Abstract: Stem cells have been increasingly recognized as a potential tool to replace or support cells damaged by the neurodegenerative process that underlies Parkinson's disease (PD). In this frame, human adult mesenchymal stem cells (hMSCs) have been proposed as an attractive alternative to heterologous embryonic or neural precursor cells. To address this issue, in this study we implanted undifferentiated hMSCs into the striatum of rats bearing a lesion of the nigrostriatal pathway induced by local injection of 6-hydroxydopamine (6-OHDA), a widely recognized rodent model of PD. Before grafting, cultured hMSCs expressed markers of both undifferentiated and committed neural cells, including nestin, GAP-43, NSE, beta-tubulin III, and MAP-2, as well as several cytokine mRNAs. No glial or specific neuronal markers were detected. Following transplantation, some hMSCs acquired a glial-like phenotype, as shown by immunoreactivity for glial fibrillary acid protein (GFAP), but only in animals bearing the nigrostriatal lesion. More importantly, rats that received the striatal graft showed increased survival of both cell bodies and terminals of dopaminergic, nigrostriatal neurons, coupled with a reduction of the behavioral abnormalities (apomorphine-induced turning behavior) associated with the lesion. No differentiation of the MSCs toward a neuronal (dopaminergic) phenotype was observed in vivo. In conclusion, our results suggest that grafted hMSCs exert neuroprotective effects against nigrostriatal degeneration induced by 6-OHDA. The mechanisms underlying this effect remain to be clarified, although it is likely that the acquisition of a glial phenotype by grafted hMSCs may lead to the release of prosurvival cytokines within the lesioned striatum.
Abstract: Stimulation of endogenous repair in neurodegenerative diseases, such as Parkinson's disease (PD), appears to be a novel and promising therapeutic application of stem cells (SCs). In fact SCs could propel local microenvironmental signals to sustain active endeavors for damaged neurons substitution, normally failing in non-supportive pathological surroundings. In this study, we demonstrated that two different doses of naïve human adult mesenchymal stem cells (hMSCs), implanted in the striatum of rats lesioned with 6-hydroxydopamine (6-OHDA), positively survived 23 days after transplantation. Their fate was directly influenced by the surrounding host environment while grafted hMSCs, dose dependently, regionally sustained the survival of striatal/nigral dopaminergic terminals and enhanced neurogenesis in the Subventricular Zone (SVZ). The number of proliferative cells (Ki67/Proliferating Cell Nuclear Antigen +) as well as neuroblasts migration significantly augmented in the lesioned striatum of transplanted animals compared to controls. No SVZ astrogenesis was detected in all experimental conditions, irrespectively of graft presence. Activation of endogenous stem cell compartments and rescue of dopaminergic neurons, supported by the persistent release of specific cytokine by MSCs in vivo, appeared in principle able to contrast the neurodegenerative processes induced by the 6-OHDA lesion. Our results suggest that reciprocal influences between grafted cells and endogenous neural precursors could be important for the observed neurorescue effect on several brain regions. Altogether, our data provide remarkable cues regarding the potential of hMSCs in promoting endogenous reparative mechanisms that may prove applicable and beneficial for PD treatment.
Abstract: The loss of nigrostriatal dopaminergic neurons that characterizes Parkinson's disease (PD) causes complex functional alterations in the basal ganglia circuit. Increased glutamatergic activity at crucial points of the circuit may be central to these alterations, thereby contributing to the onset of PD motor symptoms. Signs of neuroinflammation accompanying the neuronal loss have also been observed; also in this case, glutamate-mediated mechanisms may be involved. Glutamate may therefore intervene at multiple levels in PD pathophysiology, possibly through the modulation of metabotropic receptors. To address this issue, we evaluated the effects of systemic treatment with MPEP (2-methyl-6-(phenylethynyl)-pyridine), an antagonist of metabotropic receptor mGluR5, in a rodent model of progressive nigrostriatal degeneration based on the intrastriatal injection of 6-hydroxydopamine (6-OHDA). Following 6-OHDA injection, Sprague-Dawley rats underwent a 4-week, daily treatment with MPEP (1.5mg/kg, i.p.). To investigate whether the effects varied with the progression of the lesion, subgroups of lesioned animals started the treatment at different time-points: (1) immediately, (2) 1 week, or (3) 4 weeks after the neurotoxin injection. Akinesia, dopaminergic nigrostriatal damage and neuroinflammatory response (microglial and astroglial activation) were investigated. MPEP prompted immediate amelioration of 6-OHDA-induced akinesia, as measured by the Adjusting step test, in all subgroups, regardless of the degree of nigrostriatal damage. Conversely, MPEP did not modify neuronal survival or neuroinflammatory response in the nigrostriatal pathway. In conclusion, chronic treatment with MPEP exerted a pure symptomatic effect, further supporting that mGluR5 modulation may be a viable strategy to counteract the basal ganglia functional modifications underlying PD motor symptoms.
Abstract: Alteration of key regulatory kinases may cause aberrant protein phosphorylation and aggregation in Alzheimer's disease (AD) and Parkinson's disease (PD). In this study, we investigated expression and phosphorylation status of glycogen synthase kinase 3 (GSK-3), protein kinase B (Akt) and tau protein in peripheral blood lymphocytes of 20 AD, 25 PD patients and 20 healthy controls. GSK-3 was increased in AD and PD patients. In these latter, GSK-3 levels were positively correlated with daily l-Dopa intake. Phosphorylated Akt expression was augmented in both groups; total Akt levels were increased only in AD patients and were positively correlated with disease duration and severity. Total and phosphorylated tau were increased only in AD, with phospho-tau levels being positively correlated with levels of total tau, Akt, and disease duration. No correlations between protein levels and clinical variables were found in PD patients. Investigation of peripheral changes in the expression of specific kinases may, therefore, lead to the development of innovative biomarkers of neurodegeneration, particularly for AD.
Abstract: In this study we demonstrated that neural rosettes derived from human ES cells can give rise either to neural crest precursors, following expansion in presence of bFGF and EGF, or to dopaminergic precursors after exposure to ventralizing factors Shh and FGF8. Both regionalised precursors are capable of extensive proliferation and differentiation towards the corresponding terminally differentiated cell types. In particular, peripheral neurons, cartilage, bone, smooth muscle cells and also pigmented cells were obtained from neural crest precursors while tyrosine hydroxylase and Nurr1 positive dopaminergic neurons were derived from FGF8 and Shh primed rosette cells. Gene expression and immunocytochemistry analyses confirmed the expression of dorsal and neural crest genes such as Sox10, Slug, p75, FoxD3, Pax7 in neural precursors from bFGF-EGF exposed rosettes. By contrast, priming of rosettes with FGF8 and Shh induced the expression of dopaminergic markers Engrailed1, Pax2, Pitx3, floor plate marker FoxA2 and radial glia markers Blbp and Glast, the latter in agreement with the origin of dopaminergic precursors from floor plate radial glia. Moreover, in vivo transplant of proliferating Shh/FGF8 primed precursors in parkinsonian rats demonstrated engraftment and terminal dopaminergic differentiation. In conclusion, we demonstrated the derivation of long-term self-renewing precursors of selected regional identity as potential cell reservoirs for cell therapy applications, such as CNS degenerative diseases, or for the development of toxicological tests.
Abstract: Long-term treatment of Parkinson disease (PD) is frequently associated with l-3,4-dihydroxyphenylalanine (L-DOPA)-induced dyskinesias (LIDs). L-DOPA-induced dyskinesias are likely due to changes in the signal transduction pathways, at the striatal level, related to pulsatile stimulation of dopamine receptors. We investigated whether markers of this phenomenon can also be detected peripherally. We analyzed mRNA expression for D5 (D1-like) and D3 (D2-like) receptors and levels of second messengers, such as cAMP and free intracellular Ca2+ ([Ca2+]i), in peripheral blood lymphocytes of PD patients with (LID+) or without LIDs (LID-). Patients with PD showed depressed [Ca2+]i rise in response to mitogen-induced activation. The defect was more pronounced in LID+ (-33% with respect to healthy controls) than in LID- patients (-20%). Peripheral blood lymphocyte levels of cAMP were decreased in both LID+ (3.8 +/- 2.9 pmol/10 cells) and LID- patients (4.2 +/- 2.4 pmol/10(6) cells), with respect to controls (6 +/- 2.6 pmol/10(6) cells). No differences were found in dopamine receptor mRNA expression. Our results demonstrate that second messenger levels are altered in the peripheral blood lymphocytes of PD patients treated with dopaminergic agents and that patients with LIDs show further alterations in the regulation of [Ca2+]i homeostasis. This may represent a distinctive trait of patients prone to develop dyskinetic movements.
Abstract: Altered glutamatergic neurotransmission is central to the expression of Parkinson's disease (PD) symptoms and may underlie l-DOPA-induced dyskinesias. Drugs acting on glutamate metabotropic receptors (mGluR) of group I can modulate subthalamic nucleus (STN) overactivity, which plays a pivotal role in these phenomena, and may counteract dyskinesias. To address these issues, we investigated the effects of a 3-week treatment with mGluR5 antagonist 2-methyl-6-(phenylethynyl)-pyridine (MPEP), or of a subthalamic lesion, on abnormal involuntary movements (AIMs) and associated striatal expression of transcription factor FosB/Delta FosB caused by chronic l-DOPA administration, in rats with a nigrostriatal lesion. MPEP virtually abolished AIMs and reduced, dramatically, striatal expression of FosB/Delta FosB. Reduced FosB/Delta FosB expression, coupled with nonsignificant reduction of AIMs, was also observed in STN-lesioned rats. Our data confirm the role of glutamatergic neurotransmission in the pathogenesis of dyskinesias and the potential of mGluR5 antagonists in the treatment of l-DOPA-induced dyskinesias.
Abstract: Abnormal deposition of protein aggregates and increased susceptibility to apoptotic cell death may result from defects in the activity of the ubiquitin-proteasome system (UPS); neurotoxicity related to UPS defects seems to require dopamine to be fully expressed. The aim of this study was to investigate the pro-apoptotic effects caused by proteasomal activity inhibition, as well as the synergistic effect of dopaminergic stimulation in human lymphocytes isolated from healthy volunteers. Cells were incubated 20 h at 37 degrees C, with: (1) lactacystin, (2) increasing concentrations of dopamine or (3) mixture of dopamine and lactacystin. Activities of proteasome 20S and pro-apoptotic caspases-3 and -9 and levels of anti-apoptotic Bcl-2 were measured with fluorimetric or immunochemical assays, while a "DNA diffusion" assay was used to determine the apoptosis. Incubation of lymphocytes with lactacystin, which caused reduction of proteasomal activity, was associated with activation of caspases. A clear, dose-dependent reduction of proteasomal activity was also seen in the presence of increasing doses of dopamine, which was accompanied by a slight dose-dependent increase of caspases activities and Bcl-2 levels. Both effects on proteasome and caspase activities were enhanced when cells were simultaneously exposed to lactacystin and elevated concentrations of dopamine. Apoptosis was detected in all treated samples, but not in controls, without significant differences among the treatment groups; however, the association of dopamine and lactacystin induced a clear reduction in the number of cells being analyzed, pointing to marked cytotoxicity. Our data confirm the potentiation of cytotoxicity related to proteasome inhibition, in the presence of dopaminergic stimulation.
Abstract: The noradrenergic nucleus Locus Coeruleus (LC) densely innervates limbic structures. In rats, the damage to LC by the neurotoxin DSP-4, converts episodic limbic seizures induced by bicuculline infusion in the anterior piriform cortex (APC) into self-sustaining status epilepticus (SE). SE induced by this approach is similar to SE induced by co-infusing cyclothiazide and bicuculline into APC in rats bearing an intact LC. As opposed to other commonly used rat SE models (e.g. systemic kainate or pilocarpine), this approach allows one to analyze the effects of SE on brain regions which are solely due to spreading of seizure activity, rather than to direct effect of systemic chemoconvulsant. We evaluated the expression of Fos protein (an immediate early gene product), and the local cerebral metabolic rates for [14C] 2-deoxyglucose (lCMRglc), in rats following SE induced either by cyclothiazide+bicuculline or by DSP-4+bicuculline. We demonstrated that regional Fos expression after SE does not parallel the increase in lCMRglc, in LC-lesioned rats. In DSP-4+bicuculline rats there is an overall lower expression of the protein as compared with the cyclothiazide+bicuculline or bicuculline alone groups; even more, such a difference co-exists with an higher lCMRglc in the DSP-4+bicuculline-treated rats in some regions, as compared with the other groups. These data show that LC neurons play an important role in determining immediate early genes expression even in conditions of strong pathological activation, such as limbic SE. This might have relevant effects in the plastic mechanisms related with epileptogenesis.
Abstract: Chronic reduction of the caloric intake is associated with extended lifespan, in rodents, and has been proposed to counteract neuronal loss in animal models of neurodegeneration. To test this hypothesis, we investigated the effect of dietary restriction (DR) in a rodent model of Parkinson's disease, based on the intrastriatal infusion of 6-hydroxydopamine. We could not confirm the neuroprotective effect of DR previously suggested: histological and behavioral measures indicated similar degrees of dopaminergic neuron loss in rats maintained on DR--for two or eight weeks prior to the lesion--or with free access to food.
Abstract: The investigation of pathogenic and pathophysiological mechanisms of Parkinson's disease relies on experimental models reproducing, in the animal, the pathological and behavioural features of the disease. Despite the availability of innovative models, 6-hydroxydopamine (6-OHDA) remains the most widely used tool to induce a nigrostriatal lesion in the animal (rat). This is due to (1) the relatively low complexity and cost of the procedure, (2) the fact that the 6-OHDA-induced lesion is highly reproducible, and (3) the versatility of the procedure, which can yield varying degrees of nigrostriatal lesions that develop with different temporal profiles, depending on the site chosen for the toxin injection.
Abstract: Experimental evidence shows that dopaminergic transmission within the basal ganglia is involved in the modulation of nociceptive information. Epidemiological studies show that in some disease states inherent pathophysiological mechanisms that involve degenerative changes (Parkinson's disease; PD) can also impact negatively on other unrelated functional systems (i.e. nociception). Delayed Fos expression in response to nitroglycerin (NTG) administration is a procedure used to identify the neuroanatomical substrates of the migraine condition. In this study, we investigated the influence of dopaminergic nigrostriatal denervation, obtained by intrastriatal injection of 6-hydroxydopamine (6-OHDA), on this response in Sprague-Dawley rats. We also explored the effects on the NTG-induced hyperalgesic response to painful stimuli (formalin and tail-flick tests). Nigrostriatal lesion prevented the neuronal activation typically induced by NTG in sub-cortical areas involved in pain perception, autonomic control and neuroendocrine functions, such as hypothalamic nuclei, periaqueductal grey, parabrachial nucleus and the medullary nuclei. In addition, 6-OHDA-induced lesion inhibited NTG-induced hyperalgesia. Our data show that integrity of central dopaminergic neurotransmission is required for the NTG-induced activation of sub-cortical areas involved in the expression of migraine symptoms, as well as for the hyperalgesic response to painful stimuli elicited by the drug.
Abstract: Despite the progressive development of innovative animal models for Parkinson's disease, the intracerebral infusion of neurotoxin 6-hydroxydopamine (6-OHDA) remains the most widely used means to induce an experimental lesion of the nigrostriatal pathway in the animal, due to its relatively low complexity and cost, coupled with the high reproducibility of the lesion obtained. To gain new information from such a classic model, we studied the time-course of the nigrostriatal damage, metabolic changes in the basal ganglia nuclei (cytochrome oxidase activity) and behavioural modifications (rotational response to apomorphine) following unilateral injection of 6-OHDA into the corpus striatum of rat, over a 4-week period. Striatal infusion of 6-OHDA caused early damage of dopaminergic terminals, followed by a slowly evolving loss of dopaminergic cell bodies in the substantia nigra pars compacta, which became apparent during the second week post-injection and peaked at the 28th day post-infusion; the rotational response to apomorphine was already present at the first time point considered (Day 1), and remained substantially stable throughout the 4-week period of observation. The evolution of the nigrostriatal lesion was accompanied by complex changes in the metabolic activity of the other basal ganglia nuclei investigated (substantia nigra pars reticulata, entopeduncular nucleus, globus pallidus and subthalamic nucleus), which led, ultimately, to a generalized, metabolic hyperactivity, ipsilaterally to the lesion. However, peculiar patterns of metabolic activation, or inhibition, characterized the post-lesional responses of each nucleus, in the early and intermediate phases, with peculiar response profiles that varied closely related to the functional position occupied within the basal ganglia circuitry.
Abstract: Cyclooxygenase-2 (COX-2) may increase prostaglandin E(2) (PGE(2)) production in central nervous system (CNS) and contribute to the severity of pain responses in inflammatory pain. In this chapter, we sought to evaluate the possible role of COX-2 induction and prostaglandins (PGs) synthesis within neuronal areas proposed to be involved in migraine genesis in the animal model of migraine based on the administration of systemic nitroglycerin (NTG). Male Sprague-Dawley rats were injected with NTG (10mg/kg, i.p.) or vehicle and sacrificed 2 and 4h later. The hypothalamus and the lower brain stem were dissected out and utilized for the evaluation of COX-2 expression by means of Western blotting and for the determination of PGE(2) levels by means of ELISA immunoassay. COX-2 expression increased in the hypothalamus at 2h and in the lower brain stem at 4h. PGE(2) levels showed an opposite pattern of change with a decrease in PGE(2) levels at 2h in the hypothalamus and an increase at 4h in the lower brain stem. These data support the hypothesis that NTG administration is capable of activating the COX-2 pathway within cerebral areas. This activity may explain the pronociceptive effect of NTG described in animal and human models of pain. Most importantly, these findings point to mediators and areas that may be relevant for migraine pathogenesis and treatment.
Abstract: In Parkinson's disease (PD), the motor dysfunction caused by degeneration of the nigrostriatal pathway is often associated with alterations of pain perception. This is likely related to the role that the nigrostriatal system may play in the processing of noxious, somatosensory stimuli. To further address this issue, we used a rodent model of PD, based on the unilateral, intrastriatal injection of neurotoxin 6-hydroxydopamine (6-OHDA). We investigated the effects of the nigrostriatal lesion on behavioral responses to pain tests designed to explore different aspects of nociception, such as the formalin test and the tail flick test; we also explored modifications in the expression of Fos protein, a marker of neuronal activation, in supraspinal nuclei involved in the integration of pain perception and stress-related behavior. Rats bearing the nigrostriatal lesion showed complex alterations in pain perception, including hyperalgesic responses to the tonic, inflammatory pain elicited by formalin injection, but only when the stimulus was delivered ipsilaterally to the lesion. This phenomenon was associated with delayed responses to the phasic, thermal stimulus induced by the tail flick test. The hyperalgesic response to the formalin test was accompanied by reduced Fos expression in the paraventricular nucleus of the hypothalamus, which is part of a network (the medial pain system) that mediates motivational-affective aspects of pain. Our results confirm that a unilateral alteration of central dopaminergic transmission disrupts the neural mechanisms underlying proper integration of painful stimuli, particularly in the hemibody ipsilateral to the dopaminergic denervation.
Abstract: We compared the neuroprotective and metabolic effects of chronic treatment with ionotropic or metabotropic glutamate receptor antagonists, in rats bearing a unilateral nigrostriatal lesion induced by 6-hydroxydopamine (6-OHDA). The ionotropic, N-methyl-D-aspartate receptor antagonist MK-801 increased cell survival in the substantia nigra pars compacta (SNc) and corrected the metabolic hyperactivity (increased cytochrome oxidase activity) of the ipsilateral substantia nigra pars reticulata (SNr) associated with the lesion, but showed no effects on the 6-OHDA-induced hyperactivity of the subthalamic nucleus (STN). Significant-although less pronounced-protection of SNc neurons was also observed following treatment with the metabotropic glutamate receptor (mGluR5) antagonist 2-methyl-6-(phenylehtynyl)-pyridine (MPEP). As opposed to MK-801, MPEP abolished the STN metabolic hyperactivity associated with the nigrostriatal lesion, without affecting SNr activity. Specific modulation of STN hyperactivity obtained with mGluR5 blockade may, therefore, open interesting perspectives for the use of this class of compounds in the treatment of Parkinson's disease.
Abstract: We investigated the effects of a selective lesion of the substantia nigra pars reticulata (SNr), obtained by stereotaxic injection of ibotenic acid, on the cortical expression of Fos protein induced by striatal infusion of dopamine, D1-like agonist SKF 38393, in Sprague-Dawley rats. The specific aim was to clarify the role of the basal ganglia output structures - SNr in particular - in the cortical activation that follows a D1-dependent activation of the striatofugal, direct pathway, in freely moving animals. The striatal, unilateral infusion of 30 mM SKF 38393 induced consistent Fos expression throughout the whole ipsilateral cerebral cortex, including motor, sensorimotor, associative, and limbic areas; such expression was dramatically reduced by excitotoxic lesion of the ipsilateral SNr. These findings confirm the prominent role of the SNr in the transmission of striatofugal signals to functionally different cortical areas.
Abstract: Reducing subthalamic nucleus (STN) activity has been proposed as an anti-epileptic procedure. Here we show that, on the contrary, a unilateral lesion of the STN causes slight (nonsignificant) increases in the severity of limbic seizures evoked by bicuculline infusion into the piriform cortex, associated with marked Fos expression throughout the cerebral cortex. Abolishing the STN control over the basal ganglia output may therefore play a facilitatory role on cortical activation associated with limbic seizures.
Abstract: Complete Freund's adjuvant (CFA), a pro-inflammatory agent, was inoculated, subcutaneously, to Sprague-Dawley rats prior to the intrastriatal injection of 6-hydroxydopamine (6-OHDA). Animals were sacrificed 7 and 28 days following 6-OHDA injection; neuronal damage, glial activation and cytokine levels, within the nigrostriatal system, were then investigated. Nigrostriatal degeneration induced by 6-OHDA was accompanied by early microglial and astroglial activation, which preceded the onset of dopaminergic cell loss, in the SNc, without significant changes in cytokine levels. CFA pretreatment markedly reduced the SNc neuronal loss and associated microglial activation, as well as the rotational response to apomorphine. These changes were associated with moderate, transient increases in the nigrostriatal levels of glial-cell-derived neurotrophic factor (GDNF) and pro-inflammatory cytokines, including interleukin (IL)-1alpha, IL-1beta and IL-6. Our results show that prior delivery of a peripheral, pro-inflammatory stimulus induces neuroprotection, in a rodent model of Parkinson's disease, possibly through the modulation of cytokine production at the nigrostriatal level.
Abstract: Wilson's disease (WD) is an inherited disorder, characterized by selective copper deposition in liver and brain, chronic hepatitis and extra-pyramidal signs. In this study, we investigated changes of biochemical markers of oxidative stress and apoptosis in liver, striatum and cerebral cortex homogenates from Long-Evans Cinnamon (LEC) rats, a mutant strain isolated from Long Evans (LE) rats, in whom spontaneous hepatitis develops shortly after birth. LEC and control (LE) rats at 11 and 14 weeks of age were used. We determined tissue levels of glutathione (GSH/GSSG ratio), lipid peroxides, protein-thiols (P-SH), nitric oxide metabolites, activities of caspase-3 and total superoxide-dismutase (SOD), striatal levels of monoamines and serum levels of hepatic amino-transferases. We observed a decrease of protein-thiols, GSH/GSSG ratio and nitrogen species associated to increased lipid peroxidation in the liver and striatum - but not in the cerebral cortex - of LEC rats, accompanied by dramatic increase in serum amino-transferases and decrease of striatal catecholamines. Conversely, SOD and caspase-3 activity increased consistently only in the cortex of LEC rats. Hence, we assume that enhanced oxidative stress may play a central role in the cell degeneration in WD, at the main sites of copper deposition, with discrete pro-apoptotic conditions developing in distal areas.
Abstract: Homocysteine (Hcy) is a nonprotein-forming sulphur amino acid that plays an important role in remethylation and trans-sulphuration processes. In recent years, it has been suggested that increased levels of plasma Hcy may play a role in the pathogenesis of various diseases, particularly at the cardiovascular level. The pathogenic mechanism of hyperhomocysteinemia, however, has not been clarified. Because oxygen radicals can be generated by the auto-oxidation of this amino acid, it has been suggested that Hcy may cause cellular damage through oxidative mechanisms, ultimately leading to apoptotic cell death. In this study, we sought to investigate the effects of Hcy on oxidative damage and antioxidant agent levels, as well as on apoptosis-related proteins and apoptosis occurrence in human cells. For this purpose, we measured levels of Bcl-2, caspase-3 and caspase-9 activity, Cu/Zn superoxide dismutase, reduced glutathione, lipid peroxidation [malondialdehyde and 4-hydroxy-2 (E)-nonenal concentrations], apoptotic single-stranded DNA and nuclear changes in human isolated lymphocytes exposed to increasing concentrations of Hcy. Incubation with Hcy did not induce significant changes in any of these biomarkers. Therefore, our results do not support the existence of a direct link between increased levels of Hcy and the occurrence of a pro-apoptotic state mediated by enhanced oxidative stress.
Abstract: Sprague-Dawley rats received a unilateral injection of 6-hydroxydopamine (6-OHDA) into the striatum and were treated daily for 6 weeks with increasing doses of monoamine oxidase type B inhibitor rasagiline [R(+)-N-propargyl-1-aminoindane] or saline (controls). Both doses of rasagiline markedly increased the survival of dopaminergic neurons in the lesioned substantia nigra, compared to controls (+97% and +119%, respectively). Treatment with the lower dose of rasagiline also abolished the motor stereotypies associated with nigrostriatal lesion. Our study supports the neuroprotective potential of chronic rasagiline administration in an experimental model of Parkinson's disease (PD).
Abstract: In rats, intrastriatal injection of malonate, a reversible inhibitor of the mitochondrial enzyme succinate dehydrogenase, induces a lesion similar to that observed following focal ischemia or in Huntington's disease. In this study we used the malonate model to explore the neuroprotective potential of dopamine agonists. Rats were injected intraperitoneally with increasing concentrations of D1, D2, or mixed D1/D2 dopamine agonists prior to intrastriatal injection of malonate. Administration of increasing doses of the D2-specific agonist quinpirole resulted in increased protection against malonate toxicity. Conversely, the D1-specific agonist SKF-38393, as well as the mixed D1/D2 agonist apomorphine, conferred higher neuroprotection at lower than at higher drug concentrations. Our data suggest that malonate- induced striatal toxicity can be attenuated by systemic administration of dopamine agonists, with D1 and D2 agonists showing different profiles of efficacy.
Abstract: Mitochondrial bioenergetic defects are involved in neurological disorders associated with neuronal damage in the striatum, such as Huntington's disease and cerebral ischemia. The striatal release of neurotransmitters, in particular dopamine, may contribute to the development of the neuronal damage. Recent studies have shown that dopamine agonists may exert neuroprotective effects via multiple mechanisms, including modulation of dopamine release from nigrostriatal dopaminergic terminals. In rats, intrastriatal injection of malonate, a reversible inhibitor of the mitochondrial enzyme succinate dehydrogenase, induces a lesion similar to that observed following focal ischemia or in Huntington's disease. In this study, we used the malonate model to explore the neuroprotective potential of dopamine agonists. Sprague-Dawley rats were injected systemically with increasing concentrations of D(1), D(2), or mixed D(1)/D(2) dopamine agonists prior to malonate intrastriatal insult. Administration of increasing doses of the D(2)-specific agonist quinpirole resulted in increased protection against malonate toxicity. Conversely, the D(1)-specific agonist SKF-38393, as well as the mixed D(1)/D(2) agonist apomorphine, conferred higher neuroprotection at lower than at higher concentrations. Our data suggest that malonate-induced striatal toxicity can be attenuated by systemic administration of dopamine agonists, with D(1) and D(2) agonists showing different profiles of efficacy.
Abstract: Epstein-Barr virus (EBV)-induced lymphoproliferative disease (lpd) is a B cell neoplasm that affects patients who are immunosuppressed in the context of organ transplantation or HIV infection. A model for the aggressive form of this entity was generated by xenotransplantation of SCID mice with human peripheral blood leukocytes from individuals with prior contact with EBV. This model, where large B cell lymphoma occurs, was used to test the hypothesis that IL-6 has a major role in EBV-induced B cell tumorigenesis. IL-6 is known to differentiate B cells into immunoglobulin-secreting plasma cells and induce EBV replication, and xenochimeric animals have detectable serum levels of human IL-6. Human IL-6 inhibition with a neutralizing monoclonal antibody decreased tumor incidence from 62 % to 27 %. In addition, anti-IL-6 treatment significantly improved xenotransplanted animal survival, with median survival at > 245 days when compared to that of controls at 132 days. In conclusion, IL-6 plays a critical role in the pathogenesis of EBV-induced human lpd, and IL-6 inhibition may represent a new and promising preventive or therapeutic approach against this malignancy.
Abstract: OBJECTIVE: To generate a human-mouse xenochimeric model where human cells remain clustered in the animal to optimize their interactions and recovery. MATERIALS AND METHODS: Severe combined immune deficient mice (SCID) were xenografted subcutaneously with human adult tonsil pieces (hu-ton-SCID mice). Such animals were: (a) compared with those receiving tonsil cells in suspension, and (b) immunized with de novo and recall antigens. RESULTS: Human tonsil pieces survived a long period of time in SCID mice, while polyclonal human T- and B-lymphocytes persisted in close vicinity within the implantation area; however, little or no graft-versus-host disease was detectable. Not surprisingly, local development of lymphoproliferative disease was often observed in animals receiving lymphoid implants from donors previously infected by the Epstein-Barr virus. One month after surgery, higher serum levels of human IgG were found in SCID mice transplanted with tonsil pieces (2x10(7) cells/animal) than in animals injected with 5x10(7) tonsil cells in suspension (1.9 vs. 0.3 mg/mL, p < 0.002). Importantly, the production of human IgG in hu-ton-SCID mice remained polyclonal for at least 6 months and was linked to the presence of cells within the implants. Immunization of hu-ton-SCID mice with hepatitis B core, a de novo antigen, did not produce a significant IgG immune response; however immunization with tetanus toxoid (TT), a thymus-dependent recall antigen, yielded high (> 700-fold increase in anti-TT IgG levels) and long-lasting (> 6 months) secondary immune responses. CONCLUSION: The hu-ton-SCID mouse xenochimeric model described in this report may improve our understanding of human lymphoid cell interactions, secondary immune responses, and lymphomagenesis.
Abstract: The Epstein-Barr virus (EBV) exists in most humans as a lifelong latent infection established in host B cells after a primary viral encounter. In immunosuppressed individuals, such as post-transplant patients, the presence of EBV-infected B cells may lead to lymphoproliferative disease. Injection of human peripheral blood lymphocytes from EBV-positive donors into mice with severe combined immunodeficiency induces human lymphoproliferative disease in the recipient closely resembling that of human post-transplant patients. This xenochimeric human-mouse model is increasingly being used to elucidate the mechanisms of EBV-specific lymphomagenesis and to assess novel therapeutic approaches.
Abstract: The nuclear factor I (NFI) family consists of sequence-specific DNA-binding proteins that activate both transcription and adenovirus DNA replication. We have characterized three new members of the NFI family that belong to the Xenopus laevis NFI-X subtype and differ in their C-termini. We show that these polypeptides can activate transcription in HeLa and Drosophila Schneider line 2 cells, using an activation domain that is subdivided into adjacent variable and subtype-specific domains each having independent activation properties in chimeric proteins. Together, these two domains constitute the full NFI-X transactivation potential. In addition, we find that the X. laevis NFI-X proteins are capable of activating adenovirus DNA replication through their conserved N-terminal DNA-binding domains. Surprisingly, their in vitro DNA-binding activities are specifically inhibited by a novel repressor domain contained within the C-terminal part, while the dimerization and replication functions per se are not affected. However, inhibition of DNA-binding activity in vitro is relieved within the cell, as transcriptional activation occurs irrespective of the presence of the repressor domain. Moreover, the region comprising the repressor domain participates in transactivation. Mechanisms that may allow the relief of DNA-binding inhibition in vivo and trigger transcriptional activation are discussed.
Abstract: Efficient initiation by the DNA polymerase of adenovirus type 2 requires nuclear factor I (NFI), a cellular sequence-specific transcription factor. Three functions of NFI--dimerization, DNA binding, and activation of DNA replication--are colocalized within the N-terminal portion of the protein. To define more precisely the role of NFI in viral DNA replication, a series of site-directed mutations within the N-terminal domain have been generated, thus allowing the separation of all three functions contained within this region. Impairment of the dimerization function prevents sequence-specific DNA binding and in turn abolishes the NFI-mediated activation of DNA replication. NFI DNA-binding activity, although necessary, is not sufficient to activate the initiation of adenovirus replication. A distinct class of NFI mutations that abolish the recruitment of the viral DNA polymerase to the origin also prevent the activation of replication. Thus, a direct interaction of NFI with the viral DNA polymerase complex is required to form a stable and active preinitiation complex on the origin and is responsible for the activation of replication by NFI.
