death in native and neuronal PC12 cells, while in neuronal PC12 cells also quercetin and E2 could reverse MPP+ neurotoxic effet. Western blot analysis demonstrated that MPP+ injuries might modulate dopamine transporter (DAT) protein expression but not estrogen receptor beta (ERbeta) protein expression. EGb 761 and Cp 202 also modulate DAT and ERbeta protein expression in neuronal cells. From these studies, we outline the importance of testing estrogen-like plant-derived molecules as potent antioxidants and examine their effect on protein expression.

A large body of experimental evidence supports a role for oxidative stress as a mediator of nerve cell death in Parkinson's disease. To better understand the cellular insult of oxidative stress on dopaminergic neurons, we studied the cytotoxic effect of the 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) metabolite, 1-methyl-4-phenyl pyridium (MPP(+)), on several parameters of cell distress using neuronal PC12 cells. We also measured the level of protein expression for the dopamine transporter and the estrogen receptors alpha and beta. Since estrogens have been reported to prevent neuronal degeneration caused by increased oxidative burden, we investigated the ability of 17beta-estradiol, the stereoisomer 17alpha-estradiol, and several phytoestrogens to rescue neuronal PC12 cells submitted to MPP(+)-induced cytotoxicity. Our results consistently show a protective effect of 17alpha-estradiol, 17beta-estradiol and certain phytoestrogens such as quercetin and resveratrol, in neuronal PC12 cells treated with MPP(+). In our cellular paradigm, phytoestrogens coumestrol, genistein, and kaempferol did not revert MPP(+)-induced cellular death. By Western blot, we demonstrated that administration of MPP(+) alone decrease dopamine transporter expression, while treatments with MPP(+) together with 17alpha-estradiol, 17beta-estradiol, quercetin, or resveratrol could restore dopamine transporter protein expression to control levels. Moreover, the same treatments did not modulate alpha estrogen receptor or beta estrogen receptor expression. By these studies, we aim to provide more evidence for the involvement of phytoestrogens in the process of neuroprotection and to test our hypothesis that some of these compounds may act as neuroprotective molecules and have a lesser hormonal effect than estrogens., (Copyright 2002 Wiley-Liss, Inc.)

It is now well recognized that estrogenic signaling mechanisms are far more complex than once thought. Several crosstalks between the estrogen receptor and other signaling pathways may influence the estrogenic stimulation of cell growth. Thus, the estrogenic effects of several environmental contaminants, now suspected to act as endocrine disrupters, may be influenced by a simultaneous stimulation of other signaling pathways. The aim of this study was to investigate whether the growth response of two estrogen-responsive cell lines, MCF-7 and GH3, treated with xenoestrogens might be affected by the addition of growth factors to their culture medium. Cells were treated with two known xenoestrogens, endosulfan and chlordane, alone or in the presence of insulin-like growth factor-1 and epidermal growth factor, respectively, and their growth was measured using the 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxyanilide proliferation assay. Our results show that treatment with endosulfan or chlordane as well as treatment with growth factors increased cell growth, while the administration of xenoestrogens together with growth factors triggered a partly additive response with no antagonist or synergistic effect. These results sustain a role for xenoestrogens in cellular growth.

Xenoestrogens are widely used environmental chemicals that have recently been under scrutiny because of their possible role as endocrine disrupters. Among them are endosulfan and chlordane, two persistent insecticides suspected to act as estrogens in living organisms. To test and better understand the potential estrogenic activity of these chemicals, we used a pituitary cell line (GH(3)) known to respond to estrogens by increasing its secretion of prolactin (PRL), a hormone that is well known for its many physiological functions, especially in fetal growth, development, and reproduction. We measured the levels of PRL secretion and PRL mRNA transcription using immunometric tests, Northern blots, and relative quantitative RT-PCR. We also employed the XTT proliferation assay to compare the growth of GH(3) cells stimulated with 17-beta estradiol and endosulfan or chlordane. Our results show that endosulfan and chlordane are able to induce a substantial increase of PRL expression while these two chemicals do not increase cell growth. Together, our results suggest that endosulfan and chlordane could indeed modulate an estrogen-inducible gene such as PRL, possibly acting via second messenger-mediated cellular mechanisms instead of solely competing with estrogens for the nuclear estrogen receptor sites., ((c) 2002 Elsevier Science (USA))

Neurofilament proteins are highly phosphorylated molecules in the axonal compartment of the adult nervous system. We report the structural analysis of neurofilament proteins after oxidative damage. SDS-PAGE, immunoblotting, circular dichroism, and Fourier transform infrared spectroscopy were used to investigate the relative sensitivity of neurofilaments to oxidative stress and to identify changes in their molecular organization. An ascorbate-Fe+3-O2 buffer system as well as catechols were used to generate free radicals on a substrate of phosphorylated and dephosphorylated neurofilaments. By Fourier Transform Infrared spectroscopy and circular dichroism, we established that the neurofilament secondary structure is mainly composed of alpha-helices and that after free radical damage of the peptide backbone of neurofilaments, those helices are partly modified into beta-sheet and random coil structures. These characteristic reorganizations of the neurofilament structure after oxidative exposure suggest that free radical activity might play an important role in the biogenesis of the cytoplasmic inclusions found in several neurodegenerative diseases.

In adult rats, environmental enrichment has been shown to selectively increase -AMPA binding in the hippocampus but the molecular mechanisms underlying this effect remain unknown. We used in situ hybridization with antisense oligonucleotides to determine possible changes in the hippocampal expression of messenger RNAs for different subunits of AMPA receptors in adult rats following exposure to an enriched environment. Quantitative analysis revealed that mRNA levels for three subtypes of AMPA glutamate receptors (GluR1-3; Flip and Flop variants) were not modified in any hippocampal region after environmental enrichment. In addition, no differences were detected in the levels of GluR1 and GluR2/3 proteins in Western blots of hippocampal membranes from enriched rats. Nevertheless, quantitative ligand binding autoradiography indicated that environmental enrichment evoked a significant and uniform decrease in the capacity of calcium or phosphatidylserine (PS) to up-regulate -AMPA binding in various hippocampal regions but not in the cerebral cortex. These findings support previous observations suggesting that post-translational changes in AMPA receptor properties, as a result of the activation of calcium-dependent processes, may represent an important mechanism underlying long-term modifications of synaptic efficacy in the rat hippocampus., (Copyright 1998 All rights reserved.)

The biochemical mechanisms by which diabetes modulates cognitive function are not well established. Here, we determined the effects of streptozotocin (STZ) administration on the binding properties of alpha-amino-3-hydroxy-5-methylisoxazolepropionic acid (AMPA) and N-methyl-D-aspartate (NMDA) subtypes of glutamate receptors in rats, using quantitative autoradiographic analysis of (3)H-AMPA and [(3)H]glutamate binding on brain tissue sections. The STZ injection (70 mg/kg intraperitoneally) produced a reduction of (3)H-AMPA binding in various brain regions, an effect that is due to a decrease in receptor affinity. The STZ-induced reduction of (3)H-AMPA binding varied in different brain structures, being more pronounced in the striatum, cerebral cortex, and hippocampus and almost absent in the cerebellum. Western blots performed on hippocampal membranes revealed that the decrease in (3)H-AMPA binding is possibly associated with changes in immunologic properties for one glutamate receptor subunit (GluR1). Finally, the effect of STZ-induced diabetes appeared to be specific to the AMPA subtype of glutamate receptors, as the same treatment did not modify [(3)H]glutamate binding to NMDA receptors. These changes in AMPA receptor properties may have important implications for understanding the biochemical mechanisms underlying cognitive impairment in diabetes.

Apolipoprotein E (ApoE) genotype has been found to affect the expression of a variety of neuropsychiatric disorders. We determined ApoE genotype frequencies and their relationship to clinical and pathological features in a diverse cohort of individuals with schizophrenia. There were no differences in ApoE genotype frequencies between schizophrenics and controls. However, the ApoE epsilon 4 genotype was associated with a younger age of onset of schizophrenia, and in an elderly subsample, individuals with the epsilon 4 allele more frequently exhibited co-existent dementia and had more neurofibrillary pathology (although none of the cases met criteria for Alzheimer's disease). This examination of ApoE in relation to clinical and neurobiological features of schizophrenia suggests that it modifies the phenotypic expression of the disease.

Pineal melatonin hormonally transduces photoperiod to influence daily and seasonal cycles in most vertebrates (1, 2). Evidence of melatonin receptors throughout the brain of several fish species (3-5), particularly in retinorecipient structures, also indicates a role in visual processing. Despite the absence of solar light many deepsea organisms show seasonality (6-8). The presence of central melatonin receptors was investigated by quantitative in vitro autoradiography in the deep-sea fish Coryphaenoides (Nematonurus) armatus. Specific, time-dependent, saturable, high affinity and guanine nucleotide sensitive, 2-[125I]iodomelatonin binding was found over the mid-brain tegmentum and hindbrain. Competing ligand potency was iodomelatonin > melatonin >> 5-HT. Although C.(N.) armatus has well developed eyes no 2-[125I]iodomelatonin binding occurred in optic tectum, cerebellum or hypothalamus. Thus melatonin involvement in processing of visual information and control of seasonal physiology via hypothalamic areas appears to be absent in this species. The presence of central G-protein coupled receptors indicates a function for melatonin unrelated to solar light.

Intermediate filament proteins belong to a multigene family and constitute an important cytoskeletal component of most vertebrate cells. Their pattern of expression is tissue specific and is highly controlled during embryonic development. Numerous pathologies are known to be associated with modifications of intermediate filament organisation, although their precise role has not yet been elucidated. The present review focuses on the most recent data concerning the possible causes of intermediate filaments disorganization in specific pathologic conditions affecting the epidermis, the liver, and the nervous system. We discuss the formation of abnormal intermediate filament networks that arise as a consequence of mutations that directly affect intermediate filament structure or are induced by multifactorial causes such as modifications of post-translational processes and changes in the levels of expression.

Apolipoprotein E (APOE) is a lipoprotein expressed in liver and brain as one of three isoforms (APOE 2, APOE 3 and APOE 4). Recent findings suggest that the presence of APOE 4 is associated with an increased risk for both familial Alzheimer's disease and late-onset Alzheimer's disease. We extended these observations by determining the frequency of APOE alleles in patients with pathologically confirmed Alzheimer's Disease (AD), Parkinson's disease (PD), diffuse Lewy Body disease (DLBD), AD with concomitant PD pathology, demented PD patients without or with concomitant AD pathology and in schizophrenics with a progressive dementia (SCHIZ+DEM). The APOE genotype was determined by restriction digestion of polymerase chain reaction-amplified DNA isolated from frozen brain samples. The frequency of the APOE epsilon 4 allele was highest among sporadic AD and DLBD patients (0.30 and 0.38, respectively) and lowest in the SCHIZ+DEM and non-demented PD patients (0.06 and 0.1, respectively). Thus, the APOE epsilon 4 allele is over-represented selectively in patients with dementias associated with plaques and tangles and/or cortical Lewy bodies, but not in demented schizophrenics or non-demented PD patients.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder primarily affecting motor neurons. The etiology of the majority of cases remains unknown. Recent findings from several laboratories suggest a role for neurofilaments in the development of motor neuron disorders. The C-terminal region of the human neurofilament heavy subunit (NEFH) contains a unique functional domain consisting of 43 repeat motifs of the amino acids Lys-Ser-Pro (KSP). This C-terminal region of NEFH forms the sidearm projections which cross-link the neurofilaments. Previously, we have demonstrated polymorphism in the C-terminal region of the human NEFH: an allelic variant of a slightly larger molecular size, containing an additional KSP phosphorylation motif. Novel mutations in this region were found in five ALS patients. We propose that changes in the KSP-repeat domain may affect the cross-linking properties of the heavy neurofilament subunit and perhaps contribute to the development of neurofilamentous swellings in motor neurons, a hallmark of ALS.

We have identified and characterized a new orphan member of the nuclear hormone receptor superfamily, called MB67, which is predominantly expressed in liver. MB67 binds and transactivates the retinoic acid response elements that control expression of the retinoic acid receptor beta 2 and alcohol dehydrogenase 3 genes, both of which consist of a direct repeat hexamers related to the consensus AGGTCA, separated by 5 bp. MB67 binds these elements as a heterodimer with the 9-cis-retinoic acid receptor, RXR. However, MB67 does not bind or activate other retinoic acid response elements with alternative hexamer arrangements or any of several other wild-type and synthetic hormone response elements examined. The transactivation of retinoic acid response elements by MB67 is weaker than that conferred by the retinoic acid receptors but does not require the presence of all-trans retinoic acid, 9-cis-retinoic acid, or any exogenously added ligand. We propose that MB67 plays an important role in the complex network of proteins that govern response to retinoic acid and its metabolites.

Differentiation of neural and muscle cells is characterized by a switch in the expression of the type of intermediate filament protein subunit. In these lineages, vimentin is transiently expressed in the initial stages of development and is gradually replaced by a tissue specific protein. We have identified a giant developmentally regulated antigen (IFAPa-400) which colocalizes with vimentin in the precursor cells of the neurogenic and myogenic lineages of the chick embryo [Chabot and Vincent (1990) Dev. Brain Res. 54, 195-204; Cossette and Vincent (1991) J. Cell Sci. 98, 251-260]. Based on the expression of this protein during neurogenesis and myogenesis, we hypothesize that IFAPa-400 and vimentin define a special intermediate filament network, common to the non-differentiated cells derived from the neuroectoderm and those of the myogenic tissues. We report here the isolation and sequence of partial cDNAs encoding more than 400 amino acids of the carboxy-terminus of this protein. RNA blot analysis and in situ hybridization indicate that IFAPa-400 represents a bona fide developmentally regulated gene product. These results further confirm that IFAPa-400 mRNA transcripts are limited to the early precursor cells of both neurogenic and myogenic lineages.

A large variety of proto-oncogenes are known to be of key importance in cellular growth and differentiation during embryonic development. Using quantitative in situ hybridization, we studied in detail the levels of the proto-oncogenes Ha-ras and c-myc mRNA in embryos and extraembryonic tissues (maternal and embryonic placentas, trophoblast, and endometrial epithelium) during prenatal life of rabbit. cDNA probes encoding for Ha-ras (fragment Kpn 1-BstE II of 883 bp) and c-myc (fragment Pst 1-Pst 1 of 490 bp) were used to detect specific transcripts in fixed cryostat sections. High levels of Ha-ras and c-myc mRNA were detected in the rabbit embryo as well as in the decidua and in the trophoblast as early as day 9 of gestation. At 12 and 15 days of gestation, Ha-ras and c-myc mRNA levels decreased in both embryonic and maternal placenta while in the embryo a significant increase of Ha-ras and c-myc expression was detected with particular evidence in the central nervous system. Finally, at 25 days of gestation the expression of the two proto-oncogenes, Ha-ras and c-myc, was greatly decreased in both the embryo and extraembryonic tissues, and was undetectable by 30 days of gestation. These results show that in rabbit the expression of the two proto-oncogenes Ha-ras and c-myc is localized in the same tissues with similar intensity and follows an unparallel temporal modulation in the embryo and in the extraembryonic tissues during prenatal development.

We have investigated the variations in growth hormone (GH) and somatostatin (SOM) mRNAs in adult, middle-aged and aging rats of both sexes using quantitative in situ hybridization. cDNA probes complementary to GH mRNA and SOM mRNA were used on fixed pituitary and hypothalamic sections, respectively. A clear sexual dimorphism in GH gene expression was observed in all age groups examined, mRNA levels always being higher in males than in females. In male rats, GH mRNA levels reached a maximum at 7 months of age and then dramatically decreased in middle-aged and aging animals. In female animals, the highest expression of GH was observed at 9 months of age followed by a gradual and constant decline during the aging period. SOM mRNA levels in the periventricular nucleus of the hypothalamus also showed a sexual dimorphism in adult rats, being higher in males than females. A gradual decline in SOM mRNA was observed in middle-aged and aging rats of both sexes. These results suggest that middle age and aging are critical periods for the control of GH and SOM gene expression and that the decrease in GH mRNA levels observed during aging is probably not a consequence of an increase of SOM activity.

Insulin-like growth factors (IGFs) are well known as peptide mitogens and important growth factors in fetal as well as in early postnatal development. In particular, IGF II is strongly expressed during fetal life and in neonatal animals. Very little is known about the regulation of IGF II gene expression. In order to study in detail the regulation of IGF II mRNA levels in the liver by the potent nonaromatizable androgen dihydrotestosterone (DHT), we have used quantitative in situ hybridization to detect the mRNA encoding for this growth factor. Pups were separated into 4 groups and injected twice a day immediately after birth with 3 different doses of DHT: 0.1 mg DHT/day, 0.25 mg DHT/day, 0.5 mg DHT/day for 4 and 7 days, and the control groups were injected with the vehicle alone. Animals were perfused with 4% paraformaldehyde and sections from the liver, heart, kidneys and brain were cut with a cryostat. A [35S]-labeled cDNA probe was used to detect IGF II mRNA levels. After hybridization, sections were autoradiographed with X-ray films and then coated with liquid photographic emulsion. Densitometric measurement revealed that, at 4 days of age, IGF II mRNA levels were lower in DHT-treated rats than in control animals. No statistically significant differences in IGF II mRNA levels were observed among the three groups treated with the different doses of DHT, thus revealing that even the lowest dose of DHT (0.1 mg/day) used was sufficient to inhibit IGF II gene expression in neonatal rats. Moreover, at 7 days of age, DHT-treated rats showed the same levels of IGF II mRNA as those observed in rats treated with DHT for 4 days. These results suggest that DHT may play an important role in the regulation of IGF II gene expression in the rat liver during the neonatal period.