development-unilaterally-lesioned-6-ohda-mouse-model-parkinson-s

Development of a Unilaterally-lesioned 6-OHDA Mouse Model of Parkinson's Disease

The unilaterally lesioned 6-hyroxydopamine (6-OHDA)-lesioned rat model of Parkinson's disease (PD) has proved to be invaluable in advancing our ...

SAP97 is a modular protein composed of three PDZ domains, an SH3 domain, and a guanylate kinase-like domain. It has been implicated functionally in the assembly and structural stability of synaptic junctions as well as in the trafficking, recruitment, and localization of specific ion channels and neurotransmitter receptors. The N terminus of SAP97 (S97N) has been shown to play a key role in the selection of binding partners and the localization of SAP97 at adhesion sites, as well as the clustering of ion channels in heterologous cells. Using the S97N domain as bait in a yeast two-hybrid screen, we identified the minus-end-directed actin-based motor, myosin VI, as an S97N binding partner. Moreover, in light membrane fractions prepared from rat brain, we found that myosin VI and SAP97 form a trimeric complex with the alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptor subunit, GluR1. These data suggest that SAP97 may serve as a molecular link between GluR1 and the actin-dependent motor protein myosin VI during the dynamic translocation of AMPA receptors to and from the postsynaptic plasma membrane.

Interaction of SAP97 with minus-end-directed actin motor myosin VI. Implications for AMPA receptor trafficking.

Interaktion von SAP97 mit minus-End-gerichtete Aktin-Myosin-Motor VI. Implikationen f&#xFC;r die AMPA-Rezeptor Menschenhandel.

Treatments for Parkinson's disease based on replacement of lost dopamine have several problems. Following loss of dopamine, enhanced N-methyl-D-aspartate (NMDA) receptor-mediated transmission in the striatum is thought to be part of the cascade of events leading to the generation of parkinsonian symptoms. We determined the localisation and pharmacological characteristics of NMDA receptors that play a role in generating parkinsonian symptoms within the striatum. Rats were lesioned unilaterally with 6-hydroxydopamine (6-OHDA), and cannulae implanted bilaterally to allow injection of a range of NMDA receptor antagonists at different striatal sites. When injected rostrally into the dopamine-depleted striatum, the glycine site partial agonist, (+)-HA-966 (44-400 nmol) caused a dose-dependent contraversive rotational response consistent with an antiparkinsonian action. (+)-HA-966 (400 nmol) had no effect when infused into more caudal regions of the dopamine-depleted striatum, or following injection into any striatal region on the dopamine-intact side. To determine the pharmacological profile of NMDA receptors involved in inducing parkinsonism in 6-OHDA-lesioned rats, a range of NMDA receptor antagonists was infused directly into the rostral striatum. Ifenprodil (100 nmol) and 7-chlorokynurenate (37 nmol), but not MK-801 (15 nmol) or D-APV (25 nmol) elicited a dramatic rotational response when injected into the dopamine-depleted striatum. This pharmacological profile is not consistent with an effect mediated via blocking NR2B-containing NMDA receptors. The effect of intrastriatal injection of ifenprodil was increased in animals previously treated with levodopa (L-dopa) methyl ester. This was seen as an increase in on-time and in peak rotational response. We propose that stimulation of NR2B-containing NMDA receptors in the rostral striatum underlies the generation of parkinsonian symptoms. These studies are in line with previous findings suggesting that administration of NR2B-selective NMDA receptor antagonists may be therapeutically beneficial for parkinsonian patients, when given de novo and following L-dopa treatment.

Characterisation of striatal NMDA receptors involved in the generation of parkinsonian symptoms: intrastriatal microinjection studies in the 6-OHDA-lesioned rat.

Charakterisierung der striatalen NMDA-Rezeptoren bei der Erzeugung der Parkinson-Symptome beteiligt: &#x200B;&#x200B;intrastriatalen Mikroinjektionsstudien in der 6-OHDA-l&#xE4;dierte Ratte.

To serve and protect? Interventions in the subthalamic nucleus for Parkinson's disease. Commentary on "Ablation of the subthalamic nucleus protects dopaminergic phenotype but not cell survival in a rat model of Parkinson's disease".

To Serve and Protect? Interventionen im subthalamischen Nukleus f&#xFC;r die Parkinson-Krankheit. Kommentar zum &#x26;quot;Ablation des Nucleus subthalamicus sch&#xFC;tzt dopaminergen Ph&#xE4;notyp, aber nicht das &#xDC;berleben der Zelle in einem Rattenmodell der Parkinson-Krankheit&#x26;quot;.

Myosin VI is an actin-based motor protein that is enriched at the postsynaptic density and appears to interact with alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate-type glutamate receptors (AMPARs) via synapse associated protein 97 (SAP97). Here, we find that a Flag epitope-tagged dominant negative construct that inhibits the interaction between SAP97 and myosin VI (Flag-myoVI-DN) causes a dramatic reduction in the number of synapses and the surface expression of AMPARs in cultured hippocampal neurons. Furthermore, we find that Flag-myoVI-DN also prevents the rapid delivery of AMPARs to synapses that can be induced by the transient activation of N-methyl-d-aspartate receptors. The Flag-myoVI-DN induced decrease in surface AMPARs is not because of reduced AMPAR subunit protein synthesis. Using whole-cell recording, we show that Flag-myoVI-DN also prevents the activity-induced increase in miniature excitatory postsynaptic current frequency that is normally associated with recruitment of AMPARs to the cell surface at synaptic sites that lack these receptors (i.e. 'silent' synapses). Together, these results indicate that myosin VI/SAP97 plays an important role in trafficking and activity-dependent recruitment of AMPARs to synapses.

Disruption of the interaction between myosin VI and SAP97 is associated with a reduction in the number of AMPARs at hippocampal synapses.

Myosin VI ist ein Aktin-basierte motor-Protein, das ist auf die postsynaptisches Dichte angereichert und erscheint Interaktion mit alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionat-Typ-Glutamat-Rezeptoren (AMPARs) &uuml;ber Synapse Protein 97 (SAP97) verbunden. Hier finden wir, dass ein Flag Epitop-Tags dominante negative Konstrukt, das die Interaktion zwischen SAP97 und Myosin VI (Flag-MyoVI-DN) hemmt eine drastische Reduzierung der Zahl der Synapsen und die Oberfl&auml;che Expression von AMPARs in bewirkt, dass Neuronen kultiviert. Dar&uuml;ber hinaus finden wir, dass die Flagge-MyoVI-DN verhindert auch die schnelle Lieferung von AMPARs an Synapsen, die durch die vor&uuml;bergehende Aktivierung von N-Methyl-d-Aspartat-Rezeptoren induziert werden k&ouml;nnen. Der Flag-MyoVI-DN verursachte R&uuml;ckgang der Oberfl&auml;che AMPARs ist nicht wegen eingeschr&auml;nkter AMPAR Untereinheit Proteinsynthese. Anhand ganz-Zell-Aufnahme zeigen wir, dass die Flagge-MyoVI-DN verhindert auch die Aktivit&auml;t-induzierte Steigerung in Miniatur exzitatorischen postsynaptisches aktuelle Frequenz, die Rekrutierung von AMPARs an der Zelloberfl&auml;che an synaptischen Standorten normalerweise zugeordnet ist, die diese Rezeptoren (d. h. &#39;stumme&#39; Synapsen) fehlt. Zusammen zeigen diese Ergebnisse, dass Myosin VI/SAP97 spielt eine wichtige Rolle bei Drogenhandel und leistungsabh&auml;ngige Rekrutierung von AMPARs zu Synapsen.

St&ouml;rung der Interaktion zwischen Myosin VI und SAP97 ist mit einer Verringerung der Zahl der AMPARs auf hippocampal Synapsen.

In Parkinson's disease, chronic striatal dopamine depletion results in over-activity and under-activity of the indirect and direct striatal output pathways respectively. In this study, we investigated changes in the function of glutamatergic cortico-striatal synapses that contribute to abnormalities in striatal efferents.

Altered function of glutamatergic cortico-striatal synapses causes output pathway abnormalities in a chronic model of parkinsonism.

Parkinson-Krankheit f&uuml;hrt chronische Striatale Dopamin Ersch&ouml;pfung bzw. in Over-activity und Under-activity der indirekten und direkten Striatale Ausgabe Bahnen. In dieser Studie untersuchten wir &Auml;nderungen in der Funktion von Glutamat Kortikosteroide-Striatale Synapsen, die zu Anomalien im Striatale Efferents beitragen.

Ver&auml;nderte Funktion des Glutamat Kortikosteroide-Striatale Synapsen Ursachen Ausgabe Stoffwechselweg Anomalien in einem chronischen Modell des Parkinsonismus.

The use of hippocampal dissociated neuronal cultures has enabled the study of molecular changes in endogenous native proteins associated with long-term potentiation. Using immunofluorescence labelling of the active (Thr286-phosphorylated) alpha-Ca(2+) /calmodulin-dependent protein kinase II (CaMKII) we found that CaMKII activity was increased by transient (3â€ƒÃ—â€ƒ1â€ƒs) depolarisation in 18- to 21-day-old cultures but not in 9- to 11-day-old cultures. The increase in Thr286 phosphorylation of CaMKII required the activation of NMDA receptors and was greatly attenuated by the CaMKII inhibitor KN-62. We compared the effects of transient depolarisation on the surface expression of GluA1 and GluA2 subunits of the alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate receptor and found a preferential recruitment of the GluA1 subunit. CaMKII inhibition prevented this NMDA receptor-dependent delivery of GluA1 to the cell surface. CaMKII activation is therefore an important factor in the activity-dependent recruitment of native GluA1 subunit-containing alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate receptors to the cell surface of hippocampal neurons.

LTP in hippocampal neurons is associated with a CaMKII-mediated increase in GluA1 surface expression.

Die Verwendung der hippocampal getrennten neuronale Kulturen hat das Studium der molekularen Ver&auml;nderungen in endogene native Proteine zugeordnete langfristige Erm&ouml;glichung aktiviert. Mit Immunfluoreszenz Kennzeichnung der aktiven (Thr286-phosphoryliert) alpha-Ca(2+)-/calmodulin-dependent-Proteinkinase II (CaMKII) fanden wir, dass CaMKII Aktivit&auml;t durch vor&uuml;bergehende Depolarisierung (3 &times; 1 s) in 18, 21-Tage alten Kulturen jedoch nicht in 9 bis 11-Tag-alte Kulturen verst&auml;rkt wurde. Die Zunahme der Thr286 Phosphorylierung von CaMKII ben&ouml;tigt die Aktivierung der NMDA-Rezeptoren und wurde durch den CaMKII wird KN-62 stark abgeschw&auml;cht. Wir verglichen die Wirkung von Transienten Depolarisierung auf der Oberfl&auml;che Ausdruck der GluA1 und GluA2-Untereinheiten des Rezeptors alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionat und fand eine bevorzugte Einstellung von der GluA1-Untereinheit. CaMKII Hemmung verhindert diese NMDA-Rezeptor-abh&auml;ngige-Auslieferung von GluA1 an der Zelloberfl&auml;che. CaMKII Aktivierung ist daher ein wichtiger Faktor f&uuml;r die leistungsabh&auml;ngige Rekrutierung von native GluA1 Untereinheit-haltigen alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionat-Rezeptoren an der Zelloberfl&auml;che von Neuronen.

LTP in Neuronen ist einen CaMKII-vermittelten Anstieg der GluA1 Oberfl&auml;che Ausdruck zugeordnet.

Prolonged use of the dopamine precursor l-DOPA for the treatment of Parkinson's disease commonly results in abnormal involuntary movements, which are termed l-DOPA-induced dyskinesia (LID). Over-activity at corticostriatal synapses onto neurons of the direct and indirect striatal output pathways has been implicated in the development of dyskinesia, but it has proved difficult to investigate the pathways separately due to their morphological similarities. The recent development of bacterial artificial chromosome mice that express green fluorescent protein in either the direct or indirect pathway allows visual identification of the output neurons in each pathway. Here we describe the use of two different strains of these transgenic mice (pure FVB and FVB crossed with C57B1/6) in the development of mouse models of l-DOPA-induced dyskinesia. This model will allow the direct and indirect pathways to be studied individually to delineate the cellular and molecular mechanisms underlying dyskinesias. These studies demonstrate that mouse strain impacts on behavioural responses and l-DOPA sensitivity. Therefore, when generating mouse models of LID, strain must be taken into consideration when choosing the l-DOPA dosing regimen.

Generation of a model of L-DOPA-induced dyskinesia in two different mouse strains.

L&auml;ngere Anwendung von l-DOPA die Dopamin-Vorstufe zur Behandlung der Parkinsonschen Krankheit f&uuml;hrt h&auml;ufig zu abnorme unwillk&uuml;rliche Bewegungen, die l-DOPA-induzierte Dyskinesien (LID) bezeichnet werden. Over-Activity am Corticostriatal Synapsen auf Neuronen von den direkten und indirekten Striatale Ausgang Wege hat verwickelt wurden, bei der Entwicklung von Dyskinesien, aber es erwies sich schwer, die Bahnen getrennt aufgrund ihrer morphologischen &Auml;hnlichkeiten zu untersuchen. Die aktuelle Entwicklung der bacterial artificial Chromosome-M&auml;use, die gr&uuml;nes fluoreszierendes Protein in beiden Ausdr&uuml;cken erm&ouml;glicht die direkte oder indirekte Pathway visuellen Identifikation von der Ausgabeneuronen in jeder Weg. Hier beschreiben wir die Verwendung von zwei verschiedenen St&auml;mmen von diese transgenen M&auml;usen (reine FVB und FVB gekreuzt mit C57B1/6) bei der Entwicklung von Mausmodelle von l-DOPA-induzierte Dyskinesien. Dieses Modell erlaubt die direkte und indirekte Wege in einzeln untersucht werden, um die zellul&auml;ren und molekularen Mechanismen Dyskinesien abzugrenzen. Diese Studien zeigen, dass die Auswirkungen von Maus-Stamm auf Verhaltensst&ouml;rungen Antworten und l-DOPA-Empfindlichkeit. Daher muss beim Generieren von Mausmodelle des LIDES Belastung ber&uuml;cksichtigt werden bei der Wahl der Dosierung l-DOPA-Therapie.

Article	Year
Interaction of SAP97 with minus-end-directed actin motor myosin VI. Implications for AMPA receptor trafficking. The Journal of biological chemistry\| PubMed ID: 12050163	2002
Characterisation of striatal NMDA receptors involved in the generation of parkinsonian symptoms: intrastriatal microinjection studies in the 6-OHDA-lesioned rat. Movement disorders : official journal of the Movement Disorder Society\| PubMed ID: 12112191	2002
To serve and protect? Interventions in the subthalamic nucleus for Parkinson's disease. Commentary on "Ablation of the subthalamic nucleus protects dopaminergic phenotype but not cell surviv... Experimental neurology\| PubMed ID: 14736500	2004
Disruption of the interaction between myosin VI and SAP97 is associated with a reduction in the number of AMPARs at hippocampal synapses. Journal of neurochemistry\| PubMed ID: 19895665	2010
Altered function of glutamatergic cortico-striatal synapses causes output pathway abnormalities in a chronic model of parkinsonism. Neurobiology of disease\| PubMed ID: 20971190	2011
LTP in hippocampal neurons is associated with a CaMKII-mediated increase in GluA1 surface expression. Journal of neurochemistry\| PubMed ID: 21143596	2011
Generation of a model of L-DOPA-induced dyskinesia in two different mouse strains. Journal of neuroscience methods\| PubMed ID: 21352853	2011

Joanne E. Nash

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