Tesis sobre el tema "Dopaminergic neurons"

Lo sviluppo dei neuroni dopaminergici mesencefalici (mDA) è un fenomeno complesso e non ancora pienamente compreso. Molti studi hanno focalizzato la loro attenzione sul ruolo svolto da diversi fattori di trascrizione specifici e ben noti. L'obiettivo della mia tesi di dottorato è focalizzato su una classe relativamente nuova di regolatori post-trascrizionali denominati microRNA (miRNAs), in grado di regolare l'espressione genica legando le sequenze parzialmente complementari nelle regioni 3' non tradotte (UTR) degli mRNAs target. Per studiare il ruolo svolto dai miRNAs durante la differenziazione dei neuroni mDA, abbiamo scelto di analizzare il profilo di espressione dei miRNA usando piattaforme di Array. A tale scopo, abbiamo utilizzato un protocollo ottimizzato da cellule staminali di epiblasto di topo (epiSC) differenziate in neuroni mDA (Jeager et al.,2011). Dall'analisi bioinformatica dei dati dell'array, ottenuti dalle epiSC differenziate in neuroni mDA, abbiamo identificato alcuni candidati molto probabilmente implicati nella differenziazione e nella funzione dei neuroni DA. I miRNA candidati sono stati sottoposti a screening per la loro capacità di indurre il fenotipo DA. A questo scopo, ho generato vettori lentivirali inducibili per ciascun miRNAs e ho infettato colture primarie mesencefaliche di topo allo stadio E12.5. Tra tutti i miRNA candidati, miR-218 e miR-34b/c aumentano il numero di cellule TH + positive, suggerendo il loro possibile contributo nei neuroni mDA. Inoltre, miR-218 e miR-34b/c, risultano arricchiti sia nel mesencefalo dei topi (E13.5) che nelle cellule GFP + sortate al FACS, isolate da embrioni E13.5 Pitx3-GFP di topo, rispetto al controllo. I dati ottenuti dal saggio di Luciferasi e dal saggio di reporter a doppia fluorescenza suggeriscono che miR-34b/c legano e sopprimono la 3'UTR di Wnt1 e viene espresso durante la differenziazione dei neuroni mDA. Tramite analisi di ibridazione in situ e dati d’ immunoistochimica ho potuto verificare che miR-218 è espresso in particolare nel mesencefalo di topo allo stadio E14, dove co-localizza rostralmente con Isl-1 (marcatore di motoneuroni) e caudalmente con TH, Pitx3, Lmx1a (marcatori dopaminergico). Questi dati suggeriscono che miR-218 è espresso anche nei motoneuroni craniali, come descritto in altri recenti studi (Thiebes, K.P. et al., 2014; Amin, N.D et al., 2015). Per comprendere ulteriormente il ruolo di miR-218 nello sviluppo e nella funzione dei neuroni dopaminergici ho generato topi knock-out condizionali (cKO) per miR-218-2. Accoppiando miR-218-2 flox / flox con topi En1Cre /+ che esprimono Cre sotto il controllo del promotore di Engrailed 1 (En1, marker pro-dopaminergico), sarò in grado di comprendere il contributo di miR-218 nel sistema dopaminergico. I topi miR-218-2 flox / flox En1Cre /+ da osservazioni preliminari, hanno mostrato un fenotipo con danno motorio, ma per confermare questi dati sto attualmente effettuando test comportamentali e analisi in vivo. Attraverso il profilo di espressione di miRNAs, siamo in grado di comprendere il meccanismo e la funzione del sistema dopaminergico, poiché i miRNAs sono regolatori chiave nelle reti di espressione genica, possono influenzare molti processi biologici e in futuro potrebbero essere utilizzati come biomarkers per diagnosticare patologie legate al sistema nervoso.
Midbrain dopaminergic neurons (mDA) development is a complex and still not fully understood phenomenon. Many studies till now concentrated their attention on the roles played by several, specific and well-known transcription factors. The aim of my PhD thesis is focus on a relatively new class of post-transcriptional regulators named microRNAs (miRNAs) able to regulate gene expression by targeting partially complementary sequences in the 3’untranslated regions (UTRs) of the target mRNAs. To investigate the role played by miRNAs during mDA differentiation, we choose to analyze miRNAs expression profile by using miRNA Array platforms. To this purpose we used an optimized protocol from mouse Epiblast stem cells (epiSC) differentiated into DA neurons (Jeager et al. 2011). By bioinformatics analysis of the array data, obtained from epiSC differentiated into mDA neurons, we identified few candidates most likely implicated in the DA neurons differentiation and function. The candidate miRNAs were screened for their ability to induce DA phenotype. To this purpose, I generated inducible lentiviral vectors for each miRNA and I have infected mesencephalic primary cultures from mice at stage E12.5. Among all candidate miRNAs, miR-218 and miR-34b/c increase the number of TH+ positive cells, showing their possible contribution in the mDA neurons. Moreover, miR-218 and miR-34b/c, were enriched both in midbrain of mice (E13.5) and in FACS sorted GFP+ cells isolated from E13.5 Pitx3-GFP mice embryos when compared with control. Data obtained from Luciferase Assay and Dual Fluorescence Reporter Assay suggest that miR-34b/c target and suppress Wnt1 3’UTR and it is expressed during DA neurons differentiation. By performing In situ hybridization analysis and immunohistochemistry, I was able to detect miR-218 in particular in the mouse midbrain at stage E14, where co-localize rostrally with Isl-1 (motor neuron marker) and caudally with TH, Pitx3, Lmx1a (dopaminergic marker). This data suggests that miR-218 is expressed also in cranial motor neurons, as described in others recent studies (Thiebes, K.P. et al. 2014; Amin, N.D et al. 2015). To further understand the role of miR-218 in development and function of dopaminergic neurons I have generated the conditional knock-out (cKO) mice for miR-218-2. By mating miR-218-2 flox/flox with En1Cre/+ mice expressing the Cre under Engrailed 1 promoter (En1 is a pro-dopaminergic marker) I will be able to investigate the contribution of miR-218 in dopaminergic system. Preliminary observations on miR-218-2 flox/flox En1Cre/+ mice shown motor impairment phenotype, but to confirm this data I’m currently performing behavior tests and in vivo analysis. Through miRNA expression profiling we be able understand mechanism and function of dopaminergic system, because miRNAs are as key regulators in gene expression networks, can influence many biological processes and have also shown promise as biomarkers for neuro-disorders.

TRPM7 (Transient Receptor Potential Melastatin-like 7) is an ion channel necessary for the proper development of many cell types. Insight into the precise role of the channel in different cells has been hampered by the lethality of knocking out the gene in model organisms such as the mouse. Here I examine a zebrafish that has a loss-of-function mutation in the gene encoding Trpm7. First, I show that trpm7 is important for the function of developing dopaminergic neurons in the zebrafish. Second, I examine the interaction between trpm7 and the related gene vmat2 in order to develop a cellular mechanism of trpm7 function in presynaptic dopaminergic neurons. Finally, I investigate the necessity of the kinase and ion channel domains of trpm7 in their ability to promote pigmentation in melanophores as a model cell type. Based on the results from these experiments and observations from other researchers, I form a new hypothesis for Trpm7 function in protein sorting. These studies provide a detailed and novel analysis of the function of an ion channel that is necessary for life.

Thesis (M.S.)--West Virginia University, 2001.
Title from document title page. Document formatted into pages; contains vii, 79 p. : ill. (some col.). Vita. Includes abstract. Includes bibliographical references (p. 70-78).

L’activité électrique des neurones est déterminée par des interactions complexes entre leurs propriétés morphologiques et biophysiques. Les neurones dopaminergiques (DA) de la substance noire compacte (SNc) présentent une caractéristique morphologique peu commune parmi les neurones de mammifères: leur axone émerge fréquemment d’une dendrite à une distance très variable du soma. Malgré cette importante variabilité dans la localisation de l’axone, peu d’articles ont étudié un lien potentiel entre morphologie neuronale et activité électrique dans ces cellules. Dans un premier article, nous avons exploré l’importante variabilité observée dans les neurones DA en caractérisant de nombreux paramètres morphologiques et biophysiques. Nos résultats suggèrent que la géométrie de l’AIS n’affecte pas significativement la forme du potentiel d’action ni l’activité pacemaker. En revanche, l’activité électrique est influencée par la morphologie et les conductances somatodendritiques. Dans une seconde étude, nous avons caractérisé le développement morphologique des neurones DA au cours des trois premières semaines post-natales. Nous avons observé une croissance asymétrique de l’arbre dendritique: la dendrite portant l’axone semble se complexifier plus que les autres dendrites. Cette asymétrie est associée à une contribution différente de la dendrite portant l’axone et des dendrites ne portant pas l’axone à la forme du potentiel d’action. Ces résultats suggèrent que les neurones DA de la SNc sont robustes aux variations morphologiques de l’axone et que les particularités morphologiques et biophysiques de leur arbre dendritique minimisent l’influence de l’AIS sur leur activité électrique
Neuronal output is defined by the complex interplay between the biophysical and morphological properties of neurons. Dopaminergic (DA) neurons of the substantia nigra pars compacta (SNc) are spontaneously active and generate a regular pacemaking activity. While most mammalian neurons have an axon emerging from the soma, the axon of DA neurons often arises from a dendrite at highly variable distances from the soma. Despite this large cell-to-cell variation in axon location, few studies have tried to unravel the potential link between neuronal morphology and electrical behaviour in this cell type. In a first article, we explored the high degree of cell-to-cell variability found in DA neurons by characterising several morphological and biophysical parameters. While AIS geometry did not seem to significantly affect action potential shape or pacemaking activity, we found that the electrical behaviour of DA neurons was particularly sensitive to somatodendritic morphology and conductances. In a second study, we characterised the morphological development of DA neurons during the first three post-natal weeks. We observed an asymmetric development of the dendritic tree, favouring the elongation and complexity of the axon-bearing dendrite. This asymmetry is associated with different contributions of the axon-bearing and non-axon bearing dendrites to action potential shape. Overall, the two studies suggest that DA neurons of the SNc are highly robust to cell-to-cell variations in axonal morphology. The peculiar morphological and biophysical profile of the dendritic arborization attenuates the role of the AIS in shaping electrical behaviour in this neuronal type

Parkinson's disease (PD) is characterised by the loss of dopaminergic neurons in the Substantia Nigra pars compacta in the midbrain and the presence of intracellular aggregates, known as Lewy bodies (LBs), in the surviving neurons. The aetiology of PD is unknown but a causative role for α-Synuclein (SNCA) has been proposed. Although the function of αSyn is not well understood, a number of pathological mechanisms associated with αSyn toxicity have been proposed. In this study, nine induced pluripotent stem cells (iPSCs) lines from healthy individuals and PD patients carrying the A53T SNCA mutation or a triplication of SNCA were differentiated to dopaminergic neurons (iDAn). All iPSC lines differentiated with similar efficiency to iDAn, indicating that they could be used for phenotypic analysis. Quantification of αSyn expression showed increased αSyn intracellular staining and the novel detection of increased αSyn oligomerization in PD iDAn. Analysis of mitochondrial respiration found a decrease in basal respiration, maximal respiration, ATP production and spare capacity in PD iDAn, but not in undifferentiated iPSCs, indicating the cell-type specificity of these defects. Decreased phosphorylation of dynamin-1-like protein at Ser616 (DRP1^Ser616) and increased levels of Peroxisome proliferator-activated receptor gamma coactivator 1-α (PGC-1α) in A53T SNCA iDAn suggest a new pathological mechanism linking αSyn to the imbalance in mitochondria homeostasis. Markers of endoplasmic reticulum (ER) stress were found to be up-regulated, along with increased β- Glucocerebrosidase (GBA) activity, perturbation of autophagy and decreased expression of fatty acids binding protein 7 (FAPB7) in PD iDAn. Lastly, lentiviral vectors for RNAi-mediated knockdown of αSyn were developed and these reduced αSyn protein levels in iDAn, resulting in increased expression of FABP7. These results describe a novel functional link between αSyn and FABP7. This work demonstrates that iDAn are a promising and relevant in vitro cell model for studying cellular dysfunctions in PD pathology, and the phenotypic analysis of A53T SNCA and SNCA triplication iDAn enabled the detection of novel pathological mechanisms associated with PD.

Kyoto University (京都大学)
0048
新制・課程博士
博士(工学)
甲第12290号
工博第2619号
新制||工||1369(附属図書館)
24126
UT51-2006-J283
京都大学大学院工学研究科高分子化学専攻
(主査)教授 岩田 博夫, 教授 田畑 泰彦, 教授 木村 俊作
学位規則第4条第1項該当

Parkinson's disease (PD) is a neurological disorder characterized by the lateralization of motor manifestations, that is still evident late in the course of disease, corresponding to the unilateral predominance of the nigrostriatal degenerative process. The diagnosis is essentially clinical, supported by the evidence of a good clinical dopaminergic response. However the diagnosis is sometimes difficult also in a movement disorders' specialist setting, especially with vascular parkinsonism (VP) due to the overlap in clinical presentation and the lack of specificity at neuroimaging. We studied 20 PD, 20 VP and 20 Essential Tremor (ET) patients as control group, who had undergone a cerebral [123I] FP-CIT SPECT that is the best tecnique, to date, for in vivo assessment of dopaminergic depletion. On SPECT results a semiquantitative analisis has been performed; the binding of the ligand in the most affected side resulted significantly lower in VP than in ET patients but higher compared to PD patients. We calculate also the Striatal Asymmetry Index (SAI), that resulted significantly higher in PD compared to VP (p<0.001) and ET (p<0.001) groups. We found that a cut-off of SAI greater than 14.08 could differentiate PD from VP with a 100% specificity and a 60% sensitivity. Our work has provided an important glimpse about the possible role of functional imaging in differentiating with a good degree of certainty VP from PD, suggesting that SAI detected using [123I]FP-CIT SPECT could be used to this purpose. Moving from this consideration, we wondered if asymmetry can be predictive also of the response to levodopa (LD) at short term LD-test. In fact, up to 40% of drug nave patients are initially not responsive to dopaminergic drugs, but LD responsiveness is one of the best predictors of a correct diagnosis of PD. We performed a study enrolling 20 PD patients never previously exposed to levodopa, who had undergone a short term LD-test with levodopa/carbidopa 250/25 mg to quantify the clinical response to dopaminergic therapy, and a [123I] FP-CIT SPECT. We estimated the amplitude (mean percentual reduction of UPDRS motor score) and the duration (time necessary for the UPDRS motor score to return to the 50% of the maximal improvement) of the response at acute LD-test. At SPECT study, as expected, mean caudate and putamen uptake were lower controlaterally to the most affected side. A significant correlation between the Striatal Asymmetry Index and the magnitude of the response was found (r 0.64, p <0.002). Asymmetry appeared to predict the responsiveness to levodopa, that can be crucial in the management of therapy, helping phisicians in the choice of best therapy.

Parkinson’s disease is the second most common neurodegenerative disease after Alzheimer’s disease. One of the most evident pathological hallmarks in PD is the selective loss of mesencephalic dopaminergic (mDA) neurons with the consequent decrease of dopamine in the brain. mDA neurons present two main groups of projecting cells: the A9 neurons of the Substantia Nigra (SN) and the A10 cells of the Ventral Tegmental Area (VTA). A9 neurons controls the nigrostriatal pathway and are involved in regulation of voluntary movements and postural reflexes. Their selective degeneration leads to Parkinson’s disease (PD) and the loss of DA synapses in the striatum is believed to be primary cause for the disruption of the ability to control movements. Until now, the causes of the degeneration of A9 neurons in PD are still unknown and a lot of efforts have been done to determine the molecular differences between the dopaminergic cell subpopulations that could explain the selective susceptibility of A9 neurons. Thanks to new technologies developed in the last decade, like nanoCAGE, it was possible to investigate in depth the gene expression profiling of dopaminergic neurons. NanoCAGE and Affimetrix Exon Array technologies applied on A9 and A10 cells collected with Laser Capture Microdissection, revealed the presence of a subset of olfactory receptors in mDA cells. Expression of mDA-specific ORs was validated by PCR on RNA extracted from mouse midbrain and from LCM-purified A9 and A10 mDA neurons. Furthermore, In situ hybridization confirmed the selected expression of ORs in subpopulations of DA cells. Mesencephalic ORs were classified in a genetic tree for their sequence/structure similarities and their potential ligands were identified by homology modelling. Full length ORs were cloned from the midbrain and ectopically expressed on the cell surface of HEK cells to test their responses to odorants. We observed that a subset of chemical odors stimulated mDA-ORs expressed in heterologous cells. Moreover, performing Ca++ imaging experiments on dopaminergic cells isolated from mouse ventral midbrain, I demonstrated that endogenous ORs are expressed in these neurons and importantly, they are functional and respond to odor stimulation. Altogether these results indicate that odorant receptors might contribute to the normal physiology of dopaminergic cells and open new interesting questions about the role of these receptors in the pathology of mDA neurons in Parkinson’s disease. According to this, odour molecules could be used as agonists to trigger ORs activation in DA neurons that might be new targets of therapeutic intervention.

The zebrafish has an amazing capacity for regeneration which includes regeneration of neurons within the central nervous system (CNS) both during development and into adulthood. This attribute makes the zebrafish a valuable tool in the study of regeneration. In this thesis, the research focussed on the regeneration of a specific type of cell in the CNS, dopaminergic (DA) neurons. The DA system of the zebrafish is believed to be evolutionarily conserved with comparable DA populations found in the brain of mammals. Dissimilar to mammals, however, the zebrafish is capable of regenerating various types of neurons and their axons. Thus, the zebrafish DA system provides an excellent model to study replacement of this specific and important cell type in the adult CNS. We have developed a novel toxin ablation paradigm to specifically ablate select groups of DA neurons in the adult zebrafish diencephalon, leaving other DA populations unaffected. To do this a selective DA toxin, 6-hydroxydopamine, was used. One of the ablated DA diencephalic populations is the only source of dopaminergic spinal innervation in the zebrafish. Their ablation leads to a loss of DA spinal axons following our toxin ablation. The ascending projection of the diencephalic population ablated by the toxin has been suggested as the most likely candidate for a zebrafish equivalent of the mammalian nigro-striatal pathway. The loss of cells is very specific and reproducible, indicating that these cells are particularly vulnerable to the toxin. Quantification of affected populations at various time-points post ablation was carried out to determine the capacity for regeneration of DA neurons in the CNS of zebrafish. This revealed that in some populations neuron numbers returned to those seen in controls. However, in other populations neuron numbers only partially recovered even at late time points. We have shown that this recovery is due to neurogenesis; furthermore, by inducing inflammation after the toxin treatment the recovery of DA cell numbers was accelerated by 50%. Regenerated cells originated from Olig2 positive ependymo-radial glial cells found bordering the diencephalic ventricle. We aimed to investigate the function of this group of ablated neurons through a battery of behavioural tests. These tests revealed deficits in the toxin treated animals’ fine movement, such as is necessary for maintaining shoal cohesion and breeding behaviours, whereas general movement behaviours were not found to be impaired. Zebrafish embryos also present as a great resource in the screening of drugs. Their fast and well characterised early development makes them an ideal tool for investigating previously untested neuroprotectants. A reproducible ablation paradigm similar to that established in the adults was also established in the zebrafish embryo. This was then used as a tool to investigate potential novel neuroprotectants. This screen revealed two new flavonoid compounds which had the ability to induce full protection of the affected dopaminergic cells in the zebrafish embryonic brain. The embryonic ablation model therefore represents a vertebrate in vivo model system for future high throughput screening of neuroprotective compounds against toxin induced DA cell loss. Ultimately, understanding how zebrafish functionally regenerate dopaminergic neurons using this ablation model will likely provide a useful tool into the research of neurodegenerative diseases, such as PD.

Vascular endothelial growth factor B (VEGF-B) has shown potential for therapeutic implementation as a neuroprotective factor in the treatment of neurodegenerative disorders, including Parkinson's disease (PD). The goals of this study were two-fold: (i) to determine the neurotrophic effect of both known VEGF-B isoforms on primary neurite length of dopaminergic (DA) neurons in culture, and (ii) to compare this effect against the neurotrophic effects of other growth factors known to increase neurite outgrowth in culture. Glial-derived neurotrophic factor (GDNF), vascular endothelial growth factor A (VEGF-A165), pigment epithelium derived factor (PEDF), VEGF-B186, and VEGF-B167 were added individually to primary midbrain cultures at various concentrations, with untreated midbrain cultures serving as controls. As expected, GDNF, PEDF, and VEGF-A165 all had a significant effect on primary neurite outgrowth at one or more of their tested concentrations (1ng/mL; 5ng/mL; 1ng/mL and 5ng/mL, respectively) relative to control measurements. VEGF-B186 was shown to have a significant effect on primary neurite outgrowth at all three tested concentrations (5ng/mL; 10 ng/mL; 20 ng/mL), while VEGF-B167 showed a strong neurotrophic trend at both concentrations (5ng/mL; 10ng/mL) that failed to reach significance. Curiously, the effects of GDNF, PEDF, and VEGF-A165 on neurite outgrowth were not as pronounced as those previously observed. However, comparison between both VEGF-B isoforms and the other factors suggests a comparable neurotrophic capacity. We conclude that the growth factor VEGF-B186 increases primary neurite outgrowth of dopaminergic neurons in culture.

Recent advances in studying the mammalian transcriptome arised new questions about how genes are organized and what is the function of noncoding RNAs. Furthermore, the discovery of large amounts of polyA- transcripts and antisense transcription proved that a portion of the transcriptome has still to be characterized. The complex anatomo-functional organization of the brain has prevented a comprehensive analysis of the transcriptional landscape of this tissue. New techniques must be developed to approach neuronal heterogeneity. In this study we combined Laser Capture Microdissection (LCM) and nanoCAGE, based on Cap Analysis of Gene Expression (CAGE), to describe expressed genes and map their transcription start sites (TSS) in two specific populations, A9 and A10, of mouse mesencephalic dopaminergic cells. Although sharing common dopaminergic marker genes, these two populations are part of different midbrain anatomical structures, substantia nigra (SN) for A9 and ventral tegmental area (VTA) for A10, project to relatively distinct areas, participate to distinct ascending dopaminergic pathways, exhibit different electrophysiological properties and different susceptibility to neurodegeneration in Parkinson`s disease. Specific neurons were identified by the expression of Green Fluorescent Protein driven by a celltype specific promoter in transgenic mice. High-quality RNAs were purified from 1000-2500 cells collected by LCM. We adapted the CAGE technique to analyze limiting amounts of RNAs (nanoCAGE). We took advantage of the cap-switching properties of the reverse transcriptase to specifically tag the 5`end of transcripts with a sequence containing a class III restriction site for EcoP15I. By creating 32bp 5`tags, we considerably improved the TSS mapping rate on the genome. A semi-suppressive PCR strategy was used to prevent primer dimers formation. The use of random priming in the 1st strand synthesis allowed to capture poly(A)- RNAs. 5`tags were sequenced with Illumina-Solexa platform. Here we show that this new nanoCAGE technology ensures a true high-throughput coverage of the transcriptome of a small number of identified neurons and can be used as an effective mean for gene discovery in the noncoding RNAs, to uncover putative alternative promoters associated to variants of protein coding transcripts and to detect potentially regulatory antisense transcripts. A further experimental validation by 5`RACE (Rapid Amplification of cDNA Ends) and RT-PCR on few candidate genes, have confirmed the existence in vivo of alternative TSS in the case of key regulatory genes involved in specifying and maintaining the dopaminergic phenotype of these neurons such as α-synuclein (Snca), dopamine transporter (Dat), vescicular monoamine transporter 2 (Vmat2), catechol-O-methyltransferase (Comt). Furthermore the differential expression of an antisense transcript overlapping to the polyubiquitin (Ubc) gene was detected as potentially interesting candidate gene accounting for differences in the ubiquitin-proteasome system (UPS) function in the two neuron populations. The potential implications deriving from these newly discovered alternative promoters and transcripts are discussed, considering also the potential consequences for the corresponding protein isoforms.

Medications that treat diseases such as Parkinson???s disease work by regulating dopamine transmission at synapses. Surprisingly, little is known about the mechanisms regulating dopamine release at synapses. In this thesis, we study mechanisms that regulate vesicle recycling in axons and dendrites of dopamine neurons. Key questions we addressed were: (1) Are vesicles in axons and dendrites associated with the same regulatory proteins, and thus by implication the same regulatory mechanisms, as in excitatory neurons; (2) Do vesicles undergo recycling, and (3) if so, are they characterised by a distinct pool size and rate of recycling. To study this, we cultured dopamine neurons and used immunocytochemistry to detect vesicular monoamine transporter 2 (VMAT2) and identify axons, dendrites and synaptic proteins, combined with labelling of recycling vesicles using FM 1-43. Vesicles in axons, but not in dendrites, were associated with presynaptic proteins such as Synaptophysin and Bassoon. We identified two kinds of presynaptic sites in axons: ???synaptic??? (located close to soma and dendrites??? and ???orphan???. The recycling vesicle pool size was smaller at orphan sites than at synaptic sites, and the initial rate of vesicle pool release was also lower at orphan sites. Both synaptic and orphan sites exhibited lower rates of vesicle pool release compared to hippocampal synapses, suggesting functional differences in presynaptic physiology between dopamine neurons and hippocampal neurons. In somatodendritic regions, VMAT2 was localised to the endoplasmic reticulum, Golgi, endosome, and large dense-core vesicles, suggesting that these vesicles might function as a part of the regulated secretory pathway in mediating dopamine release. None of the synaptic vesicle proteins we studied were detected in these regions, although some preliminary evidence of vesicle turnover was detected using FM 1-43 labelling. This thesis provides a detailed analysis of neurotransmitter release mechanisms in dopamine neurons. Our data suggests that presynaptic release of dopamine is mediated by mechanisms similar to those observed in excitatory neurons. In somatodendritic regions, our data suggests that VMAT2 is localised to organelles in secretory pathways, and that distinct mechanisms of release might be present at somatodendritic sites to those present in presynaptic sites. This thesis provides novel methods for analysing vesicle recycling in dopamine neurons, which provides the basis for further studies examining presynaptic function of dopamine neurons in normal brain function, disease, and therapeutic approaches.

Thesis (M.S.)--West Virginia University, 2006.
Title from document title page. Document formatted into pages; contains vi, 86 p. : ill. (some col.). Vita. Includes abstract. Includes bibliographical references (p. 75-85).

1. Introduzione I neuroni dopaminergici (DA) della substantia nigra pars compacta (SNc) sono tra i più studiati nel sistema nervoso centrale per la loro implicazione nella malattia del Parkinson. Essi presentano un ampio corredo di correnti voltaggio-dipendenti, tra le quali emerge una tipica corrente attivata da iperpolarizzazione, la Ih. Diversamente dalla maggior parte delle cellule nervose, i neuroni dopaminergici della SNc presentano una attività spontanea regolare dopo isolamento o riduzione degli input sinaptici, e non è, quindi, sorprendente che numerosi lavori abbiano indagato il ruolo della Ih nell'attività spontanea. Tuttavia il ruolo della Ih non è stato ancora ben compreso, dal momento che il blocco di questa corrente non sembra comportare nessuna alterazione significativa della frequenza di scarica. Abbiamo, allora, riesaminato il problema studiando la corrente h, in fette sottili di cervello, in condizioni sperimentali che si differenziano dalla maggior parte degli studi precedenti per tre aspetti fondamentali: i) abbiamo utilizzato topi transgenici che esprimono una proteina reporter (GFP) sotto il promotore tirosina idrossilasi (TH), per identificare i neuroni DA della SNc; ii) abbiamo effettuato le registrazioni elettrofisiologiche a 37°C; iii) abbiamo eseguito la maggior parte degli esperimenti in condizioni di patch perforato al fine di lasciare inalterato l’ambiente fisiologico intracellulare. 2. Risultati Il nostro primo obiettivo è stato quello di effettuare un'analisi dettagliata della dipendenza della cinetica e dell’ampiezza della Ih dalla temperatura. Il protocollo di attivazione della corrente h prevedeva una serie di comandi iperpolarizzanti della durata di 4s e le registrazioni erano effettuate a 27°C e 37°C. Abbiamo calcolato che il coefficiente di temperatura (Q10) per la variazione di ampiezza della corrente h è pari 3,73, mentre i valori di Q10 relativi alle velocità di attivazione e deattivazione sono rispettivamente pari a 10,8 e 3,17. Il V50 è di -94,9 ± 1,07 mV a 27°C (n = 13) e -84,2 ± 1,31 mV a 37°C (n = 18). Abbiamo, successivamente, esaminato la modulazione da parte dei nucleotidi ciclici in condizioni di patch perforato a 37°C, in presenza di forskolina (10 μM), un attivatore della adenilato ciclasi, e IBMX (0.1 mM), un inibitore delle fosfodiesterasi, i quali, insieme, inducono un aumento della concentrazione intracellulare di adenosina monofosfato ciclico (cAMP). In queste condizioni abbiamo registrato un aumento dell'ampiezza Ih (da -178,53 ± 23,48 pA in condizioni di controllo a -227,01 ± 34,17 pA con forskolina a -130 mV, n = 8), uno spostamento del V50 di + 4,80 ± 0,68 mV (n = 8) e una riduzione delle costanti di tempo di attivazione di circa il 25%. Dato che questa modulazione è il risultato di un’interazione diretta del cAMP con il canale, abbiamo studiato gli effetti sulla Ih di diversi neurotrasmettitori accoppiati a proteine Gi o Gs, in particolare abbiamo testato la dopamina, la serotonina (5-HT) e la noradrenalina (NA). Il quinpirolo, un agonista dei recettori dopaminergici D2 (30 μM, dopo 3 minuti di applicazione nel bagno), ha indotto una diminuzione dell'ampiezza della Ih del 15% a -130 mV (n = 6), mentre il sulpiride, un antagonista dei recettori dopaminergici D2 (20 μM), ne ha determinato un aumento (n = 5). L'effetto sulla Ih della 5HT (100 μM), dopo 3 minuti di applicazione nel bagno, è stato una riduzione dell’ampiezza del 20% (n = 5); al contrario, l'applicazione nel bagno della NA (100 μM), ne ha indotto un aumento di circa il 12% (n = 8). Infine, abbiamo analizzato il ruolo della corrente h sull’autoritmicità. L'ivabradina (10 μM), bloccante del Ih, ha determinato una marcata iperpolarizzazione (circa -10 mV), che di fatto ha silenziato le cellule; tuttavia, questo effetto sull’attività spontanea era indiretto, poiché se la membrana era ripolarizzata, l’autoritmicità si ripristinava. 3. Conclusioni Gli studi eseguiti a temperatura ambiente sul ruolo e le proprietà della corrente h nei neuroni dopaminergici della SNc sono scarsamente informativi perché, in queste condizioni sperimentali, la corrente è sottovalutato in ampiezza, velocità e, in ultima analisi, nella sua capacità di svolgere alcun ruolo a potenziali fisiologici. Le registrazioni elettrofisiologiche a 37°C, invece, restituiscono un profilo più accurato e veritiero della Ih. La modulazione della corrente h ad opera di sistemi a secondo messaggero, un processo scarsamente esplorato nei neuroni dopaminergici della SNc, sembra essere rilevante, e suggerisce l'esistenza di diversi pathways importanti per il controllo dell’eccitabilità neuronale. La corrente h è, in ultima analisi, molto importante nell’autorimicità perché stabilizza il potenziale di membrana di riposo dei neuroni dopaminergici della SNc in uno stato depolarizzato, ma non ricopre un ruolo di principale nell’attività pacemaker. Questi risultati sono interessanti perché aprono nuove prospettive sul ruolo del canale HCN nei neuroni dopaminergici della substantia nigra pars compacta.

The effects of repeated, once daily exposure to either tail pinch or restraint stress on extracellular DA levels in nucleus accumbens (NAcc), prefrontal cortex (PFC), and striatum (STR) was monitored in conscious rats using high-speed chronoamperometry, an electrochemical detection technique. The first exposure to either stress reliably and consistently elevated DA levels in the extracellular space of NAcc, PFC, and STR; the increases observed in PFC were of a greater magnitude than those observed in NAcc and STR. These data are consistent with those of previous studies suggesting a higher responsiveness of the meso-PFC system to stress. However, with repeated exposure increases in DA levels elicited by restraint became progressively larger in NAcc, and to a lesser extent also in STR, but not in PFC. Apomorphine, injected at autoreceptor selective doses, attenuated tail pinch and restraint stimulated increases in DA levels in NAcc but not in PFC, a finding consistent with the drug's action on impulse-modulating receptors of meso-NAcc DA neurons and with the known absense of such receptors on meso-PFC DA neurons. That DA was the primary contributor to the electrochemical signals was confirmed by the potentiating effect of GBR-12909, a selective DA uptake inhibitor, on restraint-elicited electrochemical responses in PFC and NAcc.
Taken together, the results of the present study indicate that with repeated exposure the meso-NAcc DA response to subsequent exposure to stress is enhanced. The data indicate that this pathway, which is thought to mediate the positive reinforcing effects of rewards, is also activated during behaviors motivated by aversive stimuli.

Thesis (Ph. D.)--Illinois State University, 2001.
Title from title page screen, viewed April 13, 2006. Dissertation Committee: Paul A. Garris (chair), John E. Baur, Hou T. Cheung, Maarten E.A. Reith, David L. Williams. Includes bibliographical references (leaves 170-186) and abstract. Also available in print.

Dopamine exerts an important role in the regulation of motor activity in humans. During the progression of Parkinson’s disease, patients are faced with the progressive neurodegeneration of nigro-striatal dopamine neurons resulting in an array of pathological symptoms characteristic of the disease. Current treatment relies on targeting symptomatic aspects of the disease but currently Parkinson’s disease is incurable. Targeting the regeneration of DA neurons in PD patients could offer an alternative therapeutic approach that could stall and perhaps even revert the progression of the disease and improve the quality of life for patients. Here, I describe the generation of a transgenic zebrafish line for the non-invasive, conditional and specific ablation of dopaminergic neurons in both larval and adult zebrafish. Understanding the endogenous regenerative ability of the zebrafish may in the future contribute to the development of novel therapeutic approaches targeting DA neuron regeneration in humans. The Tg(dat:CFP-NTR) line efficiently labels and ablates most clusters of DA neurons in both the larval and the adult zebrafish brain. Neuronal ablation is followed by a locomotor and tail bend phenotype as well as by an increase in exploratory behavior. Using double transgenic larvae, we showed through live imaging that loss of DA neurons induces an increase in nestin expression; in addition we show an increase in the number of proliferating cells and an up regulation of genes involved in neurogenesis and tissue repair. Adult zebrafish were able to fully recover their DA neuronal population in the olfactory bulb within 45 days post ablation. Overall the Tg(dat:CFP-NTR) zebrafish offers a novel tool for the study of the molecular and cellular mechanisms driving the regeneration of DA neurons in the zebrafish brain and will be a useful tool for the field of regenerative medicine.

The striatum is the major input structure of the basal ganglia, and is composed of two major populations of spiny projection neurons (MSNs), which give rise to the socalled direct and indirect pathways, and several types of interneuron. Dopaminergic inputs to striatum are critical for its proper function. Indeed, loss of dopaminergic neurons in Parkinsonism leads to motor disturbances, grossly disturbs striatal activity, and is associated with the emergence of excessively-synchronized network oscillations at beta frequencies (15-30 Hz) throughout the basal ganglia. How the distinct structural, neurochemical and other properties of striatal neurons are reflected in their firing rates and patterns in vivo is poorly defined, as are their possible cell-type-selective contributions to the aberrant oscillations arising in the Parkinsonian brain. To address these issues, I first used multi-electrode arrays to record the spontaneous firing of ensembles of neurons in dorsal striatum in both anaesthetised dopamine-intact and Parkinsonian (6-hydroxydopamine-lesioned) rats during two well-defined brain states, slow-wave activity (SWA) and spontaneous activation. The chronic loss of dopamine led to an overall increase in the average firing rates of striatal neurons, irrespective of brain state. However, many neurons in the Parkinsonian striatum still exhibited the low firing rates and irregular firing patterns typical of neurons in the dopamine-intact striatum. During SWA in Parkinsonian rats, the firing of striatal neurons was more strongly synchronized at low frequencies, in time with cortical slow (~1 Hz) oscillations. During spontaneous cortical activation in Parkinsonian rats, more striatal neurons engaged in synchronized firing in time with cortical beta oscillations. Under the same experimental conditions, I then recorded the spontaneous firing of individual striatal neurons and juxtacellularly labelled the same neurons to verify their cell types, and locations; indirect pathway and direct pathway MSNs were distinguished by the expression (and lack of expression respectively), of the neuropeptide precursor preproenkephalin (PPE). After chronic dopamine loss, and on average, only indirect pathway (PPE+) MSNs significantly increased their firing rates during both brain states, and engaged in widespread, synchronized firing in the beta-frequency range. This did not hold true for all PPE+ MSNs; the Parkinsonian striatum contained many MSNs that were virtually quiescent, which were just as likely to belong to the indirect pathway as the direct pathway. Direct pathway (PPE-) MSNs increased their firing only during SWA after chronic dopamine loss and rarely engaged in aberrant beta oscillations. Taken together, these data suggest that (1) the firing patterns, as well as the firing rates of many striatal neurons are grossly disturbed by chronic loss of dopamine and (2) that the pathological synchronization of the rhythmic firing of a subpopulation of indirect pathway MSNs could contribute to the propagation of aberrant beta-frequency oscillations to downstream basal ganglia nuclei in Parkinsonism.

Lmx1a et Lmx1b sont des facteurs de transcription connus pour leur rôle au cours du développement des neurones dopaminergiques du mésencéphale (mDA). Ils ont été montrés comme essentiels à chacune des étapes de différentiation des progéniteurs en neurones dopaminergiques matures. Des études récentes ont également mis en évidence l'importance de ces deux facteurs de transcription dans les neurones dopaminergiques chez l'adulte. Lmx1a/b sont impliqués dans la régulation de gènes mitochondriaux ainsi que dans l'autophagie. Cependant, jusqu'à présent, rien n'est connu sur le rôle de Lmx1a/b dans les neurones dopaminergiques post-mitotiques. Le but de cette thèse est d'élucider le rôle de Lmx1a/b dans les neurones dopaminergiques matures. L'analyse des projections axonales dopaminergiques de souris doubles conditionnelles mutantes (cKO) pour Lmx1a/b a mis en évidence un défaut de guidage axonal confirmant le rôle essentiel de ces deux facteurs de transcription dans la formation des circuits dopaminergiques. Afin d’identifier précisément les molécules impliquées dans la régulation du système dopaminergique des techniques adaptées doivent être développées pour déterminer les principaux acteurs régulés par Lmx1a/b. À cette fin, nous avons mis au point une technique de marquage immunohistochimique rapide de la tyrosine hydroxylase (TH, enzyme nécessaire à la synthèse de la dopamine) sur des sections de mésencéphale de souris afin de délimiter la région d’intérêt. Par la suite, nous utilisons une technique de microdissection au laser afin de spécifiquement récolter les cellules dopaminergiques du mésencéphale pour réaliser un profil d'expression génique. Un premier article de méthodologie a été publié concernant cette technique. Cette procédure menée sur des souris cKO pour Lmx1a et Lmx1b et leurs contrôles associés a permis de mettre en évidence des gènes régulés par Lmx1a et Lmx1b tels que Plxnc1. Plxnc1 est une protéine de guidage axonal ayant pour ligand la sémaphorine 7a (Sema7a). Afin d'observer si la régulation de Plxnc1 par Lmx1a/b est à l'origine du défaut de guidage axonal observé chez les souris cKO pour Lmx1a/b, nous avons réalisé une analyse in vitro de l’effet de la Sema7a sur les axones d'explants mDA. Notre étude a montré un effet chimiorépulsif de la Sema7a pour les axones des neurones mDA exprimant Plxnc1. De plus, l’étude de souris Sema7a KO montre une augmentation de l’innervation DA dans la partie dorsale du striatum, partie exprimant Sema7a chez des souris contrôles. Ce phénotype met en évidence une chimiorépulsion induite par l’interaction Sema7a/Plxnc1. L’étude de souris surexprimant Plxnc1 a, quant à elle, montré une perte d’innervation DA dans la partie dorsale du striatum. En effet, la majorité des cellules du mésencéphale se mettent à exprimer Plxnc1, les rendant ainsi sensibles à la chimiorépulsion induite par Sema7a. L’ensemble de ces résultats met en évidence l’importance de la régulation de la protéine de guidage axonal Plxnc1 par Lmx1a/b pour l'innervation des cibles du mésencéphale. La répression de Plxnc1 dans les neurones dopaminergiques de la substance noire pars compacta (SNpc) semble nécessaire à l’innervation du striatum dorsal riche en Sema7a. Cette étude est la première à identifier les bases moléculaires du guidage axonal expliquant la ségrégation des voies mDA nigrostriée et mésolimbique, et devrait contribuer à améliorer l'efficacité des thérapies cellulaires pour la maladie de Parkinson. Un second article sera soumis prochainement sur le rôle des facteurs de transcription Lmx1a/b dans les neurones dopaminergiques post-mitotiques du mésencéphale. La principale caractéristique histopathologique de la maladie de Parkinson est la dégénérescence des neurones mDA de la SNpc. La thérapie de remplacement cellulaire utilisant des neurones dopaminergiques nouvellement générés à partir de cellules souches représente une thérapie prometteuse. Cependant, la mauvaise innervation des neurones nouvellement greffés limite le succès des études de transplantation. L’identification de facteurs régulant la connectivité des neurones mDA devient primordiale pour élucider les mécanismes impliqués dans la mise en place du système dopaminergique. C'est pourquoi, dans une derniere partie, afin d'illustrer cette possibilité d'amélioration d'une thérapie de remplacement cellulaire, j’ai réalisé l’implantation de cellules souches différenciées en neurones dopaminergiques dans un modèle de souris lésées à la 6-hydroxydopamine (6OHDA). Les cellules nouvellement réimplantées sont de type SNpc, en raison de l'infection par un vecteur viral induisant l'inhibition de l'expression de Plxnc1.
Lmx1a et Lmx1b sont des facteurs de transcription connus pour leur rôle au cours du développement des neurones dopaminergiques du mésencéphale (mDA). Ils ont été montrés comme essentiels à chacune des étapes de différentiation des progéniteurs en neurones dopaminergiques matures. Des études récentes ont également mis en évidence l'importance de ces deux facteurs de transcription dans les neurones dopaminergiques chez l'adulte. Lmx1a/b sont impliqués dans la régulation de gènes mitochondriaux ainsi que dans l'autophagie. Cependant, jusqu'à présent, rien n'est connu sur le rôle de Lmx1a/b dans les neurones dopaminergiques post-mitotiques. Le but de cette thèse est d'élucider le rôle de Lmx1a/b dans les neurones dopaminergiques matures. L'analyse des projections axonales dopaminergiques de souris doubles conditionnelles mutantes (cKO) pour Lmx1a/b a mis en évidence un défaut de guidage axonal confirmant le rôle essentiel de ces deux facteurs de transcription dans la formation des circuits dopaminergiques. Afin d’identifier précisément les molécules impliquées dans la régulation du système dopaminergique des techniques adaptées doivent être développées pour déterminer les principaux acteurs régulés par Lmx1a/b. À cette fin, nous avons mis au point une technique de marquage immunohistochimique rapide de la tyrosine hydroxylase (TH, enzyme nécessaire à la synthèse de la dopamine) sur des sections de mésencéphale de souris afin de délimiter la région d’intérêt. Par la suite, nous utilisons une technique de microdissection au laser afin de spécifiquement récolter les cellules dopaminergiques du mésencéphale pour réaliser un profil d'expression génique. Un premier article de méthodologie a été publié concernant cette technique. Cette procédure menée sur des souris cKO pour Lmx1a et Lmx1b et leurs contrôles associés a permis de mettre en évidence des gènes régulés par Lmx1a et Lmx1b tels que Plxnc1. Plxnc1 est une protéine de guidage axonal ayant pour ligand la sémaphorine 7a (Sema7a). Afin d'observer si la régulation de Plxnc1 par Lmx1a/b est à l'origine du défaut de guidage axonal observé chez les souris cKO pour Lmx1a/b, nous avons réalisé une analyse in vitro de l’effet de la Sema7a sur les axones d'explants mDA. Notre étude a montré un effet chimiorépulsif de la Sema7a pour les axones des neurones mDA exprimant Plxnc1. De plus, l’étude de souris Sema7a KO montre une augmentation de l’innervation DA dans la partie dorsale du striatum, partie exprimant Sema7a chez des souris contrôles. Ce phénotype met en évidence une chimiorépulsion induite par l’interaction Sema7a/Plxnc1. L’étude de souris surexprimant Plxnc1 a, quant à elle, montré une perte d’innervation DA dans la partie dorsale du striatum. En effet, la majorité des cellules du mésencéphale se mettent à exprimer Plxnc1, les rendant ainsi sensibles à la chimiorépulsion induite par Sema7a. L’ensemble de ces résultats met en évidence l’importance de la régulation de la protéine de guidage axonal Plxnc1 par Lmx1a/b pour l'innervation des cibles du mésencéphale. La répression de Plxnc1 dans les neurones dopaminergiques de la substance noire pars compacta (SNpc) semble nécessaire à l’innervation du striatum dorsal riche en Sema7a. Cette étude est la première à identifier les bases moléculaires du guidage axonal expliquant la ségrégation des voies mDA nigrostriée et mésolimbique, et devrait contribuer à améliorer l'efficacité des thérapies cellulaires pour la maladie de Parkinson. Un second article sera soumis prochainement sur le rôle des facteurs de transcription Lmx1a/b dans les neurones dopaminergiques post-mitotiques du mésencéphale. La principale caractéristique histopathologique de la maladie de Parkinson est la dégénérescence des neurones mDA de la SNpc. La thérapie de remplacement cellulaire utilisant des neurones dopaminergiques nouvellement générés à partir de cellules souches représente une thérapie prometteuse. Cependant, la mauvaise innervation des neurones nouvellement greffés limite le succès des études de transplantation. L’identification de facteurs régulant la connectivité des neurones mDA devient primordiale pour élucider les mécanismes impliqués dans la mise en place du système dopaminergique. C'est pourquoi, dans une derniere partie, afin d'illustrer cette possibilité d'amélioration d'une thérapie de remplacement cellulaire, j’ai réalisé l’implantation de cellules souches différenciées en neurones dopaminergiques dans un modèle de souris lésées à la 6-hydroxydopamine (6OHDA). Les cellules nouvellement réimplantées sont de type SNpc, en raison de l'infection par un vecteur viral induisant l'inhibition de l'expression de Plxnc1.
Lmx1a and Lmx1b are transcription factors known for their role in the development of midbrain dopamine neurons (mDA). They were shown as essential for each stage of differentiation from progenitors to mature dopaminergic neurons. Recent studies have also highlighted the importance of these two transcription factors in dopaminergic neurons in adult mice. Lmx1a/b are involved in the regulation of mitochondrial genes and in autophagy. Although some evidence suggest that they could be involved in the formation of mDa circuit formation, their role in post-mitotic mDA neurons remains unknown. The aim of this thesis is to elucidate the role of Lmx1a/b in post-mitotic dopaminergic neurons. Analysis of dopaminergic axonal projections of double conditional mutant (cKO) mice for Lmx1a/b showed an axon guidance defect confirming the essential role of these transcription factors in the formation of dopaminergic circuits. In order to precisely identify the molecules involved in the regulation of the dopamine system, suitable techniques must be developed to identify the main genes that are regulated by Lmx1a/b. To this end we developed a new technique allowing gene profiling of brain sub-population. By combining rapid immunolabeling of mDA neurons with laser capture microdissection we manage to extract RNA from two sub-regions of mDA neurons such as ventral tegmental area (VTA) and substantia nigra pars compacta (SNpc). The advantage of this technique is to compare quickly the regulation of genes expression by studying controls and mutant mice. A first methodological article has been published regarding this procedure. We then applied this technique on cKO mice for Lmx1a/b and their associated controls to identify genes regulated by Lmx1a and Lmx1b. Among these genes, we identified Plxnc1, an axon guidance receptor for the semaphorin 7a (Sema7a). In order to verify whether the regulation of Plxnc1 by Lmx1a/b is at the origin of the axon guidance defect observed in double conditional mutant for Lmx1a/b, we have made an in vitro analysis of the effect of Sema7a on mDA explants. Our study showed a chemorepulsive effect of Sema7a on Plxnc1 positives axons. In addition, the study of knockout mice for Sema7a shows an increase of DA innervation in the dorsal part of the striatum which is the region expressing Sema7a in control mice. This phenotype reveals a chemorepulsion induced by Sema7a/Plxnc1 interaction. The study of mice overexpressing Plxnc1 shows a loss of DA innervation in the dorsal striatum. Indeed, by overexpressing Plxnc1, the majority of midbrain cells begin to express this axon guidance protein instead of only mDA neurons from the VTA. Thus, all mDA neurons including neurons from the SNpc express Plxnc1 making them sensitive to Sema7a. This interaction Sema7a/Plxnc1 leads to a chemorepulsion of axons guided away from the dorsal striatum. Overall these results highlight the importance of the regulation of the axon guidance protein Plxnc1 by Lmx1a/b for the innervation of midbrain targets. The repression of Plxnc1 expression in dopaminergic neurons of the SNpc appears necessary for the innervation of dopaminergic axons in the dorsal striatum, rich in Sema7a. This study is the first to identify the molecular basis of the development of the dopaminergic system explaining the segregation of the nigrostriatal and mesolimbic pathways. These results should help to improve the effectiveness of cell therapies for Parkinson's disease. A second article will be submitted soon about the role of Lmx1a/b transciption factors in post-mitotic midbrain dopaminergic neurons. The main histopathological feature of Parkinson's disease (PD) is the degeneration of SNpc neurons. The cell replacement therapy using newly generated dopaminergic neurons from stem cells represents a promising therapy. However, a poor innervation of the newly grafted neurons limits the success of transplantation studies. The identification of factors regulating neuronal connectivity of mDA neurons becomes essential to elucidate the mechanisms involved in the establishment of the dopaminergic system. Therefore, in a final section of this thesis, I report preliminary study about cell replacement therapy in PD mouse model. I differentiated DA neurons from stem cells, knock-down Plxnc1 expression and performed grafting in 6-hydroxydopamine (6OHDA) mouse model to illustrate the possibility of improving a cell replacement therapy.
Lmx1a and Lmx1b are transcription factors known for their role in the development of midbrain dopamine neurons (mDA). They were shown as essential for each stage of differentiation from progenitors to mature dopaminergic neurons. Recent studies have also highlighted the importance of these two transcription factors in dopaminergic neurons in adult mice. Lmx1a/b are involved in the regulation of mitochondrial genes and in autophagy. Although some evidence suggest that they could be involved in the formation of mDa circuit formation, their role in post-mitotic mDA neurons remains unknown. The aim of this thesis is to elucidate the role of Lmx1a/b in post-mitotic dopaminergic neurons. Analysis of dopaminergic axonal projections of double conditional mutant (cKO) mice for Lmx1a/b showed an axon guidance defect confirming the essential role of these transcription factors in the formation of dopaminergic circuits. In order to precisely identify the molecules involved in the regulation of the dopamine system, suitable techniques must be developed to identify the main genes that are regulated by Lmx1a/b. To this end we developed a new technique allowing gene profiling of brain sub-population. By combining rapid immunolabeling of mDA neurons with laser capture microdissection we manage to extract RNA from two sub-regions of mDA neurons such as ventral tegmental area (VTA) and substantia nigra pars compacta (SNpc). The advantage of this technique is to compare quickly the regulation of genes expression by studying controls and mutant mice. A first methodological article has been published regarding this procedure. We then applied this technique on cKO mice for Lmx1a/b and their associated controls to identify genes regulated by Lmx1a and Lmx1b. Among these genes, we identified Plxnc1, an axon guidance receptor for the semaphorin 7a (Sema7a). In order to verify whether the regulation of Plxnc1 by Lmx1a/b is at the origin of the axon guidance defect observed in double conditional mutant for Lmx1a/b, we have made an in vitro analysis of the effect of Sema7a on mDA explants. Our study showed a chemorepulsive effect of Sema7a on Plxnc1 positives axons. In addition, the study of knockout mice for Sema7a shows an increase of DA innervation in the dorsal part of the striatum which is the region expressing Sema7a in control mice. This phenotype reveals a chemorepulsion induced by Sema7a/Plxnc1 interaction. The study of mice overexpressing Plxnc1 shows a loss of DA innervation in the dorsal striatum. Indeed, by overexpressing Plxnc1, the majority of midbrain cells begin to express this axon guidance protein instead of only mDA neurons from the VTA. Thus, all mDA neurons including neurons from the SNpc express Plxnc1 making them sensitive to Sema7a. This interaction Sema7a/Plxnc1 leads to a chemorepulsion of axons guided away from the dorsal striatum. Overall these results highlight the importance of the regulation of the axon guidance protein Plxnc1 by Lmx1a/b for the innervation of midbrain targets. The repression of Plxnc1 expression in dopaminergic neurons of the SNpc appears necessary for the innervation of dopaminergic axons in the dorsal striatum, rich in Sema7a. This study is the first to identify the molecular basis of the development of the dopaminergic system explaining the segregation of the nigrostriatal and mesolimbic pathways. These results should help to improve the effectiveness of cell therapies for Parkinson's disease. A second article will be submitted soon about the role of Lmx1a/b transciption factors in post-mitotic midbrain dopaminergic neurons. The main histopathological feature of Parkinson's disease (PD) is the degeneration of SNpc neurons. The cell replacement therapy using newly generated dopaminergic neurons from stem cells represents a promising therapy. However, a poor innervation of the newly grafted neurons limits the success of transplantation studies. The identification of factors regulating neuronal connectivity of mDA neurons becomes essential to elucidate the mechanisms involved in the establishment of the dopaminergic system. Therefore, in a final section of this thesis, I report preliminary study about cell replacement therapy in PD mouse model. I differentiated DA neurons from stem cells, knock-down Plxnc1 expression and performed grafting in 6-hydroxydopamine (6OHDA) mouse model to illustrate the possibility of improving a cell replacement therapy.

Dopaminergic (DA) neurons exhibit a short-latency, phasic response to unexpected biologically salient stimuli, including rewards. Despite extensive research on this DA signal, very little is known about the sources of sensory information reaching DA neurons. Previous research has identified the superior colliculus (SC) as the primary, if not exclusive route of short latency visual input to DA neurons. However, more recent research has suggested that the phasic DA response comprises two components; a short latency (50-110 ms), stimulus insensitive component, and a longer latency component (110-260 ms) that can reflect complex stimulus characteristics including reward value – more complex than might arise from intrinsic collicular processing. A solution to this apparent paradox may be suggested by recent studies that have demonstrated longer latency colour related responses in SC neurons. As the SC does not receive direct retinal input from colour sensitive cells, but it does receive input from a wide range of cortical structures, it is possible that cortical activation might underlie longer latency responses in the SC, which may in turn underlie longer latency responses in DA neurons. The aim of the research presented in this thesis was to investigate whether the cortex was capable of modulating the activity of DA neurons, and whether the SC was the relay for this cortical influence. In the anaesthetised rat, single pulse electrical stimulation of the barrel field of the primary somatosensory cortex (S1Bf) produced a short latency, short duration response in the SC, but DA neurons were largely insensitive to the stimulus. After disinhibition of the SC with the GABAA antagonist bicuculline, responses in the SC to S1Bf stimulation were enhanced, and DA neurons became responsive to S1Bf stimulation, suggesting that the SC is the route of cortical input to DA neurons. This was confirmed in the subsequent experiment. Responses were produced in DA neurons without the need for SC disinhibition by stimulating S1Bf with a high frequency train of pulses. This response in DA neurons was suppressed or eliminated by suppressing SC activity. Finally, the contribution of cortical and subcortical input to DA neuron responses was examined by stimulating the trigeminal nucleus. Trigeminal stimulation produced responses in the SC comparable to multiwhisker deflection, and produced responses in almost all DA neurons. Disinhibition of the SC differentially modulated phases of the SC response previously demonstrated to be produced by trigeminal and cortical input, and differential changes were seen in initial and later components of DA neuron responses, which were often associated with changes in the SC response. The results of these studies suggest that cortical inputs to the SC could provide a mechanism through which responses are produced in DA neurons that can reflect complex stimulus attributes. However, research in this thesis and elsewhere suggests that the activity of DA neurons is insufficiently discriminatory to reflect the full range of potentially rewarding stimuli, and hence it is suggested that DA neurons provide a salience signal, which can be biased by a pre-saccadic estimate of previously established reward value, but which does not communicate reward value per se.

Dysfunction of cholinergic and dopaminergic systems has been implicated in memory function de cits that are core pathology and associated features of several neurological disorders. However, in order to develop more effective treatments, it is crucial to better understand how different aspects of learning and memory are modulated by these neuromodulatory systems. Using optogenetic stimulation or silencing, this thesis aims to investigate the effects of cholinergic and dopaminergic modulation on various hippocamal learning and memory processes. To understand how these neuromodulatory systems modulate hippocampal network activity, I first examined their effects on hippocampal local field potentials in urethane-anaesthetised mice. I demonstrated that optogenetic cholinergic activation suppressed slow oscillations, shifting brain activity to a state dominated by theta and gamma oscillations. In contrast, dopaminergic activation suppressed gamma oscillations. Second, to directly probe the effects of neuromodulation on different stages of spatial learning, I acutely activated or inactivated cholinergic or dopaminergic neurons during various behavioural tasks. My findings suggested that cholinergic activation, solely during the reward phase of a long-term spatial memory task, slowed place learning, highlighting the importance of temporally-precise neuromodulation. Moreover, dopaminergic stimulation may enhance place learning of a food rewarded task, supporting a role for dopamine in spatial learning. In addition, I tested the effects of cholinergic and dopaminergic modulation on reversal learning and found that cholinergic inactivation and dopaminergic activation appear to impair this process. Together, these findings emphasise the importance of cholinergic and dopaminergic modulation in learning and memory. They suggest that precise timing of neuromodulator action is critical for optimal learning and memory performance, and that acetylcholine and dopamine support complementary processes that allow for effective learning and adaptation to changing environments.

Parkinson's disease (PD) is a common neurodegenerative disorder, characterised by preferential loss of ventral midbrain dopaminergic (vmDA) neurons in the substantia nigra pars compacta (SNc). The majority of PD cases have unknown aetiology; however, between 5-10% arise due to known genetic mutations, the most common of which are found in the LRRK2 gene. LRRK2 is expressed in neurons and glia in the human brain; therefore, cell-autonomous and/or non-cell autonomous effects may participate in LRRK2-mutation-mediated degeneration of vmDA neurons. This study set out to understand the effects of LRRK2 mutations on human vmDA neurons and midbrain astrocytes, and to shed new light on the mechanisms of PD pathogenesis. To achieve this goal, differentiation protocols were generated to produce vmDA neurons and midbrain patterned (MP) astrocytes from induced pluripotent stem cells (iPSCs). iPSCs from patients carrying the LRRK2-G2019S mutation were differentiated into both cell types, and hypothesis-driven analysis of cellular functions including autophagy, mitochondrial respiration, glycolysis, and cellular migration was conducted; however, no disease phenotypes were observed. Following this, proteomics and transcriptomics techniques were then used to analyse the effects of the LRRK2-G2019S mutation in an unbiased manner. In the iPSC-derived MPastrocyte cultures, this technique highlighted stochastic X-chromosome reactivation events that led to difficulties in interpreting the resulting data; however, in the iPSC-derived vmDA-neuron cultures, LRRK2-G2019S-mediated inhibition of endocytosis and axon guidance was identified. These findings were found to be consistent in iPSC-derived vmDA-neuron cultures carrying the LRRK2-R1441C mutation, suggesting that these two mutations exert their pathogenic effects through similar mechanisms. Finally, phosphoproteomics analysis of iPSC-derived vmDA-neuron cultures was conducted to identify bone fide LRRK2-kinase substrates. Eleven potential LRRK2- kinase substrates were identified, nine of which have been previously shown to participate in endocytic vesicle trafficking, neurite outgrowth, and synaptic function. The findings of this study suggest that LRRK2 has neuron-specific functions, and that its mutations contribute to neurodegeneration in a cell-autonomous manner.

Dopaminergic neurons (DA) are an anatomically and functionally heterogeneous group of cells involved in a wide range of neuronal network activities and behaviour. Among them, mesencephalic dopaminergic neurons (mDA) are the major source of dopamine in the brain. They present two main groups of projecting cells: the A9 neurons of the Substantia Nigra (SN) and the A10 cells of the Ventral Tegmental Area (VTA). A9 neurons form the nigrostriatal pathway and are involved in regulating voluntary movements and postural reflexes. Their selective degeneration leads to Parkinson’s disease (PD) and the loss of DA synapses in the striatum is believed to be primary cause for the disruption of the ability to control movements. A10 cells constitute the mesocorticolimbic pathway playing a fundamental role in reward and attention. Their abnormal function has been linked to schizophrenia, attention deficit and addiction while they are relatively spared in PD (Meyer-Lindenberg et al., 2002). The description of the repertory of genes of mDA neurons may provide crucial information on their physiology as well as on the mechanisms of cell-type specific dysfunction. Interestingly, in previous gene expression profiling experiments, mDA cells groups presented a limited number of differentially expressed genes (Chung et al., 2005; Greene et al., 2005). By a combination of different gene expression platforms with Laser Capture Microdissection (LCM), it has been unveiled the existence of an alternative splice variant of Erythropoietin Receptor (EpoR) in A9 neurons. Moreover, the transcripts of hemoglobin alpha, adult chain 1 (Hba-a1) and hemoglobin beta, adult chain 1 (Hbb-b1) have been identified. The main goal of this study is the understanding of the role of Erythropoietin receptor (EpoR) and of alpha and β-globin in dopaminergic neurons as well as in Parkinson’s disease (PD). In blood Epo regulates erythrocyte differentiation and Hb production. Since Epo has been recently shown to protect dopaminergic neurons in neurochemical PD models and it is a very important pharmacological target, it has been analyzed by the RACE technique the expression of EpoR in spleen (as control) and in A9 mouse mesencephalic cells collected by Laser Capture Microdissection. 5’RACE analysis of DA neurons indicate the existence of an alternative transcription start site for the EpoR (referred to as DA-EpoR). EpoR expression was validated by PCR experiments and was restricted to A9 DA cells, which selectively degenerate in PD. The truncated cDNA corresponding to DA EpoR has been cloned from the A9 mesencephalic neurons, while the full-length WT EpoR has been cloned from the spleen. Both cDNAs have been expressed in HEK-T cells in order to unveil their function. DA EpoR may act as a decoy toward the WT EpoR. To unveil the role of α and β globin in the brain it has been investigated an animal model of β-Thalassemia. I took advantage of a mouse model of β-Thalassemia, in which β-globin gene is deleted in heterozigosity. The goal is to study the status of global gene expression in A9 and A10 dopaminergic neurons in a thalassemic carrier genotype. This condition is present in 3 million individuals in Italy. No brain studies have been carried out so far both in mouse models and in post-mortem human brains. I bred β-Thalassemic mice with TH-GFP mice where dopaminergic neurons are labelled. Then I took advantage of Laser Capture Microdissection to harvest A9 and A10 dopaminergic neurons from 12 months old wild type and heterozygous mice for β-chain of Hb for four replicas. Liver and cerebellum cells were used as controls, since liver presents iron accumulation in old thalassemic carriers. Sample cells have been analyzed at the Affymetrix core facility. I identified among the genes differentially expressed Atg4, a major regulator of autophagy, and Hepcidin, the hormone controlling iron metabolism. Relevant target genes have been verified by qPCR.

Parkinson’s disease (PD) is an incurable, chronically progressive neurodegenerative disease leading to premature invalidity and death. The locomotor disability of PD patients is mainly rooted in the gradual and insidious degeneration of dopaminergic neurons (DA) projecting from the midbrain substantia nigra (SN) to the basal ganglia striatum, a pathological process highlighted microscopically by the formation of insoluble cytosolic protein aggregates, known as Lewy bodies and Lewy neurites. The pathogenic mechanisms leading to PD remain poorly understood, arguably owing to the lack of suitable animal and cellular experimental models of the disease. Therefore, there is an urgent need for developing reliable experimental models that recapitulate the key features of PD. The recent development of induced pluripotent stem cell (iPSC) technology has enabled the generation of patient-specific iPSC and their use to model human diseases, although it is currently unclear whether this approach could be useful to successfully model age-related conditions. Importantly, disease modeling using iPSC largely relies on the existence of efficient protocols for the differentiation of disease-relevant cell types. Here, we first developed an efficient protocol for the differentiation of iPSC to authentic midbrain-specific DA neurons with SN properties by forced expression of LMX1A using a lentivirus-mediated gene delivery system. Next, we generated an iPSC-based cellular model of PD that recapitulates key phenotypic features of PD, such as DA neuron loss and α-synuclein accumulation in DA neurons from PD patients. Overall, our results demonstrate that we have developed a valuable tool for elucidating the pathogenic mechanisms leading to PD, as well as an experimental platform for screening new drugs that may prevent or rescue neurodegeneration in PD.
La malaltia de Parkinson (MP) és una malaltia neurodegenerativa incurable que causa invalidesa i mort prematura. Els pacients de la malaltia de Parkinson presenten alteracions motores degudes a una degeneració gradual de les neurones dopaminèrgiques que projecten des de la substància nigra fins a l’estriat. A nivell microscòpic s’observa la presència d’agregats proteics insolubles en el citosol de les neurones coneguts com cossos o neurites de Lewy. Els mecanismes patològics responsables de la MP no es coneixen bé, possiblement a causa de la manca de models animals i cel•lulars adequats. Per tant, existeix una gran necessitat de desenvolupar models experimentals fiables que recapitulin les característiques bàsiques de la MP. El recent desenvolupament de les cèl•lules mare pluripotents induïdes (iPSC) ha permès la generació de iPSC específiques de pacient i el seu ús per modelar malalties humanes, ara bé, no és clar si aquesta estratègia es pot utilitzar per modelar exitosament malalties d’origen tardà, com ara la MP. És important destacar que el modelatge de malalties utilitzant iPSC, es basa, en gran mesura en l'existència de protocols eficients per a la diferenciació de les iPSC cap al tipus cel•lular rellevant per a la malaltia. Durant aquest període, per primera vegada, s’ha desenvolupat un protocol per a l’eficient diferenciació de les iPSC cap a neurones dopaminèrgiques amb les propietats característiques de neurones dopaminèrgiques nigrostriatals, mitjançant l’expressió forçada de LMX1A utilitzant vectors lentivirals. A continuació, s’ha generat un model cel•lular usant iPSC derivades de pacients de MP que recapitula les principals característiques fenotípiques de la malaltia, com ara la pèrdua de neurones dopaminèrgiques i l'acumulació de α-sinucleïna en les neurones dopaminèrgiques. En general, els nostres resultats demostren que hem desenvolupat una eina valuosa per a l’estudi dels mecanismes patològics que condueixen a la MP, així com una nova plataforma pel descobriment de nous fàrmacs encaminats a prevenir o evitar la neurodegeneració.

Nestin is a type VI intermediate filament protein that marks proliferative cells in the central and peripheral nervous system of vertebrates during development and adulthood. Nestin is not only expressed in progenitor cells of neuronal tissues but is also present in muscle, heart, lung, pancreas and skin follicle tissues. The goal of this thesis is to investigate and characterize the nestin protein in goldfish and relate nestin expression to neuroregeneration and brain plasticity events in the adult goldfish forebrain. Currently little is known about nestin function and regulation in vertebrates, especially in fish. In this study we used Rapid amplification of cDNA ends PCR (RACE-PCR) to isolate goldfish nestin mRNA. We uncovered several different mRNA transcripts. PCR analysis and sequencing further identified three different nestin transcripts of 4003, 2446, and 2126 nucleotides with a predicted protein length of 860, 274, and 344 amino acids respectively. We next applied a multiple-antigenic peptide (MAP) strategy to generate a polyclonal goldfish-specific nestin antibody against a 23 amino acid sequence located at the N-terminal end of goldfish nestin. Western blotting revealed the existence of three different nestin protein isoforms (nestin A, B and C); the first report of nestin isoforms in teleost species. Nestin expression and distribution in the goldfish brain is complex and revealed both individual and tissue-dependent variations. The most remarkable finding following principal component analysis of the western blot data was the uniqueness of the pituitary, hypothalamus and telencephalon. These tissues are proliferative in nature containing progenitor and proliferative cellular pools that are involved in important biological axes such as the motor and reproductive axis. Interestingly, all three tissues were able to change their proliferative cellular profile of nestin protein expression to alleviate the detrimental effects of the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) upon administration. The toxin MPTP destroys dopamine neurons in the fish brain leading to motor deficits and reproductive difficulties. The incorporation of 5-bromo-2’-deoxyuriding (BrdU) into newly synthesized DNA revealed an upregulation of BrdU immunolabeling following MPTP administration in the area telencephali pars dorsalis (Vd) and along the ventricular surface area of the telencephalon suggesting the generation of new neurons in the adult central nervous system. This thesis reports novel nestin isoforms and illustrates regenerative events occurring in the goldfish telencephalon following a neurotoxic insult. This work provides a framework for future investigations of the differential roles and regulation of the nestins to better understand seasonal neuronal plasticity, neuronal regeneration and neuronal circuitry in teleost.

Parkinson’s disease (PD) is a chronic progressive neurodegenerative disorder characterised by selective loss of dopaminergic neurons in the substantia nigra. Drugs are the principal method of symptom treatment, yet remain unable to reverse underlying neurodegeneration. As PD prevalence increases with aging populations, greater emphasis falls on understanding pathogenesis to establish an effective neuroprotective strategy. Current research identifies iron accretion as a potential pathogenic factor. Iron is crucial for healthy cellular physiology, however, dysregulation can be highly neurotoxic. Since iron cannot be excreted, levels must be tightly controlled via specific iron regulatory proteins (IRP), including hepcidin, transferrin receptors, divalent metal transporter 1, ferritin, ferroportin, aconitase 1, and iron response element-binding protein 2. Iron accumulation has been established in neurodegenerative diseases, including Alzheimer’s, Multiple Sclerosis, and PD. While the instigating causes remain unknown, inflammation is a common factor. Activated microglia mediate the inflammatory response, and can release cytotoxic substances leading to iron-induced toxicity. Since dopaminergic neurons in PD are vulnerable to iron overload and inflammation, it is vital to determine what promotes changes to IRP expression leading to iron accumulation. It is hypothesised that chronic microglial activation and ensuing pro-inflammatory factors can dysregulate neuronal iron. Such changes may result from alterations in key IRP gene expression, with downstream cascades leading to neuronal degeneration. Results herein provide evidence of microglial inflammatory factor involvement on neuronal iron metabolism in an in vitro model of dopaminergic neurons. Specifically, IL6, TNF and new evidence of hydrogen peroxide instigate significant alterations in expression of HAMP, TfR and DMT1, causing iron elevations. Co-culture experiments established astrocytic iron buffering mechanisms able to provide sufficient neuroprotection to abolish observed gene expression changes. Lastly, experiments conclude that neuronal death occurs mainly via apoptotic pathways. Collectively, results support the instigative role of inflammation on altering neuronal iron handling. Such discoveries could lead to potential improvements in therapeutic strategies for PD.