Linear cyclodextrin polymer prodrugs as novel yherapeutics for Niemann-Pick type C1 disorder

Niemann-Pick Type C1 disorder (NPC) is a rare lysosomal storage disease characterized by the accumulation of cholesterol in lysosomes. NPC has no FDA approved treatments yet, however 2-hydroxypropyl-β-cyclodextrin (HPβCD) has shown efficacy for treating the disease in both mouse and feline NPC models and is currently being investigated in late stage clinical trials. Despite promising results, therapeutic use of HPβCD is limited by the need for high doses, ototoxicity and intrathecal administration. These limitations can be attributed to its poor pharmacokinetic profile. In the attempt to overcome these limitations, we have designed a β-cyclodextrin (βCD) based polymer prodrugs (ORX-301) for an enhanced pharmacokinetic and biodistribution profile, which in turn can potentially provide an improved efficacy at lower doses. We demonstrated that subcutaneously injected ORX-301 extended the mean lifespan of NPC mice at a dosage 5-fold lower (800 mg/kg, body weight) the HPβCD dose proven efficacious (4000 mg/kg). We also show that ORX-301 penetrates the blood brain barrier and counteracts neurological impairment. These properties represent a substantial improvement and appear to overcome major limitations of presently available βCD-based therapy, demonstrating that this novel prodrug is a valuable alternative/complement for existing therapies

Shortened primary cilium length and dysregulated Sonic hedgehog signaling in Niemann-Pick C1 disease

The Niemann-Pick type C1 (NPC1) disease is a neurodegenerative lysosomal storage disorder due to mutations in the NPC1 gene, encoding a transmembrane protein related to the Sonic hedgehog receptor, Patched, and involved in intracellular trafficking of cholesterol. We have recently found that the proliferation of cerebellar granule neuron precursors is significantly reduced in Npc1-/- mice due to the downregulation of Shh expression. This finding prompted us to analyze the formation of the primary cilium, a non-motile organelle that is specialized for Shh signal transduction and responsible, when defective, for several human genetic disorders. In this study, we show that the expression and subcellular localization of Shh effectors and ciliary proteins are severely disturbed in Npc1-deficient mice. The dysregulation of Shh signaling is associated with a shortening of the primary cilium length and with a reduction of the fraction of ciliated cells in Npc1-deficient mouse brains and the human fibroblasts of NPC1 patients. These defects are prevented by treatment with 2-hydroxypropyl-β-cyclodextrin, a promising therapy currently under clinical investigation. Our findings indicate that defective Shh signaling is responsible for abnormal morphogenesis of the cerebellum of Npc1-deficient mice and show, for the first time, that the formation of the primary cilium is altered in NPC1 disease

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

The interplay between TGF-β-stimulated TSC22 domain family proteins regulates cell-cycle dynamics in medulloblastoma cells

Proteins belonging to the TGFβ-stimulated clone 22 domain (TSC22D) family display a repertoire of activities, regulating cell proliferation and differentiation. The tumor suppressor activity of the first identified member of the family, TSC22D1 (formerly named TSC-22), has been extensively studied, but afterward a longer isoform encoded by the same gene turned out to play an opposite role. We have previously characterized the role of TSC22D1 and TSC22D4 in cell differentiation using granule neurons (GNs) isolated from the mouse cerebellum. However, the possibility to study the role of these factors in cell proliferation was limited by the fact that GNs readily exit from the cell-cycle and differentiate upon isolation and in vitro culture. To overcome this limitation, we have now exploited DAOY medulloblastoma cells, which are ontogenetically similar to cerebellar GNs and can be efficiently transfected with interfering RNA for gene knockdown purposes. Our findings indicate that TSC22D4–TSC22D1 short isoform heterodimers are involved in the escape from cell proliferation and exit from the cell-cycle, whereas, the TSC22D1 long isoform is required for cell proliferation, acting independently from TSC22D4. We also show that the silencing of specific expression of TSC22D4 or TSC22D1 isoforms affects the cell-cycle progression. These findings add a novel insight on the function of TSC22D proteins, with particular reference to the tumor suppressor activity of the TSC22D1 short isoform, which is re-framed within the context of a functional interplay with TSC22D4 and the mutually exclusive expression with the TSC22D1 long isoform

High Doses of D-Chiro-Inositol Alone Induce a PCO-Like Syndrome and Other Alterations in Mouse Ovaries

Administration of 1000–1500 mg/day D-Chiro-Inositol (DCIns) or a combination of Myo-Inositol (MyoIns) and DCIns in their plasma molar ratio (40:1) for three or more months are among recommended treatments for metabolic syndrome and/or Polycystic Ovary Syndrome (PCOS). We previously confirmed the efficacy of this formulation (8.2 mg/day MyoIns and 0.2 mg/day DCIns for 10 days) in a mouse PCOS model, but also observed negative effects on ovarian histology and function of formulations containing 0.4–1.6 mg/day DCIns. We therefore analyzed effects of higher doses of DCIns, 5, 10 and 20 mg/day, administered to young adult female mice for 21 days, on ovarian histology, serum testosterone levels and expression of the ovarian enzyme aromatase. Five mg/day DCIns (human correspondence: 1200 mg/day) altered ovarian histology, increased serum testosterone levels and reduced the amount of aromatase of negative controls, suggesting the induction of an androgenic PCOS model. In contrast, 10–20 mg/day DCIns (human correspondence: 2400–4800 mg/day) produced ovarian lesions resembling those typical of aged mice, and reduced serum testosterone levels without affecting aromatase amounts, suggesting a failure in steroidogenic gonadal activity. Notwithstanding physiological/biochemical differences between mice and humans, the observed pictures of toxicity for ovarian histology and function recommend caution when administering DCIns to PCOS patients at high doses and/or for periods spanning several ovulatory cycles

PATHOGENETIC MECHANISMS RESPONSIBLE FOR ALTERED DEVELOPMENTAL TRAJECTORIES IN NIEMANN PICK C DISEASE

Niemann Pick type C (NPC) disease is an autosomal recessive, neurodegenerative lysosomal storage disorder caused by the abnormal function of NPC1 or NPC2 (95% and 5% of NPC patients, respectively), proteins involved in the intracellular trafficking of endocytosed cholesterol and other lipids. The accumulation/mislocalization of cholesterol, gangliosides, sphingolipids alters signaling pathways, likely causing developmental defects. As an example, the covalent cholesterol modification of Sonic hedgehog (Shh) and its downstream effector, Smoothened, is relevant for gradient formation and downstream signaling activation [1]. Hence, we have recently demonstrated that cholesterol dyshomeostasis in NPC1 affects Shh-mediated activities, at the primary cilium. This impairs the differentiation and functional maturation of neurons and glial cells, leading to abnormal cerebellar morphogenesis [2]. Downstream from Shh, the dysregulation of Brain-Derived Neurotrophic-Factor expression patterns appears to be responsible for defective cell migration and synapse formation. In addition, our recent observations indicate that the reduced cholesterol availability at the plasma membrane affects the signaling of the endocannabinoid receptor CB1, in agreement with the influence that cholesterol content in lipid rafts exerts on the portioning and internalization of this receptor. Hydroxypropyl-β-cyclodextrin (HPβCD), represents the major treatment currently studied in both animal models and patients but it comes with several drawbacks. To overcome these limitations we have recently validated a novel polymer prodrug version of HPβCD, demonstrating that its enhanced pharmacokinetic/biodistribution profiles and longer terminal half-life leads to a significant rescue of cerebellar anomalies and neurobehavioral deficits of NPC1 mouse model, at a dose 5-fold lower than the efficacious HPβCD dose [3]

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

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

SUMOylation Regulates TDP-43 Splicing Activity and Nucleocytoplasmic Distribution

21 pags., 8 figs.The nuclear RNA-binding protein TDP-43 forms abnormal cytoplasmic aggregates in the brains of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) patients and several molecular mechanisms promoting TDP-43 cytoplasmic mislocalization and aggregation have been proposed, including defects in nucleocytoplasmic transport, stress granules (SG) disassembly and post-translational modifications (PTM). SUMOylation is a PTM which regulates a variety of cellular processes and, similarly to ubiquitination, targets lysine residues. To investigate the possible regulatory effects of SUMOylation on TDP-43 activity and trafficking, we first assessed that TDP-43 is SUMO-conjugated in the nuclear compartment both covalently and non-covalently in the RRM1 domain at the predicted lysine 136 and SUMO-interacting motif (SIM, 106–110 residues), respectively. By using the SUMO-mutant TDP-43 K136R protein, we demonstrated that SUMOylation modifies TDP-43 splicing activity, specifically exon skipping, and influences its sub-cellular localization and recruitment to SG after oxidative stress. When promoting deSUMOylation by SENP1 enzyme over-expression or by treatment with the cell-permeable SENP1 peptide TS-1, the cytoplasmic localization of TDP-43 increased, depending on its SUMOylation. Moreover, deSUMOylation by TS-1 peptide favoured the formation of small cytoplasmic aggregates of the C-terminal TDP-43 fragment p35, still containing the SUMO lysine target 136, but had no effect on the already formed p25 aggregates. Our data suggest that TDP-43 can be post-translationally modified by SUMOylation which may regulate its splicing function and trafficking, indicating a novel and druggable mechanism to explore as its dysregulation may lead to TDP-43 pathological aggregation in ALS and FTD.This study was financially supported by Fondazione Italiana di Ricerca per la SLA (AriSLA) (Grant SUMALS). M.M. is granted by La Caixa Foundation (Grant LCF/BQ/PR19/11700003).Peer reviewe