第806回 千葉医学会・第10回 歯科口腔外科例会 20.

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-β-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

GRANULE NEURON PROLIFERATION IS REDUCED IN THE DEVELOPING CEREBELLUM OF THE NPC1-/- MOUSE, A MODEL OF HUMAN NIEMANN-PICK C1 DISEASE

Niemann Pick type C (NPC) disease is an neurodegenerative lysosomal storage disorder caused by mutations in either of two genes, Npc1 and Npc2. A prominent feature of NPC1 disease in humans is massive loss of cerebellar Purkinje cells (PCs). Cerebellar granule neurons (GNs) have been little studied in mouse models of this disease, perhaps because their development takes place during the first three weeks of postnatal life, whereas signs of PC pathology occur in older mice. GNs are generated during the first two postnatal weeks from a progenitor layer named, external granule layer (EGL), where their proliferation is sustained by Shh and microglia-released growth factors that activate the Notch pathway. We have observed that postnatal development of cerebellar GNs is defective in Npc1-/- mice. Compared to age-matched wild-type, there is an accelerated disappearance of the EGL in these mice, which is due to a premature exit from the cell cycle of GN precursors. As a consequence, the si

A marked paucity of granule cells in the developing cerebellum of the Npc1(-/-) mouse is corrected by a single injection of hydroxypropyl-β-cyclodextrin.

In this study we show that postnatal development of cerebellar granule neurons (GNs) is defective in Npc1-/- mice. Compared to age-matched wild-type littermates, there is an accelerated disappearance of the external granule layer (EGL) in these mice. This is due to a premature exit from the cell cycle of GN precursors residing at the level of the EGL. As a consequence, the size of cerebellar lobules of these mice displays a 20%-25% reduction compared to that of age-matched wild-type mice. This size reduction is detectable at post-natal day 28 (PN28), when cerebellar GN development is completed while signs of neuronal atrophy are not yet apparent. Based on the analysis of EGL thickness and the determination of proliferating GN fractions at increasing developmental times (PN8-PN14), we trace the onset of this GN developmental defect during the second postnatal week. We also show that during this developmental time Shh transcripts undergo a significant reduction in Npc1-/- mice compared

Decreased neural stem cell proliferation and olfaction in mouse models of Niemann–Pick C1 disease and the response to hydroxypropyl-β-cyclodextrin

The Npc1nih/nih-null model and the Npc1nmf164/nmf164 hypomorph models of Niemann-Pick C1 (NPC1) disease show defects in olfaction. We have tested the effects of the life-prolonging treatment hydroxypropyl-beta-cyclodextrin (HPBCD) on olfaction and neural stem cell numbers when delivered either systemically or by nasal inhalation. Using the paradigm of finding a hidden cube of food after overnight food deprivation, Npc1nih/nih homozygous mice showed a highly significant delay in finding the food compared with wild-type mice. Npc1nmf164/nmf164 homozygous mice showed an early loss of olfaction which was mildly corrected by somatic delivery of HPBCD which also increased the number of neural stem cells in the mutant but did not change the number in wild-type mice. In contrast, nasal delivery of this drug, at 1/5 the dosage used for somatic delivery, to Npc1nmf164/nmf164 mutant mice delayed loss of olfaction but the control of nasal delivered saline did so as well. The nasal delivery of HPBCD to wild-type mice caused loss of olfaction but nasal delivery of saline did not. Neural stem cell counts were not improved by nasal therapy with HPBCD. We credit the delay in olfaction found with the treatment, a delay which was also found for time of death, to a large amount of stimulation the mice received with handling during the nasal delivery

Sexually dimorphic expression of reelin in a mouse model of Alzheimer disease

Reelin is a glycoprotein secreted in developing brain by Cajal-Retzius cells and mediates proper positioning of migrating neurons in cortical layers. Binding to apolipoprotein E receptor 2 (ApoER2) and very-lowdensity lipoprotein receptor (VLDLR) receptors, Reelin triggers a cytosolic kinase cascade leading to the activation of phosphatidylinositol-3-kinase (PI3K) and the inhibition of glycogen synthase kinase 3β (GSK3β). The latter is the major kinase of the microtubule- associated tau protein, which upon hyperphosphorylation aggregates forming the neurofibrillary tangles. In the adult brain, Reelin is produced by GABAergic interneurons and glutamatergic pyramidal neurons of layer II of the entorhinal cortex and regulating the activity of NMDA glutamate receptors plays a crucial role in synaptic plasticity. In agreement with this role, several studies indicate that a disregulation of the reelin-mediated signaling contribute to synaptic alteration and neuronal loss in mouse models o

Sexually dimorphic expression of reelin in the brain of a mouse model of Alzheimer disease

Recent evidence highlights the protective role of reelin against amyloid β (Aβ)-induced synaptic dysfunction and cognitive impairment in Alzheimer disease (AD). In this study, exploiting TgCRND8 mice that overexpress a mutant form of amyloid β precursor protein (AβPP) and display an early onset of AD neuropathological signs, we addressed the question whether changes of reelin expression eventually precede the appearance of Aβ-plaques in a sex-dependent manner. We show that sex-associated and brain region-specific differences in reelin expression appear long before Aβ- plaque formation. However, in spite of a downregulation of reelin expression compared to males, TgCRND8 females display fewer Aβ-plaques, suggesting that additional factors, other than sex and reelin level, influence amyloidosis in this mouse model.Recent evidence highlights the protective role of reelin against amyloid β (Aβ)-induced synaptic dysfunction and cognitive impairment in Alzheimer disease (AD). In this study, exploiting TgCRND8 mice that overexpress a mutant form of amyloid β precursor protein (AβPP) and display an early onset of AD neuropathological signs, we addressed the question whether changes of reelin expression eventually precede the appearance of Aβ-plaques in a sex-dependent manner. We show that sex-associated and brain region-specific differences in reelin expression appear long before Aβ-plaque formation. However, in spite of a downregulation of reelin expression compared to males, TgCRND8 females display fewer Aβ-plaques, suggesting that additional factors, other than sex and reelin level, influence amyloidosis in this mouse model

Complex expression of BDNF and reelin in the developing and adult brain of TgCRND8 mice

This study is aimed at characterizing molecular pathways common to neurodevelopment and neurodegeneration. Indeed, it is now evident that neurodegenerative processes can be rooted in aberrant expression of neurodevelopment-related genes. Among various genes, BDNF and reelin are good candidates as they play fundamental roles during brain development/maturation and adult functions and their expression is often altered in neurodegeneration. On the other hand, PSEN1, which is involved in A β processing, also plays crucial roles in the developing brain

The appearance of phagocytic microglia in the postnatal brain of Niemann Pick type C mice is developmentally regulated and underscores shortfalls in fine odor discrimination

The loss of NPC1 or NPC2 function results in cholesterol and sphingolipid dyshomeostasis that impairs developmental trajectories, predisposing the postnatal brain to the appearance of pathological signs, including progressive and stereotyped Purkinje cell loss and microgliosis. Despite increasing evidence reporting the activation of pro-inflammatory microglia as a cardinal event of NPC1 disease progression at symptomatic stages both in patients and preclinical models, how microglia cells respond to altered neurodevelopmental dynamics remains not completely understood. To gain an insight on this issue, we have characterized patterns of microglia activation in the early postnatal cerebellum and young adult olfactory bulb of the hypomorphic Npc1(nmf164) mouse model. Previous evidence has shown that both these areas display a number of anomalies affecting neuron and glial cell proliferation and differentiation, which largely anticipate cellular changes and clinical signs, raising our interest on how microglia interplay to these changes. Even so, to separate the contribution of cues provided by the dysfunctional microenvironment we have also studied microglia isolated from mice of increasing ages and cultured in vitro for 1 week. Our findings show that microglia of both cerebellum and olfactory bulb of Npc1(nmf164) mice adopt an activated phenotype, characterized by increased cell proliferation, enlarged soma size and de-ramified processes, as well as a robust phagocytic activity, in a time- and space-specific manner. Enhanced phagocytosis associates with a profound remodeling of gene expression signatures towards gene products involved in chemotaxis, cell recognition and engulfment, including Cd68 and Trem2. These early changes in microglia morphology and activities are induced by region-specific developmental anomalies that likely anticipate alterations in neuronal connectivity. As a proof of concept, we show that microglia activation within the granule cell layer and glomerular layer of the olfactory bulb of Npc1(nmf164) mice is associated with shortfalls in fine odor discrimination

Additional file 2: Figure S2. of Visual evoked potentials of Niemann-Pick type C1 mice reveal an impairment of the visual pathway that is rescued by 2-hydroxypropyl-ß-cyclodextrin

Survival curves of naive Npc1 −/− , sham Npc1 −/− and HPβCD Npc1 −/− mice. Kaplan-Meier plots were analyzed by Log-rank (Mantel -Cox) test: Npc1 −/− vs Npc1 −/− sham, p > 0.05; Npc1 −/− vs Npc1 −/− HPβCD, p < 0.0001. Survival (mean value ± SEM): naive Npc1 −/− , 91.3 ± 1.3; sham Npc1 −/− , 87.8 ± 0.9; HPβCD Npc1 −/− , 145 ± 3.0. (JPEG 2830 kb