Ringraziamo Hanna Warner per aver contribuito i dati per la figura 3 e il Prof. Jonas Bergquist per un contributo prezioso. Il Consiglio svedese della ricerca (Grant 522-2006-6416 (MA), 521-2007-5407 (MA); Il Wiberg Åke della Fondazione (MA, JH), L&#39;Accademia Reale Svedese delle Scienze (MA, JH), e il chimico svedese Society (JH) Si ringraziano per il sostegno finanziario.

<ol>
	<li>Obeso, J. A., Olanow, C. W., Nutt, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Levodopa+motor+complications+in+Parkinson's+disease.">Levodopa motor complications in Parkinson's disease.</a> Trends Neurosci. 23, S2-S7 (2000).</li><li>Caprioli, R. M., Farmer, T. B., Gile, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+imaging+of+biological+samples:+localization+of+peptides+and+proteins+using+MALDI-TOF+MS.">Molecular imaging of biological samples: localization of peptides and proteins using MALDI-TOF MS.</a> MALDI-TOF MS. Anal. Chem. 69, 4751-4760 (1997).</li><li>Obeso, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+evolution+and+origin+of+motor+complications+in+Parkinson's+disease.">The evolution and origin of motor complications in Parkinson's disease.</a> Neurology. 55, S13-S20 (2000).</li><li>O, W. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Noncommunicable+Diseases+and+Mental+Health+Cluster,+Noncommunicable+Disease+Prevention+and+Health+Promotion+Department,+Ageing+and+Life+Course.">Noncommunicable Diseases and Mental Health Cluster, Noncommunicable Disease Prevention and Health Promotion Department, Ageing and Life Course.</a> Active Ageing: A Policy framework. , (2002).</li><li>Schapira, A. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Movement+disorders:+advances+in+cause+and+treatment.">Movement disorders: advances in cause and treatment.</a> Lancet Neurology. , 6-7 (2010).</li><li>Obeso, J. A., Rodriguez-Oroz, M. C., Rodriguez, M., DeLong, M. R., Olanow, C. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathophysiology+of+levodopa-induced+dyskinesias+in+Parkinson's+disease:+problems+with+the+current+model.">Pathophysiology of levodopa-induced dyskinesias in Parkinson's disease: problems with the current model.</a> Ann. Neurol. 47, S22-S32 (2000).</li><li>Cenci, M. A., Lee, C. S., Bjorklund, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=L-DOPA-induced+dyskinesia+in+the+rat+is+associated+with+striatal+overexpression+of+prodynorphin-+and+glutamic+acid+decarboxylase+mRNA.">L-DOPA-induced dyskinesia in the rat is associated with striatal overexpression of prodynorphin- and glutamic acid decarboxylase mRNA.</a> Eur. J. Neurosci. 10, 2694-2706 (1998).</li><li>Andersson, M., Hilbertson, A., Cenci, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Striatal+fosB+expression+is+causally+linked+with+l-DOPA-induced+abnormal+involuntary+movements+and+the+associated+upregulation+of+striatal+prodynorphin+mRNA+in+a+rat+model+of+Parkinson's+disease.">Striatal fosB expression is causally linked with l-DOPA-induced abnormal involuntary movements and the associated upregulation of striatal prodynorphin mRNA in a rat model of Parkinson's disease.</a> Neurobiol Dis. 6, 461-474 (1999).</li><li>Hanrieder, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alterations+of+striatal+neuropeptides+revealed+by+imaging+mass+spectrometry.">Alterations of striatal neuropeptides revealed by imaging mass spectrometry.</a> Molecular &#38; Cellular Proteomics. , (2011).</li><li>Cornett, D. S., Reyzer, M. L., Chaurand, P., Caprioli, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MALDI+imaging+mass+spectrometry:+molecular+snapshots+of+biochemical+systems.">MALDI imaging mass spectrometry: molecular snapshots of biochemical systems.</a> Nat. Methods. 4, 828-833 (2007).</li><li>Ljungdahl, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+Mass+Spectrometry+Reveals+Elevated+Nigral+Levels+of+Dynorphin+Neuropeptides+in+L-DOPA-Induced+Dyskinesia+in+Rat+Model+of+Parkinson's+Disease.">Imaging Mass Spectrometry Reveals Elevated Nigral Levels of Dynorphin Neuropeptides in L-DOPA-Induced Dyskinesia in Rat Model of Parkinson's Disease.</a> PLoS ONE. 6, e25653 (2011).</li><li>Groseclose, M. R., Andersson, M., Hardesty, W. M., Caprioli, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+proteins+directly+from+tissue:+in+situ+tryptic+digestions+coupled+with+imaging+mass+spectrometry.">Identification of proteins directly from tissue: in situ tryptic digestions coupled with imaging mass spectrometry.</a> J. Mass. Spectrom. 42, 254-262 (2007).</li><li>Andersson, M., Groseclose, M. R., Deutch, A. Y., Caprioli, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+mass+spectrometry+of+proteins+and+peptides:+3D+volume+reconstruction.">Imaging mass spectrometry of proteins and peptides: 3D volume reconstruction.</a> Nat. Methods. 5, 101-108 (2008).</li><li>Deininger, S. -. O., Setou, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Imaging Mass Spectrometry. , 199-208 (2010).</li><li>Norris, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Processing+MALDI+Mass+Spectra+to+Improve+Mass+Spectral+Direct+Tissue+Analysis.">Processing MALDI Mass Spectra to Improve Mass Spectral Direct Tissue Analysis.</a> Int. J. Mass. Spectrom. 260, 212-221 (2007).</li><li>Ihaka, R., Gentleman, R. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Language+for+Data+Analysis+and+Graphics.">A Language for Data Analysis and Graphics.</a> Journal of Computational and Graphical Statistics. 5, 299-314 (1996).</li><li>Tusher, V. G., Tibshirani, R., Chu, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Significance+analysis+of+microarrays+applied+to+the+ionizing+radiation+response.">Significance analysis of microarrays applied to the ionizing radiation response.</a> Proc. Natl. Acad. Sci. U.S.A. 98, 5116-5121 (2001).</li><li>Bergstrom, L., Christensson, I., Folkesson, R., Stenstrom, B., Terenius, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+ion+exchange+chromatography+and+radioimmunoassay+procedure+for+measuring+opioid+peptides+and+substance+P.">An ion exchange chromatography and radioimmunoassay procedure for measuring opioid peptides and substance P.</a> Life. Sci. 33, 1613-1619 (1983).</li><li>Falth, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuropeptidomics+strategies+for+specific+and+sensitive+identification+of+endogenous+peptides.">Neuropeptidomics strategies for specific and sensitive identification of endogenous peptides.</a> Mol. Cell. Proteomics. 6, 1188-1197 (2007).</li><li>Falth, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SwePep,+a+database+designed+for+endogenous+peptides+and+mass+spectrometry.">SwePep, a database designed for endogenous peptides and mass spectrometry.</a> Mol. Cell. Proteomics. 5, 998-1005 (2006).</li></ol>

Gli autori non hanno nulla da rivelare.

Ci sono diversi vantaggi di spettrometria di massa MALDI impiegare immagini nello studio di neuropeptidi. L&#39;analisi dei dati imparziale MS può rivelare che nuclei cervello solo specifico, o come nei risultati qui presentati in cui solo la parte dorsolaterale dello striato è associato ad una certa condizione fisiopatologica. Mantenendo le informazioni spaziali è poi possibile ridefinire regioni di interesse per effettuare analisi statistiche con maggiore sensibilità e minore variabilità rispetto all&#39;analisi di sezioni cerebrali intere o con tradizionali studi peptidomics su estratti peptide. Inoltre, è importante realizzare MALDI IMS prontamente in grado di rilevare precedentemente sconosciuti modifiche post-traduzionali, ma analisi strutturali deve seguire per determinare le posizioni esatte amminoacidiche che vengono modificati. Errori più frequenti nella visualizzazione dei dati IMS MALDI includono la mappatura l&#39;intensità massima di picco di una scala lineare ottica da black (0%) di colore (100%) per ogni sezione della serie sperimentale (figura 3), invece di mappatura tutte le sezioni di una scala comune assoluto dove 100% è l&#39;intensità del picco massimo di tutte le sezioni (figura 5) . Quest&#39;ultimo metodo consente il confronto dei dati di gruppo e la visualizzazione delle differenze tra i gruppi di trattamento. Uno dei principali ostacoli in analisi MALDI IMS è l&#39;assegnazione di peptidi a picchi di massa specifici. On-tessuto-spettrometria di massa tandem a volte è possibile, ma risulta spesso molto difficile 13,14. Abbiamo trovato che un approccio più tradizionale comprendente un frazionamento preparativa su forte cromatografia a scambio cationico, seguito da LC-MS/MS fase inversa può essere utilizzato con successo per neuropeptidi sequenza molti peptidi e soprattutto oppioidi. Non è ancora raro ottenere una buona qualità di MS / MS che non corrispondono a tutte le voci del database che utilizzano i motori di ricerca comuni come mascotte. In questi casi de novo sequencing-mano è il only opzione. L&#39;ultima prova di identità di picco può essere ottenuta tramite MALDI IMS di sezioni di tessuto dal topo knockout appropriato, ma questo non è sempre disponibile o possibile. Un&#39;alternativa è quella di convalidare i risultati con un metodo diametralmente diverso, ad esempio mediante immunoblotting occidentale o immunoistochimica. Questo può spesso includono alzando gli anticorpi e una notevole quantità di lavoro la convalida dei nuovi anticorpi. La strategia generale illustrata in questo protocollo è ottimizzato per le grandi dimensioni neuropeptidi MALDI esperimenti IMS tra cui diverse sezioni e delle condizioni sperimentali. Il protocollo è stato ottimizzato in particolare per i peptidi oppioidi e avrà grande impatto in studi futuri, come impiegata in diversi campi di ricerca, compreso il dolore dei meccanismi alla base e la risposta ai farmaci endogena di dipendenza.

1,1 Raccolta dei campioni e la preparazione <ol><li> Isoflurano (Apoteksbolaget AB, Svezia) </li><li> Strumenti chirurgici, due paia di forbici, un Rongeur, spatola </li><li> Finemente triturato ghiaccio secco </li><li> Foglio di alluminio e sacchetti di plastica per la conservazione </li><li> Criostato (Microm, Heidelberg, Germania) </li><li> Ossido di indio-stagno (ITO) rivestite con diapositive (MALDI Daltonics Bruker, Germania) </li><li> ottimale di temperatura media di taglio (OCT) (Sakura Finetek Europa, Paesi Bassi) </li><li> essiccatore di vuoto </li></ol> 1,2 Matrix applicazione <ol><li> Acetonitrile (ACN), metanolo (MeOH), etanolo (EtOH) di pro-analisi grade (Merck, Germania). </li><li> Trifluoroacetico acido (TFA) (Sigma Aldrich, St. Louis, MO). </li><li> L&#39;acqua è stata purificata con un Milli-Q sistema di purificazione (Millipore, Bedford, MA). </li><li> CHIP-1000 (Shimadzu, Giappone) o Portrait 630 spotter (LabCyte, Sunnyvale, CA) </li></ol> 1,3 Spettrometria di massa <ol><li> UlTraflex II (Bruker Daltonics) </li><li> Software FlexImaging (2,0 v, Bruker Daltonics) </li><li> Peptide o proteina Calibration Standard (Bruker Daltonics) </li></ol> 1,4 L&#39;elaborazione dei dati <ol><li> Origine V.8.1 (OriginLab Corp, Norhampton, MA) </li><li> Matlab versione 7.9.0.529 (MathWorks, Natick, MA) </li><li> pBin ( <a href="http://www.vicc.org/biostatistics/software.php" target="_blank">http://www.vicc.org/biostatistics/software.php</a> ) </li></ol> 1.5 Identificazione neuropeptide <ol><li> Ultrasuoni ultrasuoni </li><li> 10 kDa peso molecolare cut-off filtro (Millipore, Bedford, MA) </li><li> SP Sephadex C 25 gel (Sigma, Germania) </li><li> Hypersil colonna (ThermoFisher) </li><li> Piridina, acido formico (FA), acetonitrile (ACN), metanolo (MeOH), etanolo (EtOH) di pro-analisi grade (Merck, Germania). </li><li> Trifluoroacetico acido (TFA) Sigma Aldrich (St. Louis, MO, USA). </li><li> Agilent 1100 nano LC, microWPS, colonna C18 (0.075x150mm) </li><li> Agilent 1100 Micro-fractioncollector, Prespotted anchorchip piastra segnale (Bruker Daltonics) </li><li> Biotools software (v 3,1 SR2, Bruker Daltonics) </li><li> Bio Opere software (v.3.3, Thermo Fisher; <a href="http://www.thermoscientific.com" target="_blank">www.thermoscientific.com</a> ) </li><li> Mascot software (v 2.2, MatrixScience; <a href="http://www.matrixscience.com" target="_blank">www.matrixscience.com</a> ) </li><li> Swepep database ( <a href="http://www.swepep.org" target="_blank">www.swepep.org</a> ) </li></ol>

maldi imaging mass spectrometry of neuropeptides in parkinson's disease

MALDI Imaging spettrometria di massa (IMS) è un approccio potente che facilita l&#39;analisi spaziale delle specie molecolari in campioni biologici di tessuto 2 (Fig.1). Una sezione di tessuto 12 um sottile è coperto con una matrice MALDI, che facilita desorbimento e ionizzazione di peptidi e proteine ​​intatte che possono essere rilevati con un analizzatore di massa, in genere utilizzando un MALDI TOF / spettrometro di massa TOF. Generalmente centinaia di picchi può essere valutata in una singola sezione di tessuto cerebrale di ratto. In contrasto con tecniche di imaging comunemente utilizzate, questo approccio non richiede la conoscenza preventiva delle molecole di interesse e consente un&#39;analisi incontrollato e completa di più specie molecolari mantenendo elevata specificità e sensibilità molecolare 2. Qui si descrive un approccio basato MALDI IMS per la comprensione della regione specifici profili di distribuzione di neuropeptidi nel cervello di ratto di un animale modello di malattia di Parkinson (PD). PD è una comune malattia neurodegenerativa con una prevalenza di 1% per le persone sopra i 65 anni di età 3,4. Il trattamento più comune sintomatica si basa sulla sostituzione della dopamina con L-DOPA 5. Tuttavia questa è accompagnata da gravi effetti collaterali tra cui i movimenti involontari anomali, denominati L-DOPA indotta discinesie (LID) 1,3,6. Uno dei più importanti cambiamento molecolare in LID è una sovraregolazione del mRNA oppioide prodynorphin precursore 7. I peptidi dinorfina modulano la neurotrasmissione in aree cerebrali che sono essenzialmente coinvolte nel controllo del movimento 7,8. Tuttavia, ad oggi i peptidi oppioidi esatti che provengono dalla trasformazione del precursore neuropeptide non sono state caratterizzate. Pertanto, abbiamo utilizzato MALDI IMS in un modello sperimentale animale di malattia di Parkinson e di L-DOPA discinesia indotta. Spettrometria di massa MALDI immagini dimostrata particolarmente vantaggiosa rispetto a neuropeptide caratterizzazionezione, dal momento che gli approcci comunemente usati basati su anticorpi si rivolge sequenze peptidiche noti e precedentemente osservate modificazioni post-traduzionali. Al contrario MALDI IMS possono svelare nuovi prodotti per la lavorazione del peptide e quindi rivelare nuovi meccanismi molecolari di modulazione neuropeptide di trasmissione neuronale. Mentre la quantità assoluta di neuropeptidi non può essere determinata mediante MALDI IMS, l&#39;abbondanza relativa degli ioni peptidici possono essere delineate dagli spettri di massa, dando intuizioni circa la modifica dei livelli di salute e malattia. Negli esempi qui presentati, l&#39;intensità dei picchi di dinorfina B, alpha-neoendorphin e la sostanza P sono risultati significativamente aumentati nel dorsolaterale, ma non il dorsomediale, striato di animali con discinesia grave che interessano più del viso, muscoli del tronco e orolingual (Fig. 5). Inoltre, MALDI IMS ha rivelato una correlazione tra la gravità della discinesia e dei livelli di des-tirosina alfa-neoendorphin, che rappresenta un meccanismo finora sconosciuto di inattivazione funzionalezione di dinorfine nel corpo striato, come la rimozione di N-terminale tirosina riduce la dinorfina del recettore oppioide capacità legante 9. Questo è il primo studio sulla caratterizzazione neuropeptide in LID usando MALDI IMS ed i risultati evidenziano il potenziale della tecnica per l&#39;applicazione in tutti i campi della ricerca biomedica.

Watch this Scientific Journal Video about MALDI Imaging Mass Spectrometry of Neuropeptides in Parkinson's Disease at JoVE.com

MALDI Imaging Mass Spectrometry of Neuropeptides in Parkinson&#039;s Disease

Farmacoterapia sostituzione della dopamina con L-DOPA è il trattamento più comunemente utilizzato sintomatico della malattia di Parkinson, ma è accompagnata da effetti collaterali tra cui i movimenti involontari anomali, discinesia definito<sup&gt; 1</sup&gt;. Qui, un protocollo per l&#39;imaging spettrometria di massa MALDI è presentato in grado di rilevare cambiamenti nei livelli cerebrali di ratto neuropeptidi relativi alla discinesia.

MALDI Imaging Mass Spectrometry dei neuropeptidi nella malattia di Parkinson

MALDI imaging mass spectrometry (IMS) is a powerful approach that facilitates the spatial analysis of molecular species in biological tissue samples2 ...

maldi-imaging-mass-spectrometry-neuropeptides-parkinson-s

Research

JoVE Journal

Medicine

MALDI Imaging Mass Spectrometry of Neuropeptides in Parkinson's Disease

25.8K Views.  Uppsala University. Farmacoterapia sostituzione della dopamina con L-DOPA è il trattamento più comunemente utilizzato sintomatico della malattia di Parkinson, ma è accompagnata da effetti collaterali tra cui i movimenti involontari anomali, discinesia definito 1. Qui, un protocollo per l'imaging spettrometria di massa MALDI è presentato in grado di rilevare cambiamenti nei livelli cerebrali di ratto neuropeptidi relativi alla discinesia.

Video: MALDI Imaging Mass Spectrometry of Neuropeptides in Parkinson's Disease

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 understanding of the mechanisms underlying parkinsonian symptoms, since it recapitulates the changes in basal ganglia circuitry and pharmacology observed in parkinsonian patients1-4. However, the precise cellular and molecular changes occurring at cortico-striatal synapses of the output pathways within the striatum, which is the major input region of the basal ganglia remain elusive, and this is believed to be site where pathological abnormalities underlying parkinsonian symptoms arise3,5.
In PD, understanding the mechanisms underlying changes in basal ganglia circuitry following degeneration of the nigro-striatal pathway has been greatly advanced by the development of bacterial artificial chromosome (BAC) mice over-expressing green fluorescent proteins driven by promoters specific for the two striatal output pathways (direct pathway: eGFP-D1; indirect pathway: eGFP-D2 and eGFP-A2a)8, allowing them to be studied in isolation. For example, recent studies have suggested that there are pathological changes in synaptic plasticity in parkinsonian mice9,10. However, these studies utilised juvenile mice and acute models of parkinsonism. It is unclear whether the changes described in adult rats with stable 6-OHDA lesions also occur in these models. Other groups have attempted to generate a stable unilaterally-lesioned 6-OHDA adult mouse model of PD by lesioning the medial forebrain bundle (MFB), unfortunately, the mortality rate in this study was extremely high, with only 14% surviving the surgery for 21 days or longer11. More recent studies have generated intra-nigral lesions with both a low mortality rate &gt;80% loss of dopaminergic neurons, however expression of L-DOPA induced dyskinesia11,12,13,14 was variable in these studies. Another well established mouse model of PD is the MPTP-lesioned mouse15. Whilst this model has proven useful in the assessment of potential neuroprotective agents16, it is less suitable for understanding mechanisms underlying symptoms of PD, as this model often fails to induce motor deficits, and shows a wide variability in the extent of lesion17, 18.
Here we have developed a stable unilateral 6-OHDA-lesioned mouse model of PD by direct administration of 6-OHDA into the MFB, which consistently causes &gt;95% loss of striatal dopamine (as measured by HPLC), as well as producing the behavioural imbalances observed in the well characterised unilateral 6-OHDA-lesioned rat model of PD. This newly developed mouse model of PD will prove a valuable tool in understanding the mechanisms underlying generation of parkinsonian symptoms.

Development of a Unilaterally-lesioned 6-OHDA Mouse Model of Parkinson&#039;s Disease

A protocol for performing unilateral 6-OHDA lesions of the medial forebrain bundle in mice is described. This method has a low mortality rate (13.3 %) with 89% of the surviving animals showing &#62;95% loss of striatal dopamine and 90.63&#177;-4.02 % ipsiversive rotational bias towards the side of the lesion.

Sviluppo di un unilateralmente lesionata 6-OHDA modello murino della malattia di Parkinson

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

Ricerca

Medicina

Skin Punch Biopsy Explant Culture for Derivation of Primary Human Fibroblasts

Tissues and cell lines derived from an individual with disease are ideal sources to study disease-related cellular phenotypes. Patient-derived fibroblasts in this protocol have been successfully used in the derivation of induced pluripotent stem cells to model disease1. Early passages of these fibroblasts can also be used for cell-based functional assays to study specific disease pathways, mechanisms2 and subsequent drug screening approaches. The advantage of the presented protocol over enzymatic procedures are 1) the reproducibility of the technique from small amounts of tissue derived from older patients, e.g. patients affected with Parkinson's disease, 2) the technically simple approach over more challenging methodologies using enzymatic treatments, and 3) the time consideration: this protocol takes 15-20 min and can be performed immediately after biopsy arrival. Enzymatic treatments can take up to 4 hr and have the problems of overdigestion, reduction of cell viability and subsequent attachment of cells when not handled properly. This protocol describes the dissection and preparation of a 4-mm human skin biopsy for derivation of a fibroblast culture and has a very high success rate which is important when dealing with patient-derived tissue samples. In this culture, keratinocytes migrate out of the biopsy tissue within the first week after preparation. Fibroblasts appear 7-10 days after the first outgrowth of keratinocytes. DMEM high glucose media supplemented with 20% FBS favors the growth of fibroblasts over keratinocytes and fibroblasts will overgrow the keratinocytes. After 2 passages keratinocytes have been diluted out resulting in relatively homogenous fibroblast cultures which expresses the fibroblast marker SERPINH1 (HSP-47). Using this approach, 15-20 million fibroblasts can be derived in 4-8 weeks for cell banking. The skin dissection takes about 15-20 min, cells are then monitored once a day under the microscope, and media is changed every 2-3 days after attachment and outgrowth of cells.

The fibroblast explant culture protocol from human skin punch biopsies is a technically robust and simple way to derive skin cells within 4-8 weeks for banking of about 15-20 million cells at a low passage number.

Pelle Punch Biopsia Espianto Cultura per la derivazione di fibroblasti umani primari

Tissues and cell lines derived from an individual with disease are ideal sources to study disease-related cellular phenotypes. Patient-derived fibroblasts ...

The Use of Primary Human Fibroblasts for Monitoring Mitochondrial Phenotypes in the Field of Parkinson's Disease

Parkinson's disease (PD) is the second most common movement disorder and affects 1% of people over the age of 60 1. Because ageing is the most important risk factor, cases of PD will increase during the next decades 2. Next to pathological protein folding and impaired protein degradation pathways, alterations of mitochondrial function and morphology were pointed out as further hallmark of neurodegeneration in PD 3-11.
After years of research in murine and human cancer cells as in vitro models to dissect molecular pathways of Parkinsonism, the use of human fibroblasts from patients and appropriate controls as ex vivo models has become a valuable research tool, if potential caveats are considered. Other than immortalized, rather artificial cell models, primary fibroblasts from patients carrying disease-associated mutations apparently reflect important pathological features of the human disease.
Here we delineate the procedure of taking skin biopsies, culturing human fibroblasts and using detailed protocols for essential microscopic techniques to define mitochondrial phenotypes. These were used to investigate different features associated with PD that are relevant to mitochondrial function and dynamics. Ex vivo, mitochondria can be analyzed in terms of their function, morphology, colocalization with lysosomes (the organelles degrading dysfunctional mitochondria) and degradation via the lysosomal pathway. These phenotypes are highly relevant for the identification of early signs of PD and may precede clinical motor symptoms in human disease-gene carriers. Hence, the assays presented here can be utilized as valuable tools to identify pathological features of neurodegeneration and help to define new therapeutic strategies in PD.

The Use of Primary Human Fibroblasts for Monitoring Mitochondrial Phenotypes in the Field of Parkinson&#039;s Disease

Fibroblasts from patients carrying mutations in Parkinson's disease-causing genes represent an easily accessible ex vivo model to study disease-associated phenotypes. Live cell imaging gives the opportunity to study morphological and functional parameters in living cells. Here we describe the preparation of human fibroblasts and subsequent monitoring of mitochondrial phenotypes.

L&#39;uso di fibroblasti umani primari per il monitoraggio fenotipi mitocondriali nel campo della malattia di Parkinson

Parkinson's disease (PD) is the second most  common movement disorder and affects 1% of people over the age of 60 1. Because ageing is  the most important ...

Gene-environment Interaction Models to Unmask Susceptibility Mechanisms in Parkinson's Disease

Lipoxygenase (LOX) activity has been implicated in neurodegenerative disorders such as Alzheimer&#39;s disease, but its effects in Parkinson&#39;s disease (PD) pathogenesis are less understood. Gene-environment interaction models have utility in unmasking the impact of specific cellular pathways in toxicity that may not be observed using a solely genetic or toxicant disease model alone.&#160;To evaluate if distinct LOX isozymes selectively contribute to PD-related neurodegeneration, transgenic (i.e. 5-LOX and 12/15-LOX deficient) mice can be challenged with a toxin that mimics cell injury and death in the disorder.&#160;Here we describe the use of a neurotoxin, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), which produces a nigrostriatal lesion to elucidate the distinct contributions of LOX isozymes to neurodegeneration related to PD. The use of MPTP in mouse, and nonhuman primate, is well-established to recapitulate the nigrostriatal damage in PD.&#160;The extent of MPTP-induced lesioning is measured by HPLC analysis of dopamine and its metabolites and semi-quantitative Western blot analysis of striatum for tyrosine hydroxylase (TH), the rate-limiting enzyme for the synthesis of dopamine. To assess inflammatory markers, which may demonstrate LOX isozyme-selective sensitivity, glial fibrillary acidic protein (GFAP) and Iba-1 immunohistochemistry are performed on brain sections containing substantia nigra, and GFAP Western blot analysis is performed on striatal homogenates.&#160;This experimental approach can provide novel insights into gene-environment interactions underlying nigrostriatal degeneration and PD.

Gene-environment Interaction Models to Unmask Susceptibility Mechanisms in Parkinson&#039;s Disease

Lipoxygenase (LOX) isozymes can generate products that may increase or decrease neuroinflammation and neurodegeneration.&#160;A gene-environment interaction study could identify LOX isozyme-specific effects. Using the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) model of nigrostriatal damage in two LOX isozyme-deficient transgenic lines allows for comparison of the contribution of LOX isozymes on dopaminergic integrity and inflammation.

Modelli di interazione gene-ambiente per smascherare i meccanismi di suscettibilità nel morbo di Parkinson

Lipoxygenase (LOX) activity has been implicated in neurodegenerative disorders such as Alzheimer&#39;s disease, but its effects in Parkinson&#39;s disease ...

Controlling Parkinson's Disease With Adaptive Deep Brain Stimulation

Adaptive deep brain stimulation (aDBS) has the potential to improve the treatment of Parkinson&#39;s disease by optimizing stimulation in real time according to fluctuating disease and medication state. In the present realization of adaptive DBS we record and stimulate from the DBS electrodes implanted in the subthalamic nucleus of patients with Parkinson&#39;s disease in the early post-operative period. Local field potentials are analogue filtered between 3 and 47 Hz before being passed to a data acquisition unit where they are digitally filtered again around the patient specific beta peak, rectified and smoothed to give an online reading of the beta amplitude. A threshold for beta amplitude is set heuristically, which, if crossed, passes a trigger signal to the stimulator. The stimulator then ramps up stimulation to a pre-determined clinically effective voltage over 250 msec and continues to stimulate until the beta amplitude again falls down below threshold. Stimulation continues in this manner with brief episodes of ramped DBS during periods of heightened beta power.
Clinical efficacy is assessed after a minimum period of stabilization (5 min) through the unblinded and blinded video assessment of motor function using a selection of scores from the Unified Parkinson&#39;s Rating Scale (UPDRS). Recent work has demonstrated a reduction in power consumption with aDBS as well as an improvement in clinical scores compared to conventional DBS. Chronic aDBS could now be trialed in Parkinsonism.

Controlling Parkinson&#039;s Disease With Adaptive Deep Brain Stimulation

Adaptive deep brain stimulation (aDBS) is effective for Parkinson&#8217;s disease, improving symptoms and reducing power consumption compared to conventional deep brain stimulation (cDBS). In aDBS we track a local field potential biomarker (beta oscillatory amplitude) in real time and use this to control the timing of stimulation.

Controllo della Malattia di Parkinson con Adaptive stimolazione cerebrale profonda

Adaptive deep brain stimulation (aDBS) has the potential to improve the treatment of Parkinson&#39;s disease by optimizing stimulation in real time ...

Quantitative Magnetic Resonance Imaging of Skeletal Muscle Disease

Quantitative magnetic resonance imaging (qMRI) describes the development and use of MRI to quantify physical, chemical, and/or biological properties of living systems. Neuromuscular diseases often exhibit a temporally varying, spatially heterogeneous, and multi-faceted pathology. The goal of this protocol is to characterize this pathology using qMRI methods. The MRI acquisition protocol begins with localizer images (used to locate the position of the body and tissue of interest within the MRI system), quality control measurements of relevant magnetic field distributions, and structural imaging for general anatomical characterization. The qMRI portion of the protocol includes measurements of the longitudinal and transverse relaxation time constants (T1 and T2, respectively). Also acquired are diffusion-tensor MRI data, in which water diffusivity is measured and used to infer pathological processes such as edema. Quantitative magnetization transfer imaging is used to characterize the relative tissue content of macromolecular and free water protons. Lastly, fat-water MRI methods are used to characterize fibro-adipose tissue replacement of muscle. In addition to describing the data acquisition and analysis procedures, this paper also discusses the potential problems associated with these methods, the analysis and interpretation of the data, MRI safety, and strategies for artifact reduction and protocol optimization.

Neuromuscular diseases often exhibit a temporally varying, spatially heterogeneous, and multi-faceted pathology. The goal of this protocol is to characterize this pathology using non-invasive magnetic resonance imaging methods.

Quantitative Magnetic Resonance Imaging di malattia muscolare scheletrico

Quantitative magnetic resonance imaging (qMRI) describes the development and use of MRI to quantify physical, chemical, and/or biological properties of ...

Quantitative Mass Spectrometric Profiling of Cancer-cell Proteomes Derived From Liquid and Solid Tumors

In-depth analyses of cancer cell proteomes are needed to elucidate oncogenic pathomechanisms, as well as to identify potential drug targets and diagnostic biomarkers. However, methods for quantitative proteomic characterization of patient-derived tumors and in particular their cellular subpopulations are largely lacking. Here we describe an experimental set-up that allows quantitative analysis of proteomes of cancer cell subpopulations derived from either liquid or solid tumors. This is achieved by combining cellular enrichment strategies with quantitative Super-SILAC-based mass spectrometry followed by bioinformatic data analysis. To enrich specific cellular subsets, liquid tumors are first immunophenotyped by flow cytometry followed by FACS-sorting; for solid tumors, laser-capture microdissection is used to purify specific cellular subpopulations. In a second step, proteins are extracted from the purified cells and subsequently combined with a tumor-specific, SILAC-labeled spike-in standard that enables protein quantification. The resulting protein mixture is subjected to either gel electrophoresis or Filter Aided Sample Preparation (FASP) followed by tryptic digestion. Finally, tryptic peptides are analyzed using a hybrid quadrupole-orbitrap mass spectrometer, and the data obtained are processed with bioinformatic software suites including MaxQuant. By means of the workflow presented here, up to 8,000 proteins can be identified and quantified in patient-derived samples, and the resulting protein expression profiles can be compared among patients to identify diagnostic proteomic signatures or potential drug targets.

In-depth analyses of cancer cell proteomes facilitate identification of novel drug targets and diagnostic biomarkers. We describe an experimental workflow for quantitative analysis of (phospho-)proteomes in cancer cell subpopulations derived from liquid and solid tumors. This is achieved by combining cellular enrichment strategies with quantitative Super-SILAC-based mass spectrometry.

Mass quantitativa spettrometrica Profiling di proteomi cancro-cellulari derivate da liquido e tumori solidi

In-depth analyses of cancer cell proteomes are needed to elucidate oncogenic pathomechanisms, as well as to identify potential drug targets and diagnostic ...

Development of an Alpha-synuclein Based Rat Model for Parkinson's Disease via Stereotactic Injection of a Recombinant Adeno-associated Viral Vector

In order to study the molecular pathways of Parkinson&#39;s disease (PD) and to develop novel therapeutic strategies, scientific investigators rely on animal models. The identification of PD-associated genes has led to the development of genetic PD models. Most transgenic &#945;-SYN mouse models develop gradual &#945;-SYN pathology but fail to display clear dopaminergic cell loss and dopamine-dependent behavioral deficits. This hurdle was overcome by direct targeting of the substantia nigra with viral vectors overexpressing PD-associated genes. Local gene delivery using viral vectors provides an attractive way to express transgenes in the central nervous system. Specific brain regions can be targeted (e.g. the substantia nigra), expression can be induced in the adult setting and high expression levels can be achieved. Further, different vector systems based on various viruses can be used. The protocol outlines all crucial steps to perform a viral vector injection in the substantia nigra of the rat to develop a viral vector-based alpha-synuclein animal model for Parkinson&#39;s disease.

Development of an Alpha-synuclein Based Rat Model for Parkinson&#039;s Disease via Stereotactic Injection of a Recombinant Adeno-associated Viral Vector

This manuscript describes how viral vector-mediated local gene delivery provides an attractive way to express transgenes in the central nervous system. The protocol outlines all crucial steps to perform a viral vector injection in the substantia nigra of the rat to develop a viral vector-based animal model for Parkinson's disease.

Sviluppo di un Rat modello Alfa-sinucleina per la malattia di Parkinson mediante iniezione stereotassica di un ricombinante vettore virale adeno-associato

In order to study the molecular pathways of Parkinson&#39;s disease (PD) and to develop novel therapeutic strategies, scientific investigators rely on ...

Imaging Metals in Brain Tissue by Laser Ablation - Inductively Coupled Plasma - Mass Spectrometry (LA-ICP-MS)

Metals are found ubiquitously throughout an organism, with their biological role dictated by both their chemical reactivity and abundance within a specific anatomical region. Within the brain, metals have a highly compartmentalized distribution, depending on the primary function they play within the central nervous system. Imaging the spatial distribution of metals has provided unique insight into the biochemical architecture of the brain, allowing direct correlation between neuroanatomical regions and their known function with regard to metal-dependent processes. In addition, several age-related neurological disorders feature disrupted metal homeostasis, which is often confined to small regions of the brain that are otherwise difficult to analyze. Here, we describe a comprehensive method for quantitatively imaging metals in the mouse brain, using laser ablation - inductively coupled plasma - mass spectrometry (LA-ICP-MS) and specially designed image processing software. Focusing on iron, copper and zinc, which are three of the most abundant and disease-relevant metals within the brain, we describe the essential steps in sample preparation, analysis, quantitative measurements and image processing to produce maps of metal distribution within the low micrometer resolution range. This technique, applicable to any cut tissue section, is capable of demonstrating the highly variable distribution of metals within an organ or system, and can be used to identify changes in metal homeostasis and absolute levels within fine anatomical structures.

Imaging Metals in Brain Tissue by Laser Ablation - Inductively Coupled Plasma - Mass Spectrometry LA-ICP-MS

Quantitatively mapping metals in tissue by laser ablation - inductively coupled plasma - mass spectrometry (LA-ICP-MS) is a sensitive analytical technique that can provide new insight into how metals participate in normal function and disease processes. Here, we describe a protocol for quantitatively imaging metals in thin sections of mouse neurological tissue.

Metalli di imaging in tessuto cerebrale di Laser Ablation - plasma accoppiato induttivamente - spettrometria di massa (LA-ICP-MS)

Metals are found ubiquitously throughout an organism, with their biological role dictated by both their chemical reactivity and abundance within a ...

Whole-brain Segmentation and Change-point Analysis of Anatomical Brain MRI&#8212;Application in Premanifest Huntington's Disease

Recent advances in MRI offer a variety of useful markers to identify neurodegenerative diseases. In Huntington&#39;s disease (HD), regional brain atrophy begins many years prior to the motor onset (during the &#34;premanifest&#34; period), but the spatiotemporal pattern of regional atrophy across the brain has not been fully characterized. Here we demonstrate an online cloud-computing platform, &#34;MRICloud&#34;, which provides atlas-based whole-brain segmentation of T1-weighted images at multiple granularity levels, and thereby, enables us to access the regional features of brain anatomy. We then describe a regression model that detects statistically significant inflection points, at which regional brain atrophy starts to be noticeable, i.e. the &#34;change-point&#34;, with respect to a disease progression index. We used the CAG-age product (CAP) score to index the disease progression in HD patients. Change-point analysis of the volumetric measurements from the segmentation pipeline, therefore, provides important information of the order and pattern of structural atrophy across the brain. The paper illustrates the use of these techniques on T1-weighted MRI data of premanifest HD subjects from a large multicenter PREDICT-HD study. This design potentially has wide applications in a range of neurodegenerative diseases to investigate the dynamic changes of brain anatomy.

Whole-brain Segmentation and Change-point Analysis of Anatomical Brain MRI&amp;#8212;Application in Premanifest Huntington&#039;s Disease

This paper describes a statistical model for volumetric MRI data analysis, which identifies the &#34;change-point&#34; when brain atrophy begins in premanifest Huntington&#39;s disease. Whole-brain mapping of the change-points is achieved based on brain volumes obtained using an atlas-based segmentation pipeline of T1-weighted images.

Segmentazione del intero-cervello e analisi del punto di svolta di anatomico del cervello MRI — applicazione in premanifesti sulla malattia di Huntington

Recent advances in MRI offer a variety of useful markers to identify neurodegenerative diseases. In Huntington&#39;s disease (HD), regional brain atrophy ...