The 2-50 transgenic mouse is a mutant containing the functional exons of human c-myc, an oncogene and cell cycle controller, under the control of the minimal sequence of the promoter of myelin basic protein, a component of the sheath surrounding axons of neurons. The transgene complex is expressed only in oligodendrocytes during a limited period of neonatal life, and is not detectable in large amounts. The animals suffer significant loss of oligodendrocyte precursor cells prior to myelination and onset of myelin formation is delayed. These animals elaborate only an incomplete myelin sheath in the central nervous system. Quantitative genetic analysis was used to characterize the transgene insertion and optic nerves from transgenic and non-transgenic animals were used for light and election microscopy, for electrophysiological testing and for immunohistochemical studies of glial cell subpopulations and axonal cytoskeletal components.

BACKGROUND: The lysosomal storage disorder, Niemann Pick type C1 (NPC1), presents a variable phenotype including neurovisceral and neurological symptoms. 2-Hydroxypropyl-ss-cyclodextrin (HPssCD)-based therapies are presently the most promising route of intervention. While severe cerebellar dysfunction remains the main disabling feature of NPC1, sensory functions including auditory and olfactory ones are also affected. Morphological and functional anomalies of Npc1 (-/-) mouse retina have also been observed, although the functional integrity of the visual pathway from retina to visual cortex is still unsettled. We have addressed this issue by characterizing the visual evoked potential (VEP) response of Npc1 (-/-) mice and determining if/how HPssCD administration influences the VEPs of both Npc1 (-/-) and Npc1 (+/+) mice. METHODS: VEP elicited by a brief visual stimulus were recorded from the scalp overlying the visual cortex of adult (PN, postnatal days 60, 75, 85 and 100) Npc1 (+/+) and Npc1 (-/-) mice that had received repeated injections of either HPssCD or plain vehicle. The first injection was given at PN4 and was followed by a second one at PN7 and thereafter by weekly injections up to PN49. Cholesterol accumulation and myelin loss were finally assessed by filipin staining and myelin basic protein immunohistochemistry, respectively. RESULTS AND DISCUSSION: We have found that the transmission of visual signals from retina to visual cortex is negatively influenced by the loss of Npc1 function. In fact, the VEP response of Npc1 (-/-) mice displayed a highly significant increase in the latency compared to that of Npc1 (+/+) mice. HPssCD administration fully rescued this defect and counteracted the cholesterol accumulation in retinal ganglion cells and dorsal lateral geniculate nucleus neurons, as well as the myelin loss in optic nerve fibers and axons projecting to the visual cortex observed in of Npc1 (-/-) mice. By contrast, HPssCD administration had no effect on the VEP response of Npc1 (+/+) mice, further strengthening the treatment efficacy. CONCLUSIONS: This study pinpoints the analysis of VEP response as a potentially accurate and non-invasive approach to assess neural activity and visual information processing in NPC1 patients, as well as for monitoring the progression of the disease and assessing the efficacy of potential therapies.

Niemann-Pick type C1 (NPC1) disease is a lysosomal storage disorder caused by defective intracellular trafficking of exogenous cholesterol. Purkinje cell (PC) degeneration is the main sign of cerebellar dysfunction in both NPC1 patients and animal models. It has been recently shown that a significant decrease in Sonic hedgehog (Shh) expression reduces the proliferative potential of granule neuron precursors in the developing cerebellum of Npc1(-/-) mice. Pursuing the hypothesis that this developmental defect translates into functional impairments, we have assayed Npc1-deficient pups belonging to the milder mutant mouse strain Npc1(nmf164) for sensorimotor development from postnatal day (PN) 3 to PN21. Npc1(nmf164)/Npc1(nmf164) pups displayed a 2.5-day delay in the acquisition of complex motor abilities compared to wild-type (wt) littermates, in agreement with the significant disorganization of cerebellar cortex cytoarchitecture observed between PN11 and PN15. Compared to wt, Npc1(nmf164) homozygous mice exhibited a poorer morphological differentiation of Bergmann glia (BG), as indicated by thicker radial shafts and less elaborate reticular pattern of lateral processes. Also BG functional development was defective, as indicated by the significant reduction in GLAST and Glutamine synthetase expression. A reduced VGluT2 and GAD65 expression also indicated an overall derangement of the glutamatergic/GABAergic stimulation that PCs receive by climbing/parallel fibers and basket/stellate cells, respectively. Lastly, Npc1-deficiency also affected oligodendrocyte differentiation as indicated by the strong reduction of myelin basic protein. Two sequential 2-hydroxypropyl-beta-cyclodextrin administrations at PN4 and PN7 counteract these defects, partially preventing functional impairment of BG and fully restoring the normal patterns of glutamatergic/GABAergic stimulation to PCs. These findings indicate that in Npc1(nmf164) homozygous mice the derangement of synaptic connectivity and dysmyelination during cerebellar morphogenesis largely anticipate motor deficits that are typically observed during adulthood.

Trace elements are involved in many biological processes and serve important functions to maintain the normal development of the central nervous system (CNS). In the CNS, iron (Fe), copper (Cu), and zinc (Zn) are some of the most important elements that play critical roles as catalysts, cofactors, and structural components for many cellular enzymes and proteins. The deficiency or excess of these metals may lead to various neurological disorders. Demyelination is a condition of loss of myelin and leads to neurological diseases like Multiple Sclerosis. Myelin consists of transition metals and hence it would be interesting to study concentrations of these elements in normal and demyelinated models. X-Ray Fluorescence (XRF) is a popular non-destructive technique applied in trace element studies. The principle involves exciting a sample and detecting characteristic X-rays, which provide information on elemental concentrations in the sample. In the present studies the feasibility of XRF for trace element studies was explored. A total of 120 samples of brain and spinal cord tissues were collected from Long Evans (control) and Long Evans Shaker (dysmyelinated)–an incomplete formation of myelin sheaths–rats at ages of 3 weeks and 16 weeks. The samples were excited using x-rays from an Energy Dispersive X-Ray Diffraction (EDXRF) set-up. The spectral data was collected using an Silicon Drift Detector (SDD) and the resultant data were analysed to see if statistically significant changes in concentrations were present in the samples. The results were discussed and suggestions for future work were made. / Thesis / Master of Science (MSc)

Abstract Salla disease (SD) is a rare lysosomal storage disorder, belonging to the Finnish disease heritage. The condition leads to intellectual disabilities. Two main categories of the disease have been identified – a conventional subtype and a severe subtype. The gene locus of SD has been assigned to a restricted region on the long arm of chromosome 6. The gene SLC17A5 is responsible for lysosomal-membrane sialic acid transport. The objective of this study was to describe the neurocognitive developmental spectrum of SD in a long follow-up study. In the original study (1997–1999), the sample consisted of 41 Finnish patients with Salla disease. They were examined by a paediatric neurologist, a psychologist and a speech therapist. The follow-up study (2010–2012) concerned of 27 (66%) patients from the original SD patient sample. The study included neurological and neuropsychological investigations. A case study of a mildly affected female patient was also reported. In the first study, the typical neurocognitive profile of SD was outlined and the different phenotypes confirmed. The neurocognitive profile of SD consisted of a strong motor handicap, but also well-developed skills in verbal comprehension and interaction. In the follow-up study, the main finding was that the verbal skills related to comprehension did not diminish over time. However, the skills that demanded productive speech were worsened by both dyspraxia and dysarthria, markers of dysfunction of the cerebellum. The neurocognitive and neurological status of the mildly affected female patient remained stable during the long follow-up time. In addition the MRI findings revealed mild dysfunction. The results indicate that the neurocognitive deficits related to SD are clear in childhood, but the illness does not have a rapid progressive nature after teenage years. The motor handicap is strong but the cognitive skills related to verbal comprehension, and interactive skills, do not deteriorate in adulthood. Four different neurodevelopmental periods can be outlined. / Tiivistelmä Tämän tutkimuksen tavoitteena oli kuvata Sallan tautiin liittyvä neurokognitiivisen kehityksen kulku pitkän seurantatutkimuksen aikana. Sallan tauti, erittäin harvinainen lysosomaalinen kertymäsairaus, kuuluu suomalaiseen tautiperimään. Nämä perinnölliset sairaudet ovat Suomessa yleisempiä kuin muissa maissa. Sallan tauti etenee älylliseen kehitysvammaisuuteen. Kaksi taudin päätyyppiä, tavanomainen ja vakava-asteinen fenotyyppi, on tunnistettu. Sallan taudin aiheuttavan geenin sijainti on paikallistettu kromosomiin 6. SLC17A5-geeni vastaa sialihapon kuljetuksesta solujen lysosomeissa. Ensimmäisen tutkimuksen (1997–1999) aineisto koostui 41 suomalaisesta Sallan tautia sairastavasta potilaasta. Neurologi, psykologi sekä puheterapeutti tutkivat jokaisen potilaan. Seuranta-aineisto (2010–2012) koostui 27 (66 %) potilaasta. Tutkimukseen kuului neurologin sekä neuropsykologin tutkimus. Lieväoireisen naispotilaan kehityskulku julkaistiin erillisenä tapaustutkimuksena. Ensimmäisessä tutkimuksessa selvitettiin Sallan taudille ominainen neurokognitiivinen profiili, lisäksi vahvistettiin kahden eri fenotyypin olemassaolo. Neurokognitiivisiin tyyppioireisiin kuuluivat vahvat motoriset defektit, mutta toisaalta hyvin kehittyneet kielelliset taidot puheen ymmärtämisen osalta. Myös vuorovaikutustaidot olivat vahvat. Seurantatutkimuksen päätulos oli puheen ymmärtämisen taitojen säilyminen taudin edetessä. Puheen tuottamiseen liittyvien vaikeuksien osalta sekä dyspraksia että dysartria heikensivät kielellistä toimintakykyä. Nämä kielelliset defektit liittyvät pikkuaivojen toimintahäiriöihin. Lieväoireisen naispotilaan neurologiset ja neurokognitiiviset löydökset eivät olleet edenneet pitkän seurantatutkimuksen aikana. Myös aivojen kuvantamistutkimuksen tulokset olivat lievät. Sallan tautiin liittyvät neurokognitiiviset muutokset ovat selkeät lapsuusiässä, mutta sairauden luonne aikuisiällä ei ole nopeasti etenevä. Motorisen toimintakyvyn defektit ovat vahvat, mutta kielellisen ymmärtämisen ja vuorovaikutuksen taidot eivät heikkene aikuisilla potilailla. Taudista voidaan erotella neljä erilaista kehityksellistä vaihetta.

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