Anomalies in Dopamine Transporter Expression and Primary Cilium Distribution in the Dorsal Striatum of a Mouse Model of Niemann-Pick C1 Disease

The Niemann-Pick type C1 (NPC1) is a rare genetic disease characterized by the accumulation of endocytosed cholesterol and other lipids in the endosome/lysosome compartments. In the brain, the accumulation/mislocalization of unesterified cholesterol, gangliosides and sphingolipids is responsible for the appearance of neuropathological hallmarks, and progressive neurological decline in patients. The imbalance of unesterified cholesterol and other lipids, including GM2 and GM3 gangliosides, alters a number of signaling mechanisms impacting on the overall homeostasis of neurons. In particular, lipid depletion experiments have shown that lipid rafts regulate the cell surface expression of dopamine transporter (DAT) and modulate its activity. Dysregulated dopamine transporter’s function results in imbalanced dopamine levels at synapses and severely affects dopamine-induced locomotor responses and dopamine receptor-mediated synaptic signaling. Recent studies begin to correlate dopaminergic stimulation with the length and function of the primary cilium, a non-motile organelle that coordinates numerous signaling pathways. In particular, the absence of dopaminergic D2 receptor stimulation induces the elongation of dorso-striatal neuron’s primary cilia. This study has used a mouse model of the NPC1 disease to correlate cholesterol dyshomeostasis with dorso-striatal anomalies in terms of DAT expression and primary cilium (PC) length and morphology. We found that juvenile Npc1nmf164 mice display a reduction of dorso-striatal DAT expression, with associated alterations of PC number, length-frequency distribution, and tortuosity

Sex moderates the association between the COMT Val158Met single-nucleotide polymorphism and disorderliness facet of novelty seeking

Previous studies have shown inconsistent results regarding the effect of the Val158Met polymorphism of the catechol-O-methyltransferase (COMT) gene on personality and cognition. Here, nonclinical Caucasian university students of Italian origin were administered the Temperament and Character Inventory-Revised, Tellegen Absorption Scale, Differential Attentional Processes Inventory, and Waterloo-Stanford Group Scale of Hypnotic Susceptibility. We found that the COMT Val158Met polymorphism was significantly associated with the disorderliness facet of novelty seeking (NS4) and that sex was a moderator of this association. Females with the Met/Met genotype showed higher NS4 scores compared to those with the Val/Met and Val/Val genotypes. No significant genotype effect was found for males. Additionally, we failed to find a significant effect of the COMT gene on attention and hypnotic suggestibility measures. These results provide further evidence for a sex-specific influence on the gene-behaviour associations. Polymorphisms in dopamine system genes are reported to play a crucial role in influencing various aspects of plays a crucial role in influencing various aspects of personality traits and cognitive performance; however, previous studies have shown inconsistent results on the involvment of the functional Val158Met polymorphism of the catechol-O-methyltransferase (COMT) gene. In the present study, nonclinical Caucasian university students of Italian origin were administered the Temperament and Character Inventory-Revised, Tellegen Absorption Scale, Differential Attentional Processes Inventory, and Waterloo-Stanford Group Scale of Hypnotic Susceptibility. We found that the COMT Val158Met polymorphism was significantly associated with the disorderliness facet of novelty seeking (NS4) and that sex was a moderator of this association. Females with the Met/Met genotype showed higher NS4 scores compared to those with the Val/Met and Val/Val genotypes. In contrast, no significant genotype effect was found for males. Additionally, we failed to find a significant association of COMT enzyme activity with attention and hypnotic suggestibility measures. These results provide further evidence of a sex-specific influence on the gene-behaviour association

Abnormal BDNF signaling in a mouse model of a lysosomal lipid storage disease

Lysosomal lipid storage diseases are characterized by abnormal amounts of lipid deposits in cells. When lysosomal function is impaired, degradation of intracellular material cannot proceed normally and toxic accumulation of substrates occurs. Niemann Pick C1 (NPC1) disease is a lysosomal storage disorder due to abnormal function of NPC1, a protein residing in late endosomes/lysosomes that mediates the efflux of cholesterol. The massive loss of cerebellar Purkinje cells is the prominent feature of NPC1 disease. NPC1 patients develop ataxia and neurological manifestations including dementia. Studying the development of cerebellum in NPC1 mouse models, we observed a defective proliferation of granule neurons (GNs) that affects the size of all cerebellar lobules of NPC1-deficient mice. This GNs developmental defect occurs during the second postnatal week and is concomitant with abnormal generation and reception of Shh signaling at level of the primary cilium, an organelle implicated in various signaling pathways such as Sonic-hedgehog (Shh) and brain-derived neurotrophic-factor (BDNF). The activation of Shh pathway up-regulates BDNF, which in turn regulate the migration/differentiation of various neuronal and glial cells and the formation of synapses, playing an important role in the cytoarchitecture and connectivity within the cerebellar cortex. Our studies on Npc1nmf164 , a mouse hypomorphic mutant of NPC1 disease with slower disease progression, indicate that these mice display an abnormal BDNF signaling pathway in the developing cerebellum in almost all developmental stages analyzed and alterations in the fine structure and connectivity of mossy fibers. Mossy fiber axons represent one of the major inputs to the cerebellum and contain the highest concentration of BDNF in the CNS. These results pinpoint BDNF dysregulation as a possible candidate into the molecular pathogenesis of Niemann-Pick type C disease, thereby providing new targets for therapeutic intervention

Cerebellar Brain-Derived Neurotrophic Factor (BDNF) defects in a mouse model of a lysosomal lipid storage disease

Lysosomal lipid storage diseases are characterized by abnormal amounts of lipid deposits in cells. When lysosomal function is impaired, degradation of intracellular material cannot proceed normally and toxic accumulation of substrates occurs. Niemann-Pick C1 (NPC1) disease is a lysosomal storage disorder due to abnormal function of NPC1, a transmembrane protein related to Hedgehog receptor and involved in intracellular trafficking of cholesterol. Clinically NPC1, also called “juvenile Alzheimer’s”, presents massive loss of cerebellar Purkinje cells, ataxia and neurological manifestations including dementia. Studying the development of cerebellum, we observed a defective proliferation of granule neurons that affects the size of all cerebellar lobules of NPC1-deficient mice. This defect occurs during the second postnatal week and is concomitant with abnormal generation/reception of Shh signaling at level of the primary cilium, an organelle implicated in various signaling pathways such as Shh and BDNF. The activation of Shh pathway up-regulates the BDNF expression, which in turn regulate synapse formation as well as connectivity within the cerebellar cortex. By immunohistochemistry/biochemical approaches, we have observed various defects in the cerebellar BDNF signaling of Npc1nmf164, a mouse hypomorphic mutant of NPC1 disease with slower disease progression. These mice display defects in almost all developmental stages analyzed and alterations in the fine structure and connectivity of mossy fibers. Mossy fiber axons represent one of the major inputs to the cerebellum and contain the highest concentration of BDNF in the CNS. These results pinpoint BDNF dysregulation as a possible candidate into the molecular pathogenesis of Niemann-Pick type C disease

BDNF anomalies in Niemann Pick type C disease

Niemann-Pick C1 (NPC1) disease is a lysosomal lipid storage disorder due to abnormal function of NPC1, a transmembrane protein involved in intracellular trafficking of cholesterol. Clinically, NPC1 presents massive loss of cerebellar Purkinje cells, ataxia and neurological manifestations including dementia. We have previously demonstrated that the early postnatal cerebellar development is impaired in Npc1-deficient mice. The reduced cholesterol availability affects SHH signaling at level of the primary cilium that leads to a reduction of granule neuron precursors proliferative potential. The activation of Shh pathway up-regulates the BDNF expression, which in turn regulates synapse formation as well as connectivity within the cerebellar cortex. By immunohistochemistry/biochemical approaches, we have observed various defects in the cerebellar BDNF signaling in NPC1-hypomorphic mutant mice and alterations in the fine structure and connectivity of mossy fibers which represent one of the major source of BDNF in the cerebellum. These results pinpoint BDNF dysregulation as a possible candidate into the molecular pathogenesis of Niemann-Pick type C disease

Altered development of cerebellar granule neurons in a mouse model of Niemann-Pick type C1 disease

Niemann-Pick type C1 disease (NPCD), also called “Juvenile Alzheimer’s Disease”, is a lysosomal cholesterol storage disorder due to mutations in the NPC1 gene, presenting visceral/neurological impairments associated with cognitive decline. NPC1 neurodegeneration is characterized by the presence of amyloid-β deposition and tau aggregations, alike to Alzheimer's disease. Unfortunately, the molecular mechanisms that cause neurodegeneration in this disease are currently unknown. Studying the cerebellar development in Npc1 mutant mice, we observed a defective proliferation of granule neurons (GNs)1, concomitant with the dysregulation of Sonic Hedgehog (Shh) and Brain Derived Neurotrophic factor (BDNF) expression, that affects the size of cerebellar lobules2 . Aims: In light of the relevance of BDNF in a wide range of neurophysiological processes, as neuronal migration, differentiation and degeneration3, we analyzed BDNF-TrkB signaling during the first weeks of postnatal life, in a milder mouse model, Npc1nmf164, harboring a missense mutation in a region where a high proportion of human mutations are found and associated with a slower disease progression. Materials and Methods: In order to study the expression and localization of BDNF/TrkB, we performed Western Blot and Immunofluorescence analyses in cerebellar samples of wt and Npc1nmf164mice. Cerebellar neuron responsiveness to BDNF was investigated by chemotaxis assay in in vitro culture, while neuronal differentiation was studied by detailed dendritic branching analysis of Golgi-stained GNs. Results: We observed an altered expression/localization of BDNF and its receptor TrkB, at the very early stages of cerebellar development. Furthermore, purified GNs from Npc1nmf164 mice, in the presence of exogenous BDNF, exhibit a reduced chemotaxis response, dependent on altered TrkB receptor trafficking. Using Golgi staining, we observed that mature GNs present an abnormal dendritic morphology. Discussion These data raising the possibility that morpho-functional anomalies in granule neurons largely anticipate the overt manifestation of symptoms in adult NPC1 patients. Our focus on very early morphological/ biochemical defects, which precede neurodegeneration in adults, is relevant for identifying predictive markers, useful for NPC1 disease treatment. Thus, given the potential of BDNF to protect synapses against various toxic insults, manipulation of BDNF could represent a valid therapeutic approach for a variety of neurological disorders, including NPC1 disease. Conclusions: Here, we propose that an altered BDNF signaling is a part of complex cerebellar deficits, which may be predictive of symptomatic events in NPCD, as well as the progressive neurodegeneration of Purkinje cells in adulthood

The Influence of Catechol-O-Methyltransferase (COMT) Val158Met Gene Polymorphism, Persistence, and Attentional Characteristics on Novelty Seeking

Over the last five decades, a number of biological oriented personality theories have been proposed to explain how anatomical and functional differences in the human brain are responsible for individual differences in personality. Catechol-O-methyltransferase (COMT) gene for the Val158Met single nucleotide polymorphism (rs4680) is known to influence the activity of the enzyme responsible for dopamine metabolism and has been linked with various aspect of personality dimensions and cognitive processes. In the present study, non-clinical participants (201 women and 53 men) were administered the Temperament and Character Inventory-Revised, Tellegen Absorption Scale, Differential Attentional Processes Inventory, and Waterloo-Stanford Group Scale of Hypnotic Suggestibility, Form C. Among these participants, COMT polymorphism (parameterized as a 3-level variable: 0 = Met/Met, 1 = Val/Met, 2 = Val/Val) was assessed in 117 women and 51 men. Hypnotic Suggestibility scores were significantly correlated with scores from the Absorption, Extremely Focused Attention, and Dual Attention for Physical-Cognitive task. We failed to find the expected significant association between COMT and Hypnotic Suggestibility scores. In contrast, COMT scores were significantly correlated with scores from Novelty Seeking (r = -.15, p = .049) and its Disorderliness subscale (r = -.21, p = .006). A principal component analysis (with varimax rotation), performed on personality and attention measures, yielded a four-factor solution: Factor-1 (Moderately Focused Attention, Dual Attention Cognitive-Cognitive, and Dual Attention Physical-Cognitive), Factor-2 (Novelty Seeking, Reward Dependence, and Harm Avoidance), Factor-3 (Hypnotic Suggestibility, Absorption, and Extremely Focused Attention), and Factor-4 (Persistence). These factors accounted for 18.4%, 16.3%, 16.2%, and 12.3% of the total variance, respectively. These findings guided us in the choice of the COMT, Persistence, Extremely Focused Attention, and Absorption scores as predictors of Disorderliness scores in separate multivariate regression analyses. Lower COMT activity, higher Absorption scores, and lower Persistence scores accounted for 18% of the total variance in the whole sample, and 10.8% in female sample. In male sample, higher Absorption and lower Persistence scores significantly predicted Disorderliness scores that accounted for 7.4% of the total variance. Since our male sample was relatively small, further research is needed to understand gender differences, if any, using a larger male sample

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

Altered cerebellar granule cell differentiation and synapse maturation in a mouse model of a lysosomal lipid storage disease.

The rare Niemann-Pick type C1 (NPC1) disease is a lysosomal lipid storage disorder, caused by mutations in the Npc1 gene. Since the encoded protein mediates the outflow of cholesterol from endosomal-lysosomal compartments, these mutations cause intracellular cholesterol and other lipids overload, leading to neurodegeneration, neurovisceral symptoms and early death. Both NPC1 patients and animal models develop neurological manifestations and ataxia due to Purkinje cell loss. We demonstrated a reduced proliferation of cerebellar granule neurons that influences the size of all cerebellar lobules of NPC1-deficient mice, caused by a significant decrease in the availability of Shh signaling, at the level of the primary cilium. Multiple extrinsic signals, such as Brain-Derived Neurotrophic Factor (BDNF), are needed to support post-natal cerebellar developmental processes, including migration and differentiation of granule precursors as well as connectivity within the cerebellar cortex. For instance, mossy fiber axons represent one of the main excitatory inputs to the internal granule layer and are the primary source of BDNF. Therefore, we wondered if BDNF-mediated signaling was involved in impaired cerebellar morphogenesis in hypomorphic Npc1nmf164/nmf164 mice, characterized by a slow progression of the disease. By immunohistochemistry/biochemical and morphological approaches on PN11-PN30 Npc1nmf164/nmf164 mice, we observed a strong downregulation of BDNF and its receptor, abnormal differentiation of granule neurons and alterations in the structure/connectivity of mossy fibers. In line with previous studies that demonstrated reduced cerebellar BDNF levels in autistic patients, male Npc1nmf164/nmf164 mice showed no preference for social/nonsocial stimulus in a typical task exploited to investigate autistic-like behaviour

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]