Diss. (sammanfattning) Umeå : Univ., 2008. / Härtill 5 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst., 2004. / Härtill 4 uppsatser.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2009.

During development of the nervous system, signals from specialized organizing centers generate distinct cell types. The signaling molecule, Sonic Hedgehog (SHH) is expressed by the floor plate (FP) and is sufficient to specify the ventral midbrain pattern. In the spinal cord, Bone Morphogenetic Proteins (BMPs) expressed in the roof plate (RP) specify dorsal cell-fates. The attenuation of BMP signaling is required for SHHmediated patterning of the ventral hindbrain and spinal cord, while BMP signaling is required in conjunction with SHH for ventral forebrain patterning. This thesis will focus on the function of SHH and BMPs in the midbrain by examining the molecules ability to pattern and regulate development. Midbrains of Shh[superscript -/-] mice were examined. Some ventral cell fates are specified in the Shh[superscript -/-] mouse in a Ptc1 and Gli1 independent manner. Ventral midbrain induction was observed to be Hh-independent by the existence of a Pax7-negative ventral midbrain territory before embryonic day 9. Interestingly, dorsal markers are not uniformly altered and increased cell death was seen in Shh[superscript -/-] dorsal midbrains. These results suggest specific regulation of dorsal patterning by Shh, rather than a simple deregulation. Several BMPs and their antagonists are expressed in a spatial and temporal manner in the midbrain. Expression of BMPs is seen in the RP, and rostral FP (rFP), which also expresses SHH. BMP signaling was manipulated using in vivo electroporation. NOGGIN misexpression resulted in a loss of RP and a reduction of dorsal cell-fates that was preceded by cell-shape changes, delamination of cells into the lumen and their elimination. This was accompanied by a reduction and alteration of midbrain size and shape. BMP blockade changed N-Cadherin distribution and perturbed pseudostratified morphology of the neurepithelium. Ventrally, BMP blockade resulted in a decrease of proliferation, while increasing differentiation, Notch signaling molecules at the rFP and medial FP markers. However ventral midbrain cell-fates were correctly specified. Notch-Delta signaling was examined in the Mib[superscript -/-] mouse. Different regulation of cell-fates was observed in the midbrain and spinal cord. Mib[superscript -/-] midbrains lacked a mature lateral FP, however ventral cell-fates are specified. Mib[superscript -/-] spinal cords lose Shh expression and several ventral cell-fates. / text

Parkinson’s disease is a common neurological disorder with a complex etiology. The disease is characterized by a progressive loss of dopaminergic cells in the substantia nigra, which leads to motor function and sometimes cognitive function disabilities. One of the pathological hallmarks in Parkinson’s disease is the cytoplasmic inclusions called Lewy bodies found in the dopamine neurons. The aggregated protein α-synuclein is a main component of Lewy bodies. In view of severe symptoms and the upcoming of problematic side effects that are developed by the current most commonly used treatment in Parkinson’s disease, new treatment strategies need to be elucidated. One such strategy is replacing the lost dopamine neurons with new dopamine-rich tissue. To improve survival of the implanted neurons, neurotrophic factors have been used. Glial cell line-derived neurotrophic factor (GDNF), which was discovered in 1993, improves survival of ventral mesencephalic dopamine neurons and enhances dopamine nerve fiber formation according to several studies. Thus, GDNF can be used to improve dopamine-rich graft outgrowth into the host brain as well as inducing sprouting from endogenous remaining nerve fibers. This study was performed on Gdnf gene-deleted mice to investigate the role of GDNF on the nigrostriatal dopamine system. The transplantation technique was used to create a nigrostriatal microcircuit from ventral mesencephalon (VM) and the lateral ganglionic eminence (LGE) from different Gdnf gene-deleted mice. The tissue was grafted into the lateral ventricle of wildtype mice. The results revealed that reduced concentrations of GDNF, as a consequence from the Gdnf gene deletion, had effects on survival of dopamine neurons and the dopamine innervation of the nigrostriatal microcircuit. All transplants had survived at 3 months independently of Gdnf genotype, however, the grafts derived from Gdnf gene-deleted tissue had died at 6 months. Transplants with partial Gdnf gene deletion survived up to 12 months after transplantation. Moreover, the dopaminergic innervation of striatal co-grafts was impaired in Gdnf gene-deleted tissue. These results highlight the role of GDNF for long-term maintenance of the nigrostriatal dopamine system. To further investigate the role of GDNF expression on survival and organization of the nigrostriatal dopamine system, VM and LGE as single or combined to double co-grafts created from mismatches in Gdnf genotypes were transplanted into the lateral ventricle of wildtype mice. Survival of the single grafts was monitored over one year using a 9.4T MR scanner. The size of single LGE transplants was significantly reduced by the lack of GDNF already at 2 weeks postgrafting while the size of single VM was maintained over time, independently of GDNF expression. The double grafts were evaluated at 2 months, and the results revealed that lack of GDNF in LGE reduced the dopamine cell survival, while no loss of dopamine neurons was found in VM single grafts. The dopaminergic innervation of LGE was affected by absence of GDNF, which also caused a disorganization of the striatal portion of the co-grafts. Small, cytoplasmic inclusions were frequently found in the dopamine neurons in grafts lacking GDNF expression. These inclusions were not possible to classify as Lewy bodies by immunohistochemistry and the presence of phospho-α-synuclein and ubiquitin; however, mitochondrial dysfunction could not be excluded. To further study the death of the dopamine neurons by the deprivation of GDNF, the attention was turned to how Lewy bodies are developed. With respect to the high levels of α-synuclein that was found in the striatum, this area was selected as a target to inject the small molecule – FN075, which stimulates α-synuclein aggregation, to further investigate the role of α-synuclein in the formation of cytoplasmic inclusions. The results revealed that cytoplasmic inclusions, similar to those found in the grafts, was present at 1 month after the injection, while impairment in sensorimotor function was exhibited, the number of dopamine neurons was not changed at 6 months after the injection. Injecting the templator to the substantia nigra, however, significantly reduced the number of TH-positive neurons at 3 months after injection. In conclusion, these studies elucidate the role of GDNF for maintenance and survival of the nigrostriatal dopamine system and mechanisms of dopamine cell death using small molecules that template the α-synuclein aggregation.

Thesis (Ph.D.) - University of St Andrews, April 2008.

Thesis (Ph. D.)--University of Texas at Austin, 2008. / Vita. Includes bibliographical references.

Thesis (Ph. D.) University of Missouri-Columbia, 2006. / The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Title from title screen of research.pdf file (viewed on August 9, 2007) Vita. Includes bibliographical references.

Master of Science

Parkinson’s disease, the second most common neurodegenerative disorder,is characterized by loss of nigrostriatal dopaminergic neurons. To date,there is no defined cause and cure for the disease. An ideal treatmentstrategy is to replace the lost neurons by transplanting fetal dopaminergicneurons to the brain of parkinsonian patients. Clinical trials have beenperformed and the outcome was variable where one significant obstaclewas the limited graft reinnervation of the host brain. To study this issue,organotypic tissue culture can be utilized to monitor dopaminergic nervefiber outgrowth in vitro and their association with astrocytes. Using thisculture technique, dopaminergic nerve fibers appear in twomorphologically and temporally different types. The early appearing nervefibers are formed in the absence of astrocytes, reach long distances, andare called non-glial-associated tyrosine hydroxylase (TH) -positive nervefibers. After a few days, the second sequence of nerve fibers, the glialassociatedTH-positive nerve fibers, are formed, and their growth arelimited to the presence of astrocytes, that migrate and form a monolayersurrounding the plated tissue. The aim of this thesis was to study theinteraction between nerve fiber formation and astrocytes with a specialfocus on the long-distance growing nerve fibers. Ventral mesencephalic(VM) organotypic slice cultures from embryonic day (E) 12, E14, and E18were incubated for 14, 21, 28, and 35 days in vitro (DIV). The resultsrevealed that the two morphologically different processes were found incultures from the younger stages, while no non-glial-associated growthwas found in cultures of tissue from E18. Instead neurons had migratedonto the migrating astrocytes. Astrocytes migrated longer distances intissue from older stages, and the migration reached a plateau at 21 DIV.Co-cultures of E14 VM tissue pieces and cell suspension of matureastrocytes promoted migration of neurons, as seen in E18 cultures. Thus,9the maturity of the astrocytes was an important factor for nerve fiberoutgrowth. Hence, targeting molecules secreted by astrocytes might bebeneficial for regeneration. Chondroitin sulfate proteoglycan (CSPG), amember of proteoglycan family, is produced by the astrocytes and has adual role of being permissive during development and inhibitory afterbrain injury in adult brain. Cultures were treated with chondroitinase ABC(ChABC) or methyl-umbelliferyl-β-D-xyloside (β-xyloside) in twodifferent protocols, early and late treatments. The results from the earlytreated cultures showed that both compounds inhibited the outgrowth ofnerve fibers and astrocytic migration in cultures from E14 tissue, while β-xyloside but not ChABC promoted the non-glial-associated growth incultures derived from E18 fetuses. In addition, β-xyloside but not ChABCinhibited neuronal migration in E18 cultures. Taken together, β-xylosideappeared more effective than ChABC in promoting nerve fiber growth.Another potential candidate, integrin-associated protein CD47, was studiedbecause of its role in synaptogenesis, which is important for nerve fibergrowth. Cultures from E14 CD47 knockout (CD47-/-) mice were plated andcompared to their wildtypes. CD47-/- cultures displayed a massive and longnon-glial-associated TH-positive nerve fiber outgrowth despite theirnormal astrocytic migration. Blocking either signal regulatory protein-α(SIRPα) or thrombospondin-1 (TSP-1), which bind to CD47, had nogrowth promoting effect. In conclusion, to promote nerve growth, youngertissue can grow for longer distances than older tissue, and inhibiting CSPGproduction promotes nerve growth in older tissue, while gene deletion ofCD47 makes the astrocytes permissive for a robust nerve fiber growth.