Effects of Low Luteinizing Hormone Serum Levels on Oocyte Retrieval, Fertilization Rate, and Embryo Quality during Controlled Ovarian Stimulation: Results from a Prospective Cohort Analysis.

OBJECTIVES: Luteinizing hormone (LH) plays a key role in normal follicular development and oocyte maturation in controlled ovarian stimulation. LH stimulates the proliferation and differentiation of theca cells for the secretion of androgens, synergistically increasing estrogen production. This study aimed to investigate the effects of low LH concentrations on oocyte retrieval, fertilization, and embryo development in patients undergoing in vitro fertilization/intracytoplasmic sperm injection. DESIGN: We prospectively (ClinicalTrials ID: NCT05755529) analyzed patients undergoing in vitro fertilization/intracytoplasmic sperm injection, subdividing them into three groups according to their age. Serum LH levels were evaluated on day 3, during stimulation (day 10) and before ovulation induction (day 12). PARTICIPANTS/MATERIALS, SETTING, METHODS: Forty-three consecutive women were scheduled for IVF and received ovarian stimulation with follitropin alfa (Gonal F, Merck Serono, Germany) and ganirelix (Fyremaldel, Sun Pharma, Italy). Statistical analysis was performed with InStat 3.10, GraphPad software, San Diego, CA, USA. Normal distribution was tested by the Shapiro-Wilk test. Continuous variables were expressed as the mean and standard deviation. Categorical variables are expressed as frequencies and percentages. RESULTS: Our data analysis suggests that serum LH levels progressively decrease during controlled ovarian stimulation, and this effect is more evident in the early phase of this procedure. From this perspective, circulating LH levels may significantly decrease during the late follicular phase due to the negative feedback of ovarian hormones from multiple follicular developments or after the suppressive effects of gonadotropin-releasing hormone antagonists. LIMITATIONS: Although our study confirms that exogenous LH can be considered a strategy in women with reduced LH levels during ovarian stimulation to improve oocyte quality and reproductive outcome, the generalizability of the results is limited by the low number of participants enrolled. CONCLUSIONS: Exogenous LH may be considered a strategy in women with a decrease in LH levels during ovarian stimulation to improve oocyte quality and reproductive outcome.

In Vitro Nano-Polystyrene Toxicity: Metabolic Dysfunctions and Cytoprotective Responses of Human Spermatozoa.

The ubiquitous spread of Polystyrene nanoplastics (PS-NPs) has rendered chronic human exposure an unavoidable phenomenon. The biodistribution of such particles leads to bioaccumulation in target organs including the testis, the site of sperm maturation. The purpose of this research has been to estimate the impact of PS-NPs (50 and 100 nm) on the metabolism of mature spermatozoa. The analysis of the semen parameters has revealed a higher toxicity of the smaller sized PS-NPs, which have negatively affected major organelles, leading to increased acrosomal damage, oxidative stress with the production of ROS, DNA fragmentation, and decreased mitochondrial activity. PS-NPs of 100 nm, on the other hand, have mainly affected the acrosome and induced a general state of stress. An attempt has also been made to highlight possible protective mechanisms such as the expression of HSP70s and their correlation among various parameters. The results have evinced a marked production of HSP70s in the samples exposed to the smaller PS-NPs, negatively correlated with the worsening in oxidative stress, DNA fragmentation, and mitochondrial anomalies. In conclusion, our results have confirmed the toxicity of PS-NPs on human spermatozoa but have also demonstrated the presence of mechanisms capable of counteracting at least in part these injuries.

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 Npc1nmf164 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 Npc1nmf164 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 Npc1nmf164 mice is associated with shortfalls in fine odor discrimination.

Protective Effect of Curcuma Extract in an Ex Vivo Model of Retinal Degeneration via Antioxidant Activity and Targeting the SUMOylation.

Retinal degeneration is the major and principal cause behind many incurable blindness diseases. Several studies indicated the neuroprotective effect of Curcuma longa in eye pathologies, specifically retinopathy. However, the molecular mechanism behind its effect has not been completely elucidated. Using an ex vivo model of retinal degeneration obtained from an ex vivo optic nerve cut (ONC), we demonstrated that Curcuma extract (Cur) exerted a neuroprotective effect. Importantly, Cur was able to modulate apoptosis and MAPK signaling pathway activation and prevent retinal ganglion cell (RGC) loss. Other well-known neuroprotective pharmacological tools, including memantine (Mem), citicoline (Cit), and ginkgolic acid (GA), were used to compare the potential mechanisms of Cur. The antioxidant activity of retinas treated with Cur following optic nerve cut was significantly higher than control, but Cur failed to change the retina glutamate content. Considering the antioxidant effect of Cur and taking advantage of our recent findings on the crosstalk between oxidative stress and post-translational protein modifiers, in particular, small ubiquitin-related modifier (SUMO), we were interested in exploring the effect of Cur on SUMOylation. We found that Cur significantly prevented the increase of protein SUMOylation, confirming our previous in vitro data indicating the cytoprotective effect of curcumin through modulating the oxidative stress and SUMO-JNK axis. Altogether, these results suggest that Curcuma protects the retina from degeneration via antioxidant activity and targets SUMOylation. Therefore, it might be considered for the combination therapy with other neuroprotective agents with different mechanisms in preclinical studies on retinal degeneration.

Retinal ganglion cell loss in an ex vivo mouse model of optic nerve cut is prevented by curcumin treatment.

Retinal ganglion cell (RGC) loss is a pathologic feature common to several retinopathies associated to optic nerve damage, leading to visual loss and blindness. Although several scientific efforts have been spent to understand the molecular and cellular changes occurring in retinal degeneration, an effective therapy to counteract the retinal damage is still not available. Here we show that eyeballs, enucleated with the concomitant optic nerve cut (ONC), when kept in PBS for 24 h showed retinal and optic nerve degeneration. Examining retinas and optic nerves at different time points in a temporal window of 24 h, we found a thinning of some retinal layers especially RGC's layer, observing a powerful RGC loss after 24 h correlated with an apoptotic, MAPKs and degradative pathways dysfunctions. Specifically, we detected a time-dependent increase of Caspase-3, -9 and pro-apoptotic marker levels, associated with a strong reduction of BRN3A and NeuN levels. Importantly, a powerful activation of JNK, c-Jun, and ERK signaling (MAPKs) were observed, correlated with a significant augmented SUMO-1 and UBC9 protein levels. The degradation signaling pathways was also altered, causing a significant decrease of ubiquitination level and an increased LC3B activation. Notably, it was also detected an augmented Tau protein level. Curcumin, a powerful antioxidant natural compound, prevented the alterations of apoptotic cascade, MAPKs, and SUMO-1 pathways and the degradation system, preserving the RGC survival and the retinal layer thickness. This ex vivo retinal degeneration model could be a useful method to study, in a short time window, the effect of neuroprotective tools like curcumin that could represent a potential treatment to contrast retinal cell death.

SUMOylation Regulates TDP-43 Splicing Activity and Nucleocytoplasmic Distribution.

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.

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.

Obstacles against the Marketing of Curcumin as a Drug.

Among the extensive public and scientific interest in the use of phytochemicals to prevent or treat human diseases in recent years, natural compounds have been highly investigated to elucidate their therapeutic effect on chronic human diseases including cancer, cardiovascular disease, and neurodegenerative disease. Curcumin, an active principle of the perennial herb Curcuma longa, has attracted an increasing research interest over the last half-century due to its diversity of molecular targets, including transcription factors, enzymes, protein kinases, growth factors, inflammatory cytokines, receptors, and it's interesting pharmacological activities. Despite that, the clinical effectiveness of the native curcumin is weak, owing to its low bioavailability and rapid metabolism. Preclinical data obtained from animal models and phase I clinical studies done in human volunteers confirmed a small amount of intestinal absorption, hepatic first pass effect, and some degree of intestinal metabolism, might explain its poor systemic availability when it is given via the oral route. During the last decade, researchers have attempted with new pharmaceutical methods such as nanoparticles, liposomes, micelles, solid dispersions, emulsions, and microspheres to improve the bioavailability of curcumin. As a result, a significant number of bioavailable curcumin-based formulations were introduced with a varying range of enhanced bioavailability. This manuscript critically reviews the available scientific evidence on the basic and clinical effects and molecular targets of curcumin. We also discuss its pharmacokinetic and problems for marketing curcumin as a drug.

Considerations around the SARS-CoV-2 Spike Protein with Particular Attention to COVID-19 Brain Infection and Neurological Symptoms.

Spike protein (S protein) is the virus "key" to infect cells and is able to strongly bind to the human angiotensin-converting enzyme2 (ACE2), as has been reported. In fact, Spike structure and function is known to be highly important for cell infection as well as for entering the brain. Growing evidence indicates that different types of coronaviruses not only affect the respiratory system, but they might also invade the central nervous system (CNS). However, very little evidence has been so far reported on the presence of COVID-19 in the brain, and the potential exploitation, by this virus, of the lung to brain axis to reach neurons has not been completely understood. In this Article, we assessed the SARS-CoV and SARS-CoV-2 Spike protein sequence, structure, and electrostatic potential using computational approaches. Our results showed that the S proteins of SARS-CoV-2 and SARS-CoV are highly similar, sharing a sequence identity of 77%. In addition, we found that the SARS-CoV-2 S protein is slightly more positively charged than that of SARS-CoV since it contains four more positively charged residues and five less negatively charged residues which may lead to an increased affinity to bind to negatively charged regions of other molecules through nonspecific and specific interactions. Analysis the S protein binding to the host ACE2 receptor showed a 30% higher binding energy for SARS-CoV-2 than for the SARS-CoV S protein. These results might be useful for understanding the mechanism of cell entry, blood-brain barrier crossing, and clinical features related to the CNS infection by SARS-CoV-2.

The pivotal role of SUMO-1-JNK-Tau axis in an in vitro model of oxidative stress counteracted by the protective effect of curcumin.

Oxidative stress is a toxic cellular condition, strictly related to inflammation and known to be a common feature of many neurodegenerative diseases. The imbalanced redox state modifies several molecular processes including protein SUMOylation, JNK and Tau protein activation, important actors in Alzheimer's disease. In this study, we showed a strong interaction among SUMO-1-JNK-Tau proteins and their molecular targets in an in vitro model (SHSY5Y cell line) of oxidative stress in which a significant reduction of cell viability and an augmented cell death was induced by increased doses of H2O2. The evoked oxidative stress led to a deficiency in the degradation system showing altered levels of Caspase-3, LC3BII/I and Ubiquitin. Curcumin, a natural compound with anti-oxidant and anti-inflammatory effects, demonstrated to tackle oxidative stress re-equilibrating SUMO-1, JNK and Tau functions. Importantly, 5 µM of curcumin induced an efficient recovery of cell viability, a reduction of cell death and a normalization of altered protein degradation marker levels. Interestingly, we found that H2O2 treatment induced a strong co-localization of SUMO-1-p-JNK-Tau proteins in nuclear bodies (NBs) and that curcumin was able to reduce these nuclear aggregates. These results highlight the SUMO-1-JNK-Tau axis key role in oxidative stress and the protective effect of curcumin against this pathological event, focusing on the importance of SUMO/deSUMOylation balance to regulate essential cellular processes.

Decreased membrane cholesterol in liver mitochondria of the point mutation mouse model of juvenile Niemann-Pick C1, Npc1nmf164.

It has long been known that there is decreased mitochondrial function in several tissues of Niemann-Pick C1 model mice and cultured cells. These defects contribute to the accumulation of Reactive Oxygen Species (ROS) and tissue damage. It is also well established that there is increased unesterified cholesterol, stored in late endosomes/lysosomes, in many tissues in mutant humans, mouse models, and mutant cultured cells. Using a mouse model with an NPC1 point mutation that is more typical of the most common form of the disease, and highly purified liver mitochondria, we find markedly decreased mitochondrial membrane cholesterol. This is compared to previous reports of increased mitochondrial membrane cholesterol. We also find that, although in wild-type or heterozygous mitochondria cytochrome c oxidase (COX) activity decreases with age as expected, surprisingly, COX activity in homozygous mutant mice improves with age. COX activity is less than half of wild-type amounts in young mutant mice but later reaches wild-type levels while total liver cholesterol is decreasing. Mutant mice also contain a decreased number of mitochondria that are morphologically abnormal. We suggest that the decreased mitochondrial membrane cholesterol is causative for the mitochondrial energy defects. In addition, we find that the mitochondrial stress regulator protein MNRR1 can stimulate NPC1 synthesis and is deficient in mutant mouse livers. Furthermore, the age curve of MNRR1 deficiency paralleled levels of total cholesterol. The role of such altered mitochondria in initiating the abnormal autophagy and neuroinflammation found in NPC1 mouse models is discussed.

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

The endocannabinoid system is affected by cholesterol dyshomeostasis: Insights from a murine model of Niemann Pick type C disease.

The dyshomeostasis of intracellular cholesterol trafficking is typical of the Niemann-Pick type C (NPC) disease, a fatal inherited lysosomal storage disorder presenting with progressive neurodegeneration and visceral organ involvement. In light of the well-established relevance of cholesterol in regulating the endocannabinoid (eCB) system expression and activity, this study was aimed at elucidating whether NPC disease-related cholesterol dyshomeostasis affects the functional status of the brain eCB system. To this end, we exploited a murine model of NPC deficiency for determining changes in the expression and activity of the major molecular components of the eCB signaling, including cannabinoid type-1 and type-2 (CB1 and CB2) receptors, their ligands, N-arachidonoylethanolamine (AEA) and 2-arachidonoylglycerol (2-AG), along with their main synthesizing/inactivating enzymes. We found a robust alteration of distinct components of the eCB system in various brain regions, including the cortex, hippocampus, striatum and cerebellum, of Npc1-deficient compared to wild-type pre-symptomatic mice. Changes of the eCB component expression and activity differ from one brain structure to another, although 2-AG and AEA are consistently found to decrease and increase in each structure, respectively. The thorough biochemical characterization of the eCB system was accompanied by a behavioral characterization of Npc1-deficient mice using a number of paradigms evaluating anxiety, locomotor activity, spatial learning/memory abilities, and coping response to stressful experience. Our findings provide the first description of an early and region-specific alteration of the brain eCB system in NPC and suggest that defective eCB signaling could contribute at producing and/or worsening the neurological symptoms of this disorder.

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

Proteins belonging to the TGFß-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.

Myo-inositol and D-chiro-inositol (40:1) reverse histological and functional features of polycystic ovary syndrome in a mouse model.

Mice exposed to continuous light undergo functional and histological changes that mimic those of human Polycystic Ovary Syndrome (PCOS). We herein induced the syndrome by exposing 30-day-old females to 10 weeks of permanent light. Ovarian morphology and histology, as well as reproductive parameters (time of observed pregnancy/delivery) were investigated. Ovaries of PCOS-modeled mice showed lack of tertiary follicles and corpora lutea, altered ovarian architecture, and increased thickness of the theca layer. When mice were returned to a normal light-dark regimen for 10 days, a slight, spontaneous improvement occurred, whereas a quick and almost complete recovery from PCOS signs and symptoms was obtained by treating animals with a daily supplementation of 420 mg/kg myo-inositol and D-chiro-inositol (MyoIns/DCIns) in a 40:1 molar ratio. Namely, ovaries from mice treated by this protocol recovered normal histological features and a proper ratio of theca/granulosa cell layer thickness (TGR), suggesting that the androgenic phenotype was efficiently reversed. Indeed, we identified TGR as a useful index of PCOS, as its increase in PCOS-modeled mice correlated linearly with reduced reproductive capability ( r = 0.75, p < 0.0001). Mice treated with a 40:1 formula regained low TGR values and faster recovery of their fertility, with a physiological delivery time after mating. On the other hand, a higher D-chiro-inositol treatment formula, such as MyoIns versus DCIns 5:1, was ineffective or even had a negative effect on clinical-pathological outcomes.

Linear Cyclodextrin Polymer Prodrugs as Novel Therapeutics 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-ß-cyclodextrin (HPßCD) 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 HPßCD 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 ß-cyclodextrin (ßCD) 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 HPßCD 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 ßCD-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 (Shh) 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-ß-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.

Developmental delay in motor skill acquisition in Niemann-Pick C1 mice reveals abnormal cerebellar morphogenesis.

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-ß-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.