Alpha-synuclein oligomers alter the spontaneous firing discharge of cultured midbrain neurons

The aim of this work was to monitor the effects of extracellular α-synuclein on the firing activity of midbrain neurons dissociated from substantia nigra TH-GFP mice embryos and cultured on microelectrode arrays (MEA). We monitored the spontaneous firing discharge of the network for 21 days after plating and the role of glutamatergic and GABAergic inputs in regulating burst generation and network synchronism. Addition of GABAA, AMPA and NMDA antagonists did not suppress the spontaneous activity but allowed to identify three types of neurons that exhibited different modalities of firing and response to applied L-DOPA: high-rate (HR) neurons, low-rate pacemaking (LR-p), and low-rate non-pacemaking (LR-np) neurons. Most HR neurons were insensitive to L-DOPA, while the majority of LR- p neurons responded with a decrease of the firing discharge; less defined was the response of LR-np neurons. The effect of exogenous α-synuclein (α-syn) on the firing discharge of midbrain neurons was then studied by varying the exposure time (0–48 h) and the α-syn concentration (0.3–70 μM), while the formation of α-syn oligomers was monitored by means of AFM. Independently of the applied concentration, acute exposure to α-syn monomers did not exert any effect on the spontaneous firing rate of HR, LR-p, and LR-np neurons. On the contrary, after 48 h exposure, the firing activity was drastically altered at late developmental stages (14 days in vitro, DIV, neurons): α-syn oligomers progressively reduced the spontaneous firing discharge (IC50 = 1.03 μM), impaired burst generation and network synchronism, proportionally to the increased oligomer/monomer ratio. Different effects were found on early-stage developed neurons (9 DIV), whose firing discharge remained unaltered, regardless of the applied α-syn concentration and the exposure time. Our findings unravel, for the first time, the variable effects of exogenous α-syn at different stages of midbrain network development and provide new evidence for the early detection of neuronal function impairment associated to aggregated forms of α-syn

Alpha-synuclein oligomers alter the spontaneous firing discharge of cultured midbrain neurons

The aim of this work was to monitor the effects of extracellular α-synuclein on the firing activity of midbrain neurons dissociated from substantia nigra TH-GFP mice embryos and cultured on microelectrode arrays (MEA). We monitored the spontaneous firing discharge of the network for 21 days after plating and the role of glutamatergic and GABAergic inputs in regulating burst generation and network synchronism. Addition of GABAA, AMPA and NMDA antagonists did not suppress the spontaneous activity but allowed to identify three types of neurons that exhibited different modalities of firing and response to applied L-DOPA: high-rate (HR) neurons, low-rate pacemaking (LR-p), and low-rate non-pacemaking (LR-np) neurons. Most HR neurons were insensitive to L-DOPA, while the majority of LR-p neurons responded with a decrease of the firing discharge; less defined was the response of LR-np neurons. The effect of exogenous α-synuclein (α-syn) on the firing discharge of midbrain neurons was then studied by varying the exposure time (0–48 h) and the α-syn concentration (0.3–70 μM), while the formation of α-syn oligomers was monitored by means of AFM. Independently of the applied concentration, acute exposure to α-syn monomers did not exert any effect on the spontaneous firing rate of HR, LR-p, and LR-np neurons. On the contrary, after 48 h exposure, the firing activity was drastically altered at late developmental stages (14 days in vitro, DIV, neurons): α-syn oligomers progressively reduced the spontaneous firing discharge (IC50 = 1.03 μM), impaired burst generation and network synchronism, proportionally to the increased oligomer/monomer ratio. Different effects were found on early-stage developed neurons (9 DIV), whose firing discharge remained unaltered, regardless of the applied α-syn concentration and the exposure time. Our findings unravel, for the first time, the variable effects of exogenous α-syn at different stages of midbrain network development and provide new evidence for the early detection of neuronal function impairment associated to aggregated forms of α-syn

Structure-function study of ubiquitin c-terminal hydrolase L1 (UCH-L1) by NMR spectroscopy - insights into UCH-L1 mutation's association with the risk of Parkinson's disease

Poster Presentation: P72Protein ubiquitination and deubiquitination, play important roles in many aspects of cellular mechanisms. Its defective regulation results in diseases that range from developmental abnormalities to neurodegenerative diseases and cancer. Ubiquitin carboxy-terminal hydrolase L1 (UCH-L1) is a protein of 223 amino acids, which is highly abundant in brain, constituting up to 2% of total brain proteins. Although it was originally characterized as a deubiquitinating enzyme, recent studies indicate that it also functions as a ubiquitin ligase and a mono-Ub stabilizer. Down-regulation and extensive oxidative modifications of UCH-L1 have been observed in the brains of Alzheimer’s disease and Parkinson’s disease (PD) patients. Of importance, I93M and S18Y point mutations in the UCH-L1 gene have been reported to be linked to susceptibility to and protection from PD respectively. Hence, the structure of UCH-L1 and the effects of disease associated mutations on the structure and function are of considerable interest. Our circular dichroism studies suggest that the S18Y point mutation only slightly perturbs the structure while a significant decrease in the α-helical content is observed in the I93M mutant. We have determined the solution structure of S18Y and mapping its interaction with ubiquitin by chemical shift perturbation approach. The electrostatic surface potential analysis reveals that the interaction between ubiquitin and UCH-L1-S18Y is primarily electrostatic in nature, with negatively charged residues on the surface of UCH-L1-S18Y interacting with the positively charged residues on the basic face of ubiquitin. Although the active site and the L8 loop in UCH-L1-S18Y adopts conformations similar to that observed in the crystal structure of UCH-L1-WT, both the altered hydrogen bond network and surface charge distributions have demonstrated that the S18Y substitution could lead to profound structural changes. In particular, the difference in the dimeric interfaces of the wild-type and the S18Y mutant has shown that mutation can significantly affect the distribution of the surface-exposed residues involved in the dimeric interface. Such observed difference might weaken the stability of the UCH-L1 dimer and hence may explain the reduced dimerization-dependent ligase activity of UCH-L1-S18Y in comparison to UCH-L1-WT.postprin

Protective effects of lycium barbarum polysaccharides on cerebral edema and blood-brain barrier disruption after ischemic stroke

Young Investigators Symposium I (Y3) - Di YangBACKGROUND: Ischemic stroke is a destructive cerebrovascular disease and one of the leading causes of death worldwide. The long term disability after stroke induces heavy burden both to the patients and the society. Yet, no effective neuroprotective agents are available. The polysaccharides extracted from the fruits of wolfberry, Lycium barbarum (LBP), showed neuroprotective and immune-modulative functions. We aim to evaluate the protective effects of LBP in experimental stroke using a focal cerebral ischemia/reperfusion (I/R) model. METHODS: C57BL/6N mice were subjected to 2 h of middle cerebral artery occlusion (MCAO) followed by 22 h of reperfusion. Prior to ischemia induction, animals were treated with either vehicle (PBS) or LBP daily for 7 days. Mice were evaluated for neurological deficits just before sacrifice. Brains were harvested for infarct size estimation, water content measurement and immunohistochemical analysis as well as Western blot experiments. Evans blue (EB) extravasation experiment was performed to determine blood-brain barrier (BBB) disruption after MCAO. RESULTS: LBP treatment significantly improved neurological scores and decreased infarct size, hemispheric swelling and water content as well as reduced EB extravasation. In addition, fewer apoptotic cells were identified in the LBP-treated brains by TUNEL assay. Immunoreactivity for aquaporin-4 and glial fibrillary acidic protein were also significantly decreased in LBP-treated brains. We further observed a reduction of nuclear factor-κB translocation and IκB expression after LBP treatment. CONCLUSION: Seven-day LBP pre-treatment effectively improved neurological deficits, decreased infarct size and cerebral edema as well as protected the brain from BBB disruption, aquaporin water channel up-regulation and glial activation. The protective effects of LBP might partially act through its anti-inflammatory effects. The present study suggests that LBP may be used as a preventive neuroprotectant for ischemic stroke.postprin

Aldose reductase deficiency protects the retinal neurons in a mouse model of retinopathy of prematurity

Poster Presentation: P64PURPOSE: Retinopathy of prematurity (ROP) is a common retinal disease occurred in premature babies. It is found to be related to oxidative stress while dysfunction of the neural retina has also been documented. We previously showed that genetic deletion or pharmacological inhibition of aldose reductase (AR), a rate- limiting enzyme in the polyol pathway, prevented ischemia-induced retinal ganglion cell (RGC) loss and oxidative stress. Here, we assessed the effects of AR deletion on retinal neurons using a mouse model of ROP. METHODS: Seven-day-old mouse pups were exposed to 75% oxygen for five days and returned to room air. The pathological neuronal changes were examined and compared between wild-type (WT) and AR-deficient retinae on P14 and P17 (P, postnatal). Retinal thickness was measured and immunohistochemistry for calbindin, calretinin, PKCα, Tuj1, glial fibrillary acidic protein (GFAP), nitrotyrosine (NT), as well as poly(ADP-ribose) (PAR) was performed. RESULTS: After hyperoxia exposure, significantly reduced inner nuclear layer (INL) and inner plexiform layer (IPL) thickness were found in both genotypes. The intensity of calbindin staining for horizontal cells in INL was reduced in the WT retinae but not in AR-deficient retinae. In addition, significant reduction was found in calretinin-positive amacrine cell bodies in central INL especially in WT retinae. Serious distortion was also observed in the three calretinin-positive strata along IPL in the WT retinae but not AR-deficient retinae on P17. Moreover, increased GFAP intensity across IPL indicating Müller cell processes was observed in AR-deficient retinae on P14 and in WT retinae on P17. Furthermore, increased NT immunoreactivity in INL and nuclear or para-nuclear PAR staining along GCL were observed in WT retina while these changes were not apparent in AR-deficient retina. CONCLUSION: Our observations demonstrated morphological changes of retinal neurons in the mouse model of ROP and indicated that AR deficiency showed neuronal protection in the retina, possibly through modulating glial responses and reducing oxidative stress.postprin

Regulatory role of proheparanase with peri-synaptic heparan sulfate proteoglycan and AMPA-type glutamate receptor in synaptic plasticity

Poster Presentation: P59AMPA-type glutamate receptors (AMPAR) govern excitatory synaptic transmission. Perineuronal heparan sulfates (HS) have been implicated in controlling the open-state of AMPAR. Our finding of neuronal heparanase expression in adult rats led us to test (1) if neuronal heparanase is secreted and (2) if the secreted form acts on perineuronal HS to modulate synaptic plasticity. Neuronal secretion of heparanase was triggered by phorbol ester of rat hippocampal neurons in culture. Western blot analysis of the secreted product revealed enzymatically inactive proheparanase, but not the enzymatically active heparanase. Synaptosomes prepared from phorbol ester-treated rat cortexslices showed enrichment in proheparanase; co-immunoprecipitation studies further showed association of AMPAR subunits (GluA1 and GluA2/3) with both syndecan-3 (a transmembrane HS-proteoglycan) and proheparanase, suggesting their partnership in the peri-synaptic environment. Treatment of hippocampal neurons in culture with recombinant proheparanase triggered internalization of proheparanase, perineuronal HS-proteoglycans and AMPARs, suggesting their clustering as a functional complex. Heparitinase pre-treatment of hippocampal neuron cultures reduced proheparanase-induced internalization of AMPARs, suggesting that the HS moiety is critical for effecting the partnership. Treatment of hippocampal slices with recombinant proheparanase resulted in down-regulation of both basal synaptic strength and LTP at Schaffer collateral synapses. These results reveal a novel role of neuronal proheparanase in resetting AMPAR and perineuronal HS levels at the synapse and thus the modulation of synaptic plasticity.postprin

Age-dependent hippocampal network dysfuntion in a mouse model of alpha-synucleinopathy

PhD ThesisAggregation of the protein alpha-synuclein (ASYN) is a key pathological feature of the alpha-synucleinopathies, a group of diseases including Lewy body dementia and Parkinson’s disease. A common symptom of alpha-synucleinopathy is cognitive dysfunction, and impairment in hippocampal gamma-frequency oscillations may underlie some of the cognitive deficits associated with ASYN pathology. The Thy-1 A30P mouse model overexpresses human mutant ASYN, with mice developing hippocampal spatial memory impairment by 12 months (Freichel et al. 2007). The aim of this thesis was to explore age-related hippocampal network changes in 2-4 month (A30P2+) mice and 10-13 month (A30P10+) mice to assess the effect of overexpression of mutant ASYN on hippocampal network activity in vitro. Using acute brain slice preparations of isolated hippocampi, A30P2+ mouse slices were found to exhibit excitatory/inhibitory network changes in region CA3 in the form of increased spontaneous sharp wave amplitude, increased frequency and amplitude of inhibitory postsynaptic potentials, and increased power of kainate-induced gamma-frequency oscillations. Immunohistochemistry revealed an increase in the density of parvalbuminpositive interneurons alongside a decrease in calbindin-positive interneurons. This change was accompanied by a more depolarised resting membrane potential in A30P2+ mouse CA3 pyramidal cells, and a sensitivity to interictal discharges in response to either kainate receptor agonism or GABAA receptor antagonism. With ageing, levels of excitability in A30P10+ mice were comparable to WT10+ mice. A30P10+ mice instead exhibited an impairment in cholinergic-induced, but not kainateinduced or spontaneous, gamma-frequency network oscillations. While mitochondrial dysfunction was not detectable with COX/SDH histochemistry until 15+ months in A30P mice, A30P10+ mice did show increased immunoreactivity for Iba1+ microglia. An environment of inflammation and excitotoxicity may be present in older A30P mice as a result of early network hyperexcitability, and this thesis explores early network changes in A30P mice and the wider dysfunction that followsEisai and the Medical Research Council for funding the MRC Industrial CASE Studentship

Modulation of the 5-HT3 Receptor as a Novel Anti-Dyskinetic Target in Parkinson’s Disease

La L-3,4-dihydroxyphénylalanine (L-DOPA) est le traitement le plus efficace de la maladie de Parkinson. Cependant, avec une administration chronique de L-DOPA, les patients développent des complications motrices telles que les dyskinésies. Des études antérieures ont montré que le blocage des récepteurs type 3 de la sérotonine (5-HT3) réduit les niveaux de dopamine dans les ganglions de la base, suggérant qu'il pourrait atténuer la libération de dopamine qui caractérise l'état dyskinétique. Ici, nous avons étudié les effets de l’ondansétron, un antagoniste hautement sélectif du récepteur 5-HT3 à diminuer et à prévenir le développement des dyskinésies induites par L-DOPA chez le rat lésé a la 6-hydroxydopamine. Dans la première expérience, les rats sensibilisés avec L-DOPA pour induire des mouvements involontaires anormaux (AIMs), ont reçu L-DOPA en combinaison avec l'ondansétron ou un véhicule. Dans la seconde expérience, les doses efficaces d'ondansétron ont été administrées simultanément avec L-DOPA pendant 22 jours, et la sévérité des dyskinésies a été évaluée. Après 3 jours d’élimination, L-DOPA a été administré en aigu et la sévérité des dyskinésies évaluée. Nous avons trouvé que l'ondansétron 0,0001 mg/kg en combinaison avec L-DOPA, a significativement diminué la sévérité des dyskinésies par rapport à L-DOPA seul. Ondansétron 0,0001 mg/kg, administré en même temps que L-DOPA, a retardé le développement des dyskinésies. L'action anti-dyskinétique de l'ondansétron n'a pas compromis le bénéfice thérapeutique conféré par la L-DOPA. Ces résultats suggèrent que l'antagonisme des récepteurs 5-HT3 est une stratégie thérapeutique potentiellement nouvelle et efficace pour soulager la sévérité et prévenir le développement des dyskinésies.L-3,4-dihydroxyphenylalanine (L-DOPA) is the most effective treatment for Parkinson’s disease However, with chronic administration of L-DOPA, patients develop motor complications such as dyskinesia. Previous studies have shown that 5-HT3 receptor blockade reduces dopamine levels within the basal ganglia, suggesting that it could mitigate the aberrant dopamine release that characterises the dyskinetic state. Here, we investigated the effects of the highly-selective 5-HT3 antagonist ondansetron at diminishing the expression of established, and preventing the development of L-DOPA-induced dyskinesia in the 6-hydroxydopamine-lesioned rat. In the first set of experiments, rats were primed with L-DOPA to induce abnormal involuntary movements (AIMs), after which L-DOPA was administered, in combination with ondansetron or vehicle. The effect of ondansetron on L-DOPA anti-parkinsonian action was subsequently determined by the cylinder test. In the second set of experiments, rats were administered effective doses of ondansetron, started concurrently with L-DOPA for 22 days, during which dyskinesia severity was monitored. After a 3-day washout period, an acute challenge of L-DOPA was administered and AIMs severity was assessed. We found that acute challenges of ondansetron 0.0001 mg/kg in combination with L-DOPA, significantly diminished the severity of AIMs compared to L-DOPA alone. Ondansetron 0.0001 mg/kg, when started concurrently with L-DOPA, attenuated the priming process leading to the development of dyskinesia. The anti-dyskinetic action of ondansetron did not compromise the therapeutic benefit conferred by L-DOPA. These results suggest that 5-HT3 receptor antagonism is a potentially new and effective therapeutic strategy to alleviate the severity, and prevent the development of dyskinesia

Mitochondrial Dysfunction in Dopaminergic Neurons and the Impact on Neurodegeneration in Parkinson's disease

Parkinson’s disease (PD) is a neurodegenerative disorder affecting ~1% of the population above 60 years. It is primarily characterized by severe motor deficits following the progressive and selective loss of dopaminergic neurons (DaNs) in the substantia nigra pars compacta (SNc). Non-motor symptoms, such as hyposmia and depression, further arise in a prodromal manner. Mitochondrial dysfunction plays an essential role for the loss of SNc DaNs, as evidenced by mitochondrial complex I impairing toxins, an especially high accumulation rate of mitochondrial DNA (mtDNA) deletions as well as the wide variety of mitochondrial-related gene mutations in familial forms of PD. Given that neighboring DaNs in the ventral tegmental area (VTA) are relatively spared from neurodegeneration, however, cell type specific factors must additionally contribute to the selective vulnerability in PD. In this work, data of three manuscripts are presented, confirming that distinct DaN populations respond differently to mitochondrial dysfunction following inactivation of mitochondrial transcription factor A (TFAM) in mice. In the olfactory bulb (OB), only small anaxonic DaNs (SCs) are mildly reduced in numbers, which is however associated with severe olfactory dysfunction and suggests a putative role for OB DaNs in the development of PD-related hyposmia. The midbrain reveals progressive and exclusive loss of SNc DaNs, which is accompanied by a severe decline of motor function. In contrast to the VTA, DaNs in the SNc die through a detrimental imbalance in the mitochondrial redox system, triggered by enhanced intracellular Ca2+ loads. Whereas SNc DaNs perish due to the rapid and continuous loss of mtDNA upon TFAM inactivation, they can adapt to impaired mtDNA replication, demonstrated after the expression of a mutated variant of the mitochondrial helicase TWINKLE (K320E). Despite similar severe neurodegeneration of both SNc and VTA DaNs, aged SNc DaNs preserve striatal innervation and thereby normal motor performance. Conversely, VTA DaN projections are lost, causing depressive-like behavior in these animals. Thus, identification and stimulation of ongoing compensatory mechanisms in aged SNc DaNs of K320E-TwinkleDaN mice host the potential to help the small number of remaining SNc DaNs in PD patients to escape from cell death and concurrently to recover motor performance through enhanced striatal innervation