Alpha-synuclein/synapsin III pathological interplay boosts the motor response to methylphenidate.

Loss of dopaminergic nigrostriatal neurons and fibrillary α-synuclein (α-syn) aggregation in Lewy bodies (LB) characterize Parkinson's disease (PD). We recently found that Synapsin III (Syn III), a phosphoprotein regulating dopamine (DA) release with α-syn, is another key component of LB fibrils in the brain of PD patients and acts as a crucial mediator of α-syn aggregation and toxicity. Methylphenidate (MPH), a monoamine reuptake inhibitor (MRI) efficiently counteracting freezing of gait in advanced PD patients, can bind α-syn and controls α-syn-mediated DA overflow and presynaptic compartmentalization. Interestingly, MPH results also efficient for the treatment of attention deficits and hyperactivity disorder (ADHD), a neurodevelopmental psychiatric syndrome associated with Syn III and α-syn polymorphisms and constituting a risk factor for the development of LB disorders. Here, we studied α-syn/Syn III co-deposition and longitudinal changes of α-syn, Syn III and DA transporter (DAT) striatal levels in nigrostriatal neurons of a PD model, the human C-terminally truncated (1-120) α-syn transgenic (SYN120 tg) mouse, in comparison with C57BL/6J wild type (wt) and C57BL/6JOlaHsd α-syn null littermates. Then, we analyzed the locomotor response of these animals to an acute administration of MPH (d-threo) and other MRIs: cocaine, that we previously found to stimulate Syn III-reliant DA release in the absence of α-syn, or the selective DAT blocker GBR-12935, along aging. Finally, we assessed whether these drugs modulate α-syn/Syn III interaction by fluorescence resonance energy transfer (FRET) and performed in silico studies engendering a heuristic model of the α-syn conformations stabilized upon MPH binding. We found that only MPH was able to over-stimulate a Syn III-dependent/DAT-independent locomotor activity in the aged SYN120 tg mice showing α-syn/Syn III co-aggregates. MPH enhanced full length (fl) α-syn/Syn III and even more (1-120) α-syn/Syn III interaction in cells exhibiting α-syn/Syn III inclusions. Moreover, in silico studies confirmed that MPH may reduce α-syn fibrillation by stabilizing a protein conformation with increased lipid binding predisposition. Our observations indicate that the motor-stimulating effect of MPH can be positively fostered in the presence of α-syn/Syn III co-aggregation. This evidence holds significant implications for PD and ADHD therapeutic management.

Oral and intravenous transmission of α-synuclein fibrils to mice.

Parkinson's disease and related disorders are neuropathologically characterized by cellular deposits of misfolded and aggregated α-synuclein in the CNS. Disease-associated α-synuclein adopts a conformation that causes it to form oligomers and fibrils, which have reduced solubility, become hyperphosphorylated, and contribute to the spatiotemporal spreading of pathology in the CNS. The infectious properties of disease-associated α-synuclein, e.g., by which peripheral route and with which efficiency it can be transmitted, are not fully understood. Here, we investigated the potential of α-synuclein fibrils to induce neurological disease in TgM83+/- mice expressing the A53T mutant of human α-synuclein after oral or intravenous challenge and compared it to intraperitoneal and intracerebral challenge. Oral challenge with 50 µg of α-synuclein fibrils caused neurological disease in two out of eight mice in 220 days and 350 days, and challenge with 500 µg in four out of eight mice in 384 ± 131 days, respectively. Intravenous challenge with 50 µg of α-synuclein fibrils led to disease in 208 ± 20 days in 10 out of 10 mice and was in duration comparable to intraperitoneal challenge with 50 µg of α-synuclein fibrils, which caused disease in 10 out of 10 mice in 202 ± 35 days. Ten out of 10 mice that were each intracerebrally challenged with 10 µg or 50 µg of α-synuclein fibrils developed disease in 156 ± 20 days and 133 ± 4 days, respectively. The CNS of diseased mice displayed aggregates of sarkosyl-insoluble and phosphorylated α-synuclein, which colocalized with ubiquitin and p62 and were accompanied by gliosis indicative of neuroinflammation. In contrast, none of the control mice that were challenged with bovine serum albumin via the same routes developed any neurological disease or neuropathology. These findings are important, because they show that α-synuclein fibrils can neuroinvade the CNS after a single oral or intravenous challenge and cause neuropathology and disease.

Effect of atorvastatin on behavioral alterations and neuroinflammation during epileptogenesis.

Temporal lobe epilepsy (TLE) is the most frequent and medically refractory type of epilepsy in humans. In addition to seizures, patients with TLE suffer from behavioral alterations and cognitive deficits. Poststatus epilepticus model of TLE induced by pilocarpine in rodents has enhanced the understanding of the processes leading to epilepsy and thus, of potential targets for antiepileptogenic therapies. Clinical and experimental evidence suggests that inflammatory processes in the brain may critically contribute to epileptogenesis. Statins are inhibitors of cholesterol synthesis, and present pleiotropic effects that include antiinflammatory properties. We aimed the present study to test the hypothesis that atorvastatin prevents behavioral alterations and proinflammatory state in the early period after pilocarpine-induced status epilepticus. Male and female C57BL/6 mice were subjected to status epilepticus induced by pilocarpine and treated with atorvastatin (10 or 100mg/kg) for 14days. Atorvastatin slightly improved the performance of mice in the open-field and object recognition tests. In addition, atorvastatin dose-dependently decreased basal and status epilepticus-induced levels of interleukin-1ß (IL-1ß), interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), and interferon-γ (INF-γ) and increased interleukin-10 (IL-10) levels in the hippocampus and cerebral cortex. The antiinflammatory effects of atorvastatin were qualitatively identical in both sexes. Altogether, these findings extend the range of beneficial actions of atorvastatin and indicate that its antiinflammatory effects may be useful after an epileptogenic insult.

α-synuclein and synapsin III cooperatively regulate synaptic function in dopamine neurons.

The main neuropathological features of Parkinson's disease are dopaminergic nigrostriatal neuron degeneration, and intraneuronal and intraneuritic proteinaceous inclusions named Lewy bodies and Lewy neurites, respectively, which mainly contain α-synuclein (α-syn, also known as SNCA). The neuronal phosphoprotein synapsin III (also known as SYN3), is a pivotal regulator of dopamine neuron synaptic function. Here, we show that α-syn interacts with and modulates synapsin III. The absence of α-syn causes a selective increase and redistribution of synapsin III, and changes the organization of synaptic vesicle pools in dopamine neurons. In α-syn-null mice, the alterations of synapsin III induce an increased locomotor response to the stimulation of synapsin-dependent dopamine overflow, despite this, these mice show decreased basal and depolarization-dependent striatal dopamine release. Of note, synapsin III seems to be involved in α-syn aggregation, which also coaxes its increase and redistribution. Furthermore, synapsin III accumulates in the caudate and putamen of individuals with Parkinson's disease. These findings support a reciprocal modulatory interaction of α-syn and synapsin III in the regulation of dopamine neuron synaptic function.

Depletion of Progranulin Reduces GluN2B-Containing NMDA Receptor Density, Tau Phosphorylation, and Dendritic Arborization in Mouse Primary Cortical Neurons.

Loss-of-function mutations in the progranulin (PGRN) gene are a common cause of familial frontotemporal lobar degeneration (FTLD). This age-related neurodegenerative disorder, characterized by brain atrophy in the frontal and temporal lobes and such typical symptoms as cognitive and memory impairment, profound behavioral abnormalities, and personality changes is thought to be related to connectome dysfunctions. Recently, PGRN reduction has been found to induce a behavioral phenotype reminiscent of FTLD symptoms in mice by affecting neuron spine density and morphology, suggesting that the protein can influence neuronal structural plasticity. Here, we evaluated whether a partial haploinsufficiency-like PGRN depletion, achieved by using RNA interference in primary mouse cortical neurons, could modulate GluN2B-containing N-methyl-d-aspartate (NMDA) receptors and tau phosphorylation, which are crucially involved in the regulation of the structural plasticity of these cells. In addition, we studied the effect of PGRN decrease on neuronal cell arborization both in the presence and absence of GluN2B-containing NMDA receptor stimulation. We found that PGRN decline diminished GluN2B-containing NMDA receptor levels and density as well as NMDA-dependent tau phosphorylation. These alterations were accompanied by a marked drop in neuronal arborization that was prevented by an acute GluN2B-containing NMDA receptor stimulation. Our findings support that PGRN decrease, resulting from pathogenic mutations, might compromise the trophism of cortical neurons by affecting GluN2B-contaning NMDA receptors. These mechanisms might be implicated in the pathogenesis of FTLD.

Rosmarinic acid is anticonvulsant against seizures induced by pentylenetetrazol and pilocarpine in mice.

Epilepsy is a chronic neurological disease characterized by spontaneous recurrent seizures (SRS). Current anticonvulsant drugs are ineffective in nearly one-third of patients and may cause significant adverse effects. Rosmarinic acid is a naturally occurring substance which displays several biological effects including antioxidant and neuroprotective activity. Since oxidative stress and excitotoxicity play a role in the pathophysiology of seizures, we aimed the present study to test the hypothesis that rosmarinic acid displays anticonvulsant and disease-modifying effects. Female C57BL/6 mice received rosmarinic acid (0, 3, 10, or 30mg/kg; p.o.) 60min before the injection of pentylenetetrazol (PTZ, 60mg/kg; i.p.) or pilocarpine (300mg/kg, i.p.). Myoclonic and generalized tonic-clonic seizure latencies and generalized seizure duration were analyzed by behavioral and electroencephalographic (EEG) methods. The effect of acute administration of rosmarinic acid on mice behavior in the open-field, object recognition, rotarod, and forced swim tests was also evaluated. In an independent set of experiments, we evaluated the effect of rosmarinic acid (3 or 30mg/kg, p.o. for 14days) on the development of SRS and behavioral comorbidities in the pilocarpine post-status epilepticus (SE) model of epilepsy. Rosmarinic acid dose-dependently (peak effect at 30mg/kg) increased the latency to myoclonic jerks and generalized seizures in the PTZ model and increased the latency to myoclonic jerks induced by pilocarpine. Rosmarinic acid (30mg/kg) increased the number of crossings, the time at the center of the open field, and the immobility time in the forced swim test. In the chronic epilepsy model, treatment with rosmarinic acid did not prevent the appearance of SRS or behavioral comorbidities. In summary, rosmarinic acid displayed acute anticonvulsant-like activity against seizures induced by PTZ or pilocarpine in mice, but further studies are needed to determine its epilepsy-modifying potential.

Anticonvulsant activity of ß-caryophyllene against pentylenetetrazol-induced seizures.

Increasing evidence suggests that plant-derived extracts and their isolated components are useful for treatment of seizures and, hence, constitute a valuable source of new antiepileptic drugs with improved efficacy and better adverse effect profile. ß-Caryophyllene is a natural bicyclic sesquiterpene that occurs in a wide range of plant species and displays a number of biological actions, including neuroprotective activity. In the present study, we tested the hypothesis that ß-caryophyllene displays anticonvulsant effects. In addition, we investigated the effect of ß-caryophyllene on behavioral parameters and on seizure-induced oxidative stress. Adult C57BL/6 mice received increasing doses of ß-caryophyllene (0, 10, 30, or 100mg/kg). After 60 min, we measured the latencies to myoclonic and generalized seizures induced by pentylenetetrazole (PTZ, 60 mg/kg). We found that ß-caryophyllene increased the latency to myoclonic jerks induced by PTZ. This result was confirmed by electroencephalographic analysis. In a separate set of experiments, we found that mice treated with an anticonvulsant dose of ß-caryophyllene (100mg/kg) displayed an improved recognition index in the object recognition test. This effect was not accompanied by behavioral changes in the open-field, rotarod, or forced swim tests. Administration of an anticonvulsant dose of ß-caryophyllene (100mg/kg) did not prevent PTZ-induced oxidative stress (i.e., increase in the levels of thiobarbituric acid-reactive substances or the decrease in nonprotein thiols content). Altogether, the present data suggest that ß-caryophyllene displays anticonvulsant activity against seizures induced by PTZ in mice. Since no adverse effects were observed in the same dose range of the anticonvulsant effect, ß-caryophyllene should be further evaluated in future development of new anticonvulsant drugs.

Lysine residues at the first and second KTKEGV repeats mediate α-Synuclein binding to membrane phospholipids.

While α-Synuclein (α-Syn) is mainly detected as a cytosolic protein, a portion of it is recovered bound to membranes. It is suggested that binding to membrane phospholipids controls α-Syn structure, physiology and pathogenesis. We aimed at investigating the role, of the positive charged lysine residues at the KTKEGV repeat motif, in mediating α-Syn associations with membrane phospholipids and in α-Syn oligomerization and aggregation. Specifically, two positive lysine (K) residues were replaced with two negative glutamic acid (E) residues at either the first or second KTKEGV repeat motifs. The effect of these mutations on membrane binding was determined by a quantitative phospholipid ELISA assay and compared to wild-type α-Syn and to the Parkinson's disease-causing mutations, A30P, E46K and A53T. We found that the K to E substitutions affected α-Syn binding to phospholipids. In addition, K to E substitutions resulted in a dramatically lower level of soluble α-Syn oligomers and larger intracellular inclusions. Together, our results suggest a critical role for lysine residues at the N-terminal repeat domain in the pathophysiology of α-Syn.

Atorvastatin withdrawal elicits oxidative/nitrosative damage in the rat cerebral cortex.

Statins are inhibitors of the enzyme 3-hydroxy-3-methylglutaryl coenzyme A reductase, the rate-limiting step in cholesterol biosynthesis. Statins effectively prevent and reduce the risk of coronary artery disease through lowering serum cholesterol, and also exert anti-thrombotic, anti-inflammatory and antioxidant effects independently of changes in cholesterol levels. On the other hand, clinical and experimental evidence suggests that abrupt cessation of statin treatment (i.e. statin withdrawal) is associated with a deleterious rebound phenomenon. In fact, statin withdrawal increases the risk of thrombotic vascular events, causes impairment of endothelium-dependent relaxation and facilitates experimental seizures. However, evidence for statin withdrawal-induced detrimental effects to the brain parenchyma is still lacking. In the present study adult male Wistar rats were treated with atorvastatin for seven days (10mg/kg/day) and neurochemical assays were performed in the cerebral cortex 30 min (atorvastatin treatment) or 24h (atorvastatin withdrawal) after the last atorvastatin administration. We found that atorvastatin withdrawal decreased levels of nitric oxide and mitochondrial superoxide dismutase activity, whereas increased NADPH oxidase activity and immunoreactivity for the protein nitration marker 3-nitrotyrosine in the cerebral cortex. Catalase, glutathione-S-transferase and xanthine oxidase activities were not altered by atorvastatin treatment or withdrawal, as well as protein carbonyl and 4-hydroxy-2-nonenal immunoreactivity. Immunoprecipitation of mitochondrial SOD followed by analysis of 3-nitrotyrosine revealed increased levels of nitrated mitochondrial SOD, suggesting the mechanism underlying the atorvastatin withdrawal-induced decrease in enzyme activity. Altogether, our results indicate the atorvastatin withdrawal elicits oxidative/nitrosative damage in the rat cerebral cortex, and that changes in NADPH oxidase activity and mitochondrial superoxide dismutase activities may underlie such harmful effects.

Velusetrag rescues GI dysfunction, gut inflammation and dysbiosis in a mouse model of Parkinson's disease.

In patients with Parkinson's disease (PD), constipation is common, and it appears in a prodromal stage before the hallmark motor symptoms. The present study aimed to investigate whether Velusetrag, a selective 5­HT4 receptor agonist, may be a suitable candidate to improve intestinal motility in a mouse model of PD. Five months old PrP human A53T alpha-synuclein transgenic (Tg) mice, which display severe constipation along with decreased colonic cholinergic transmission already at 3 months, were treated daily with the drug for 4 weeks. Velusetrag treatment reduced constipation by significantly stimulating both the longitudinal and circular-driven contractions and improved inflammation by reducing the level of serum and colonic IL1ß and TNF-α and by decreasing the number of GFAP-positive glia cells in the colon of treated mice. No significant downregulation of the 5-HT4 receptor was observed but instead Velusetrag seemed to improve axonal degeneration in Tgs as shown by an increase in NF-H and VAChT staining. Ultimately, Velusetrag restored a well-balanced intestinal microbial composition comparable to non-Tg mice. Based on these promising data, we are confident that Velusetrag is potentially eligible for clinical studies to treat constipation in PD patients.

Alpha-Synuclein and Parkinson's Disease Motor and Non-Motor Symptoms: What Is New?

Although it was discovered about 25 years ago, alpha-synuclein (αS) misfolding and accumulation in neuronal tissues is still recognized as one of the most crucial aspects in Parkinson's disease (PD) pathology [...].

Loss of Corticostriatal Mu-Opioid Receptors in α-Synuclein Transgenic Mouse Brains.

Ultrastructural, neurochemical, and molecular alterations within the striatum are associated with the onset and progression of Parkinson's disease (PD). In PD, the dopamine-containing neurons in the substantia nigra pars compacta (SNc) degenerate and reduce dopamine-containing innervations to the striatum. The loss of striatal dopamine is associated with enhanced corticostriatal glutamatergic plasticity at the early stages of PD. However, with disease progression, the glutamatergic corticostriatal white matter tracts (WMTs) also degenerate. We analyzed the levels of Mu opioid receptors (MORs) in the corticostriatal WMTs, as a function of α-Synuclein (α-Syn) toxicity in transgenic mouse brains. Our data show an age-dependent loss of MOR expression levels in the striatum and specifically, within the caudal striatal WMTs in α-Syn tg mouse brains. The loss of MOR expression is associated with degeneration of the myelinated axons that are localized within the corticostriatal WMTs. In brains affected with late stages of PD, we detect evidence confirming the degeneration of myelinated axons within the corticostriatal WMTs. We conclude that loss of corticostriatal MOR expression is associated with degeneration of corticostriatal WMT in α-Syn tg mice, modeling PD.

Ischemic stroke causes Parkinson's disease-like pathology and symptoms in transgenic mice overexpressing alpha-synuclein.

The etiology of Parkinson's disease is poorly understood and is most commonly associated with advancing age, genetic predisposition, or environmental toxins. Epidemiological findings suggest that patients have a higher risk of developing Parkinson's disease after ischemic stroke, but this potential causality lacks mechanistic evidence. We investigated the long-term effects of ischemic stroke on pathogenesis in hemizygous TgM83 mice, which express human α-synuclein with the familial A53T mutation without developing any neuropathology or signs of neurologic disease for more than 600 days. We induced transient focal ischemia by middle cerebral artery occlusion in 2-month-old TgM83+/- mice and monitored their behavior and health status for up to 360 days post surgery. Groups of mice were sacrificed at 14, 30, 90, 180, and 360 days after surgery for neuropathological analysis of their brains. Motor deficits first appeared 6 months after focal ischemia and worsened until 12 months afterward. Immunohistochemical analysis revealed ischemia-induced neuronal loss in the infarct region and astrogliosis and microgliosis indicative of an inflammatory response, which was most pronounced at 14 days post surgery. Infarct volume and inflammation gradually decreased in size and severity until 180 days post surgery. Surprisingly, neuronal loss and inflammation were increased again by 360 days post surgery. These changes were accompanied by a continuous increase in α-synuclein aggregation, its neuronal deposition, and a late loss of dopaminergic neurons in the substantia nigra, which we detected at 360 days post surgery. Control animals that underwent sham surgery without middle cerebral artery occlusion showed no signs of disease or neuropathology. Our results establish a mechanistic link between ischemic stroke and Parkinson's disease and provide an animal model for studying possible interventions.

A role for α-Synuclein in axon growth and its implications in corticostriatal glutamatergic plasticity in Parkinson's disease.

BACKGROUND: α-Synuclein (α-Syn) is a protein implicated in the pathogenesis of Parkinson's disease (PD). α-Syn has been shown to associate with membranes and bind acidic phospholipids. However, the physiological importance of these associations to the integrity of axons is not fully clear. METHODS: Biochemical, immunohistochemical and ultrastructural analyses in cultured neurons, transgenic mouse brains, PD and control human brains. RESULTS: We analyzed the ultrastructure of cross-sectioned axons localized to white matter tracts (WMTs), within the dorsal striatum of old and symptomatic α-Syn transgenic mouse brains. The analysis indicated a higher density of axons of thinner diameter. Our findings in cultured cortical neurons indicate a role for α-Syn in elongation of the main axon and its collaterals, resulting in enhanced axonal arborization. We show that α-Syn effect to enhance axonal outgrowth is mediated through its activity to regulate membrane levels of the acidic phosphatidylinositol 4,5-bisphosphate (PI4,5P2). Moreover, our findings link α-Syn- enhanced axonal growth with evidence for axonal injury. In relevance to disease mechanisms, we detect in human brains evidence for a higher degree of corticostriatal glutamatergic plasticity within WMTs at early stages of PD. However, at later PD stages, the respective WMTs in the caudate are degenerated with accumulation of Lewy pathology. CONCLUSIONS: Our results show that through regulating PI4,5P2 levels, α-Syn acts to elongate the main axon and collaterals, resulting in a higher density of axons in the striatal WMTs. Based on these results we suggest a role for α-Syn in compensating mechanisms, involving corticostriatal glutamatergic plasticity, taking place early in PD.

Ciliary neurotrophic factor infused intracerebroventricularly shows reduced catabolic effects when linked to the TAT protein transduction domain.

Ciliary neurotrophic factor (CNTF) regulates the differentiation and survival of a wide spectrum of developing and adult neurons, including motor neuron loss after injury. We recently described a cell-penetrant recombinant human CNTF (rhCNTF) molecule, formed by fusion with the human immunodeficiency virus-1 transactivator of transcription (TAT) protein transduction domain (TAT-CNTF) that, upon subcutaneous administration, retains full neurotrophic activity without cytokine-like side-effects. Although the CNTF receptor is present in hypothalamic nuclei, which are involved in the control of energy, rhCNTF but not TAT-CNTF stimulates signal transducers and activators of transcription 3 phosphorylation in the rat hypothalamus after subcutaneous administration. This could be due limited TAT-CNTF distribution in the hypothalamus and/or altered intracellular signaling by the fusion protein. To explore these possibilities, we examined the effect of intracerebroventricular administration of TAT-CNTF in male adult rats. TAT-CNTF-induced weight loss, although the effect was smaller than that seen with either rhCNTF or leptin (which exerts CNTF-like effects via its receptor). In contrast to rhCNTF and leptin, TAT-CNTF neither induced morphological changes in adipose tissues nor increased uncoupling protein 1 expression in brown adipose tissue, a characteristic feature of rhCNTF and leptin. Acute intracerebroventricular administration of TAT-CNTF induced a less robust phosphorylation of signal transducers and activators of transcription 3 in the hypothalamus, compared with rhCNTF. The data show that fusion of a protein transduction domain may change rhCNTF CNS distribution, while further strengthening the utility of cell-penetrating peptide technology to neurotrophic factor biology beyond the neuroscience field.

α-Synuclein in blood cells differentiates Parkinson's disease from healthy controls.

OBJECTIVE: To determine whether blood cells expressed α-Syn can differentiate Parkinson's disease (PD) from healthy controls (HC). METHODS: The concentrations of α-Syn were determined in samples of blood cell pellets using a quantitative Lipid-ELISA assay. In addition, the levels of total protein, hemoglobin, iron and H-ferritin were determined. The study includes samples from the Biofind cohort (n = 46 PD and 45 HC) and results were validated with an additional cohort (n = 35 PD and 28 HC). RESULTS: A composite biomarker consisting of the concentrations of total α-Syn, proteinase-K resistant (PKres ) α-Syn and phospho-Serine 129 α-Syn (PSer 129), is designed based on the analysis of the discovery BioFIND cohort. This composite biomarker differentiates a PD subgroup, presenting motor symptoms without dementia from a HC group, with a convincing accuracy, represented by an AUC = 0.81 (95% CI, 0.71 to 0.92). Closely similar results were obtained for the validation cohort, that is, AUC = 0.81, (95% CI, 0.70 to 0.94). INTERPRETATION: Our results demonstrate the potential usefulness of blood cells expressed α-Syn as a biomarker for PD.

Higher levels of myelin phospholipids in brains of neuronal α-Synuclein transgenic mice precede myelin loss.

α-Synuclein is a protein involved in the pathogenesis of synucleinopathies, including Parkinson's disease (PD), dementia with Lewy bodies (DLB) and multiple system atrophy (MSA). We investigated the role of neuronal α-Syn in myelin composition and abnormalities. The phospholipid content of purified myelin was determined by 31P NMR in two mouse lines modeling PD, PrP-A53T α-Syn and Thy-1 wt-α-Syn. Significantly higher levels of phospholipids were detected in myelin purified from brains of these α-Syn transgenic mouse models than in control mice. Nevertheless, myelin ultrastructure appeared intact. To further investigate the effect of α-Syn on myelin abnormalities, we systematically analyzed the striatum, a brain region associated with neurodegeneration in PD. An age and disease-dependent loss of myelin basic protein (MBP) signal was detected by immunohistochemistry in striatal striosomes (patches). The age-dependent loss of MBP signal was associated with lower P25α levels in oligodendrocytes. In addition, we found that α-Syn inhibited oligodendrocyte maturation and the formation of membranous sheets in vitro. Based on these results we concluded that neuronal α-Syn is involved in the regulation and/or maintenance of myelin phospholipid. However, axonal hypomyelination in the PD models is evident only in progressive stages of the disease and associated with α-Syn toxicity.

Anticonvulsant activity of Caryocar coriaceum Wittm. fixed pulp oil against pentylenetetrazol-induced seizures.

OBJECTIVES: Epilepsy is a common brain disease and a major worldwide public health problem. The seizures in a significant number of patients suffering from epilepsy remain inadequately controlled by currently available pharmacological treatments. Accordingly, there is a need for the discovery of new anticonvulsant approaches with improved efficacy and a better safety profile. In this context, natural products can be a valuable source of substances with potential anticonvulsant activity. In the present study, we tested the anticonvulsant potential of Caryocar coriaceum Wittm., a plant native from the Brazilian Cerrado biome (tropical savanna ecoregion). METHODS: Adult male C57BL/6 mice were treated with increasing doses of the fixed oil obtained from the pulp of Caryocar coriaceum Wittm. Seizure activity was induced by PTZ (60 mg/kg, i.p.), and evaluated by behavioral and electrographic methods. Potential adverse effects were investigated in the open-field, rotarod, forced swim, or object recognition tests. The antioxidant potential of the oil was evaluated by the DPPH scavenging assay. RESULTS: Administration of the oil at the dose of 100 mg/kg increased the latency for the first myoclonic jerk and the first generalized tonic-clonic seizures. The duration of generalized convulsions induced by PTZ was not altered. No significant behavioral adverse effects were detected in the open-field, rotarod, forced swim, or object recognition tests. Interestingly, a significant antioxidant activity of Caryocar coriaceum Wittm. fixed pulp oil was detected in the DPPH scavenging assay. DISCUSSION: Natural products can be a valuable source of substances with potential anticonvulsant activity and improved safety profile. Further studies are needed to evaluate the mechanisms underlying the anticonvulsant effects of Caryocar coriaceum Wittm. fixed pulp oil as well as the potential of the oil as a source of new anticonvulsant compounds.

A proteomic approach in the study of an animal model of Parkinson's disease.

BACKGROUND: The aetiology of Parkinson's disease (PD), an age-related disorder characterized by a progressive degeneration of dopaminergic neurons of the substantia nigra (SN) pars compacta, remains unclear. Current treatments, such as administration of L-DOPA, are only symptomatic and do not stop or delay the progressive loss of neurons. In fact, it has been suggested that the dopamine precursor L-DOPA, increases generation of reactive oxygen species (ROS) leading to further neuronal damage. A similar loss in nigrostriatal dopaminergic neurons is produced on intracerebral administration of the catecholaminergic neurotoxin 6-hydroxydopamine (6-OHDA). In an animal model of PD, termed 'the hemiparkinsonian rat', unilateral injection of 6-OHDA into the nigrostriatal pathway results in extensive loss of dopaminergic cells in the ipsolateral SN. In an attempt to identify some of the proteins that are involved in dopaminergic neuronal death, we used the proteomic methods to analyze this animal model of PD. METHODS: Five hemiparkinsonian rats were obtained by intranigral stereotaxic injection of 6-OHDA. The right 6-OHDA-lesioned substantia nigra and striatum tissues along with the left, unlesioned controlateral tissues, were excised and homogenized, using urea-based buffer, to extract the tissues protein. The separation of the protein mixtures and the visualization of the protein patterns obtained were performed using two-dimensional polyacrylamide gel electrophoresis (2D-PAGE). Protein profiles of control and treated tissues were compare by the PDQuest 2D-gel analysis software (BIO-Rad laboratory). The protein spots showing differential expression were analysed by matrix assisted laser desorption/ionizing time of flight (MALDI-TOF) mass spectrometry. RESULTS: The brain protein extraction and solubilization protocol was validated obtaining a satisfactory protein profile. In comparison to the normal rats, hemiparkinsonian animals exhibited a different expression in alpha-enolase and beta-actin in substantia nigra and striatum, respectively. CONCLUSION: The proteomic study of 6-OHDA-induced lesions in the nigrostriatial pathway allowed us to identify two proteins, alpha-enolase and beta-actin, showing increased levels in the 6-OHDA-lesioned brain tissues compared to control. Previous studies described the same proteins as oxidized and proteins in Alzheimer's disease (AD) brain. Our preliminary data could mirror those results pointing out a common mechanism of neurodegenerative diseases.

Evaluation of potential gender-related differences in behavioral and cognitive alterations following pilocarpine-induced status epilepticus in C57BL/6 mice.

Together with pharmacoresistant seizures, the quality of life of temporal lobe epilepsy (TLE) patients is negatively impacted by behavioral comorbidities including but not limited to depression, anxiety and cognitive deficits. The pilocarpine model of TLE has been widely used to study characteristics of human TLE, including behavioral comorbidities. Since the outcomes of pilocarpine-induced TLE might vary depending on several experimental factors, we sought to investigate potential gender-related differences regarding selected behavioral alterations in C57BL6 mice. We found that epileptic mice, independent of gender, displayed increased anxiety-like behavior in the open-field test. In the object recognition test, epileptic mice, regardless of gender, showed a decreased recognition index at 24 (but not at 4) hours after training. On the other hand, no significant differences were found regarding mice learning and memory performance in the Barnes maze paradigm. Motor coordination and balance as assessed by the beam walk and rotarod tests were not impaired in epileptic mice of both genders. However, female mice, independent of epilepsy, performed the beam walk and rotarod tasks better than their male counterparts. We also found that only male epileptic mice displayed disturbed behavior in the forced swim test, but the mice of both genders displayed anhedonia-like behavior in the taste preference test. Lastly, we found that the extent of hilar cell loss is similar in both genders. In summary, both genders can be successfully employed to study behavioral comorbidities of TLE; however, taking the potential gender differences into account may help choose the more appropriated gender for a given task, which may be of value for the minimization of the number of animals used during the experiments.