Intracerebroventricular Treatment with 2-Hydroxypropyl-β-Cyclodextrin Decreased Cerebellar and Hepatic Glycoprotein Nonmetastatic Melanoma Protein B (GPNMB) Expression in Niemann–Pick Disease Type C Model Mice

Niemann–Pick disease type C (NPC) is a recessive hereditary disease caused by mutation of the NPC1 or NPC2 gene. It is characterized by abnormality of cellular cholesterol trafficking with severe neuronal and hepatic injury. In this study, we investigated the potential of glycoprotein nonmetastatic melanoma protein B (GPNMB) to act as a biomarker reflecting the therapeutic effect of 2-hydroxypropyl-β-cyclodextrin (HP-β-CD) in an NPC mouse model. We measured serum, brain, and liver expression levels of GPNMB, and evaluated their therapeutic effects on NPC manifestations in the brain and liver after the intracerebroventricular administration of HP-β-CD in Npc1 gene-deficient (Npc1^−/−) mice. Intracerebroventricular HP-β-CD inhibited cerebellar Purkinje cell damage in Npc1^−/− mice and significantly reduced serum and cerebellar GPNMB levels. Interestingly, we also observed that the intracerebral administration significantly reduced hepatic GPNMB expression and elevated serum ALT in Npc1^−/− mice. Repeated doses of intracerebroventricular HP-β-CD (30 mg/kg, started at 4 weeks of age and repeated every 2 weeks) drastically extended the lifespan of Npc1^−/− mice compared with saline treatment. In summary, our results suggest that GPNMB level in serum is a potential biomarker for evaluating the attenuation of NPC pathophysiology by intracerebroventricular HP-β-CD treatment.     

Cholesterol-Dependent Energy Transfer between Fluorescent Proteins—Insights into Protein Proximity of APP and BACE1 in Different Membranes in Niemann-Pick Type C Disease Cells

Förster resonance energy transfer (FRET) -based techniques have recently been applied to study the interactions between β-site APP-cleaving enzyme-GFP (BACE1-GFP) and amyloid precursor protein-mRFP (APP-mRFP) in U373 glioblastoma cells. In this context, the role of APP-BACE1 proximity in Alzheimer’s disease (AD) pathogenesis has been discussed. FRET was found to depend on intracellular cholesterol levels and associated alterations in membrane stiffness. Here, NPC1 null cells (CHO-NPC1^−/−), exhibiting increased cholesterol levels and disturbed cholesterol transport similar to that observed in Niemann-Pick type C disease (NPC), were used to analyze the influence of altered cholesterol levels on APP-BACE1 proximity. Fluorescence lifetime measurements of whole CHO-wild type (WT) and CHO-NPC1^−/− cells (EPI-illumination microscopy), as well as their plasma membranes (total internal reflection fluorescence microscopy, TIRFM), were performed. Additionally, generalized polarization (GP) measurements of CHO-WT and CHO-NPC1^−/− cells incubated with the fluorescence marker laurdan were performed to determine membrane stiffness of plasma- and intracellular-membranes. CHO-NPC1^−/− cells showed higher membrane stiffness at intracellular- but not plasma-membranes, equivalent to cholesterol accumulation in late endosomes/lysosomes. Along with higher membrane stiffness, the FRET efficiency between BACE1-GFP and APP-mRFP was reduced at intracellular membranes, but not within the plasma membrane of CHO-NPC1^−/−. Our data show that FRET combined with TIRF is a powerful technique to determine protein proximity and membrane fluidity in cellular models of neurodegenerative diseases.     

Weekly Treatment of 2-Hydroxypropyl-β-cyclodextrin Improves Intracellular Cholesterol Levels in LDL Receptor Knockout Mice

Recently, the importance of lysosomes in the context of the metabolic syndrome has received increased attention. Increased lysosomal cholesterol storage and cholesterol crystallization inside macrophages have been linked to several metabolic diseases, such as atherosclerosis and non-alcoholic fatty liver disease (NAFLD). Two-hydroxypropyl-β-cyclodextrin (HP-B-CD) is able to redirect lysosomal cholesterol to the cytoplasm in Niemann-Pick type C1 disease, a lysosomal storage disorder. We hypothesize that HP-B-CD ameliorates liver cholesterol and intracellular cholesterol levels inside Kupffer cells (KCs). Hyperlipidemic low-density lipoprotein receptor knockout (Ldlr^−/−) mice were given weekly, subcutaneous injections with HP-B-CD or control PBS. In contrast to control injections, hyperlipidemic mice treated with HP-B-CD demonstrated a shift in intracellular cholesterol distribution towards cytoplasmic cholesteryl ester (CE) storage and a decrease in cholesterol crystallization inside KCs. Compared to untreated hyperlipidemic mice, the foamy KC appearance and liver cholesterol remained similar upon HP-B-CD administration, while hepatic campesterol and 7α-hydroxycholesterol levels were back increased. Thus, HP-B-CD could be a useful tool to improve intracellular cholesterol levels in the context of the metabolic syndrome, possibly through modulation of phyto- and oxysterols, and should be tested in the future. Additionally, these data underline the existence of a shared etiology between lysosomal storage diseases and NAFLD.     

Myelin Defects in Niemann–Pick Type C Disease: Mechanisms and Possible Therapeutic Perspectives

Niemann–Pick type C (NPC) disease is a wide-spectrum clinical condition classified as a neurovisceral disorder affecting mainly the liver and the brain. It is caused by mutations in one of two genes, NPC1 and NPC2, coding for proteins located in the lysosomes. NPC proteins are deputed to transport cholesterol within lysosomes or between late endosome/lysosome systems and other cellular compartments, such as the endoplasmic reticulum and plasma membrane. The first trait of NPC is the accumulation of unesterified cholesterol and other lipids, like sphingosine and glycosphingolipids, in the late endosomal and lysosomal compartments, which causes the blockade of autophagic flux and the impairment of mitochondrial functions. In the brain, the main consequences of NPC are cerebellar neurodegeneration, neuroinflammation, and myelin defects. This review will focus on myelin defects and the pivotal importance of cholesterol for myelination and will offer an overview of the molecular targets and the pharmacological strategies so far proposed, or an object of clinical trials for NPC. Finally, it will summarize recent data on a new and promising pharmacological perspective involving A_2A adenosine receptor stimulation in genetic and pharmacological NPC dysmyelination models.     

GBA Mutations Influence the Release and Pathological Effects of Small Extracellular Vesicles from Fibroblasts of Patients with Parkinson’s Disease

Heterozygous mutations in the GBA gene, encoding the lysosomal enzyme glucocerebrosidase (GCase), are the strongest known genetic risk factor for Parkinson’s disease (PD). The molecular mechanisms underlying the increased PD risk and the variable phenotypes observed in carriers of different GBA mutations are not yet fully elucidated. Extracellular vesicles (EVs) have gained increasing importance in neurodegenerative diseases since they can vehiculate pathological molecules potentially promoting disease propagation. Accumulating evidence showed that perturbations of the endosomal–lysosomal pathway can affect EV release and composition. Here, we investigate the impact of GCase deficiency on EV release and their effect in recipient cells. EVs were purified by ultracentrifugation from the supernatant of fibroblast cell lines derived from PD patients with or without GBA mutations and quantified by nanoparticle tracking analysis. SH-SY5Y cells over-expressing alpha-synuclein (α-syn) were used to assess the ability of patient-derived small EVs to affect α-syn expression. We observed that defective GCase activity promotes the release of EVs, independently of mutation severity. Moreover, small EVs released from PD fibroblasts carrying severe mutations increased the intra-cellular levels of phosphorylated α-syn. In summary, our work shows that the dysregulation of small EV trafficking and alpha-synuclein mishandling may play a role in GBA-associated PD.     

Xylose-Configured Cyclophellitols as Selective Inhibitors for Glucocerebrosidase

Glucocerebrosidase (GBA), a lysosomal retaining β-d -glucosidase, has recently been shown to hydrolyze β-d -xylosides and to transxylosylate cholesterol. Genetic defects in GBA cause the lysosomal storage disorder Gaucher disease (GD), and also constitute a risk factor for developing Parkinson's disease. GBA and other retaining glycosidases can be selectively visualized by activity-based protein profiling (ABPP) using fluorescent probes composed of a cyclophellitol scaffold having a configuration tailored to the targeted glycosidase family. GBA processes β-d -xylosides in addition to β-d -glucosides, this in contrast to the other two mammalian cellular retaining β-d -glucosidases, GBA2 and GBA3. Here we show that the xylopyranose preference also holds up for covalent inhibitors: xylose-configured cyclophellitol and cyclophellitol aziridines selectively react with GBA over GBA2 and GBA3 in vitro and in vivo, and that the xylose-configured cyclophellitol is more potent and more selective for GBA than the classical GBA inhibitor, conduritol B-epoxide (CBE). Both xylose-configured cyclophellitol and cyclophellitol aziridine cause accumulation of glucosylsphingosine in zebrafish embryo, a characteristic hallmark of GD, and we conclude that these compounds are well suited for creating such chemically induced GD models.     

Inhibition of Soluble Epoxide Hydrolase Ameliorates Phenotype and Cognitive Abilities in a Murine Model of Niemann Pick Type C Disease

Niemann–Pick type C (NPC) disease is a rare autosomal recessive inherited childhood neurodegenerative disease characterized by the accumulation of cholesterol and glycosphingolipids, involving the autophagy-lysosome system. Inhibition of soluble epoxide hydrolase (sEH), an enzyme that metabolizes epoxy fatty acids (EpFAs) to 12-diols, exerts beneficial effects in modulating inflammation and autophagy, critical features of the NPC disease. This study aims to evaluate the effects of UB-EV-52, an sEH inhibitor (sEHi), in an NPC mouse model (Npc) by administering it for 4 weeks (5 mg/kg/day). Behavioral and cognitive tests (open-field test (OF)), elevated plus maze (EPM), novel object recognition test (NORT) and object location test (OLT) demonstrated that the treatment produced an improvement in short- and long-term memory as well as in spatial memory. Furthermore, UB-EV-52 treatment increased body weight and lifespan by 25% and reduced gene expression of the inflammatory markers (i.e., Il-1β and Mcp1) and enhanced oxidative stress (OS) markers (iNOS and Hmox1) in the treated Npc mice group. As for autophagic markers, surprisingly, we found significantly reduced levels of LC3B-II/LC3B-I ratio and significantly reduced brain protein levels of lysosomal-associated membrane protein-1 (LAMP-1) in treated Npc mice group compared to untreated ones in hippocampal tissue. Lipid profile analysis showed a significant reduction of lipid storage in the liver and some slight changes in homogenated brain tissue in the treated NPC mice compared to the untreated groups. Therefore, our results suggest that pharmacological inhibition of sEH ameliorates most of the characteristic features of NPC mice, demonstrating that sEH can be considered a potential therapeutic target for this disease.     

Identification of pharmacological chaperones for Gaucher disease and characterization of their effects on beta-glucocerebrosidase by hydrogen/deuterium exchange mass spectrometry

Point mutations in beta-glucocerebrosidase (GCase) can result in a deficiency of both GCase activity and protein in lysosomes thereby causing Gaucher Disease (GD). Enzyme inhibitors such as isofagomine, acting as pharmacological chaperones (PCs), increase these levels by binding and stabilizing the native form of the enzyme in the endoplasmic reticulum (ER), and allow increased lysosomal transport of the enzyme. A high-throughput screen of the 50,000-compound Maybridge library identified two, non-carbohydrate-based inhibitory molecules, a 2,4-diamino-5-substituted quinazoline (IC(50) 5 microM) and a 5-substituted pyridinyl-2-furamide (IC(50) 8 microM). They raised the levels of functional GCase 1.5-2.5-fold in N370S or F213I GD fibroblasts. Immunofluorescence confirmed that treated GD fibroblasts had decreased levels of GCase in their ER and increased levels in lysosomes. Changes in protein dynamics, monitored by hydrogen/deuterium-exchange mass spectrometry, identified a domain III active-site loop (residues 243-249) as being significantly stabilized upon binding of isofagomine or either of these two new compounds; this suggests a common mechanism for PC enhancement of intracellular transport.     

Behavioral Phenotyping in a Murine Model of GBA1-Associated Parkinson Disease

Mutations in GBA1, the gene encoding glucocerebrosidase, are common genetic risk factors for Parkinson disease (PD). While the mechanism underlying this relationship is unclear, patients with GBA1-associated PD often have an earlier onset and faster progression than idiopathic PD. Previously, we modeled GBA1-associated PD by crossing gba haploinsufficient mice with mice overexpressing a human mutant α-synuclein transgene (SNCA^A53T), observing an earlier demise, shorter life span and faster symptom progression, although behavioral testing was not performed. To assess whether gba^+/−//SNCA^A53T mice exhibit a prodromal behavioral phenotype, we studied three cardinal PD features: olfactory discrimination, memory dysfunction, and motor function. The longitudinal performance of gba^+/−//SNCA^A53T (n = 8), SNCA^A53T (n = 9), gba^+/− (n = 10) and wildtype (n = 6) mice was evaluated between ages 8 and 23 months using the buried pellet test, novel object recognition test and the beam walk. Fifteen-month-old gba^+/−//SNCA^A53T mice showed more olfactory and motor deficits than wildtype mice. However, differences between gba^+/−//SNCA^A53T and SNCA^A53T mice generally did not reach statistical significance, possibly due to small sample sizes. Furthermore, while gba haploinsufficiency leads to a more rapid demise, this might not result in an earlier prodromal stage, and other factors, including aging, oxidative stress and epigenetics, may contribute to the more fulminant disease course.     

First-Generation Antipsychotic Haloperidol Alters the Functionality of the Late Endosomal/Lysosomal Compartment in Vitro

First- and second-generation antipsychotics (FGAs and SGAs, respectively), have the ability to inhibit cholesterol biosynthesis and also to interrupt the intracellular cholesterol trafficking, interfering with low-density lipoprotein (LDL)-derived cholesterol egress from late endosomes/lysosomes. In the present work, we examined the effects of FGA haloperidol on the functionality of late endosomes/lysosomes in vitro. In HepG2 hepatocarcinoma cells incubated in the presence of 1,1′-dioctadecyl-3,3,3,3′-tetramethylindocarbocyanineperchlorate (DiI)-LDL, treatment with haloperidol caused the enlargement of organelles positive for late endosome markers lysosome-associated membrane protein 2 (LAMP-2) and LBPA (lysobisphosphatidic acid), which also showed increased content of both free-cholesterol and DiI derived from LDL. This indicates the accumulation of LDL-lipids in the late endosomal/lysosomal compartment caused by haloperidol. In contrast, LDL traffic through early endosomes and the Golgi apparatus appeared to be unaffected by the antipsychotic as the distribution of both early endosome antigen 1 (EEA1) and coatomer subunit β (β-COP) were not perturbed. Notably, treatment with haloperidol significantly increased the lysosomal pH and decreased the activities of lysosomal protease and β-d-galactosidase in a dose-dependent manner. We conclude that the alkalinization of the lysosomes’ internal milieu induced by haloperidol affects lysosomal functionality.     

An Engineered sgsh Mutant Zebrafish Recapitulates Molecular and Behavioural Pathobiology of Sanfilippo Syndrome A/MPS IIIA

Mucopolysaccharidosis IIIA (MPS IIIA, Sanfilippo syndrome type A), a paediatric neurological lysosomal storage disease, is caused by impaired function of the enzyme N-sulfoglucosamine sulfohydrolase (SGSH) resulting in impaired catabolism of heparan sulfate glycosaminoglycan (HS GAG) and its accumulation in tissues. MPS IIIA represents a significant proportion of childhood dementias. This condition generally leads to patient death in the teenage years, yet no effective therapy exists for MPS IIIA and a complete understanding of the mechanisms of MPS IIIA pathogenesis is lacking. Here, we employ targeted CRISPR/Cas9 mutagenesis to generate a model of MPS IIIA in the zebrafish, a model organism with strong genetic tractability and amenity for high-throughput screening. The sgsh^Δex5−6 zebrafish mutant exhibits a complete absence of Sgsh enzymatic activity, leading to progressive accumulation of HS degradation products with age. sgsh^Δex5−6 zebrafish faithfully recapitulate diverse CNS-specific features of MPS IIIA, including neuronal lysosomal overabundance, complex behavioural phenotypes, and profound, lifelong neuroinflammation. We further demonstrate that neuroinflammation in sgsh^Δex5−6 zebrafish is largely dependent on interleukin-1β and can be attenuated via the pharmacological inhibition of Caspase-1, which partially rescues behavioural abnormalities in sgsh^Δex5−6 mutant larvae in a context-dependent manner. We expect the sgsh^Δex5−6 zebrafish mutant to be a valuable resource in gaining a better understanding of MPS IIIA pathobiology towards the development of timely and effective therapeutic interventions.     

Pathophysiological In Vitro Profile of Neuronal Differentiated Cells Derived from Niemann-Pick Disease Type C2 Patient-Specific iPSCs Carrying the NPC2 Mutations c.58G>T/c.140G>T

Niemann-Pick type C2 (NP-C2) disease is a rare hereditary disease caused by mutations in the NPC2 gene. NPC2 is a small, soluble protein consisting of 151 amino acids, primarily expressed in late endosomes and lysosomes (LE/LY). Together with NPC1, a transmembrane protein found in these organelles, NPC2 accomplishes the exclusion of cholesterol; thus, both proteins are essential to maintain cellular cholesterol homeostasis. Consequently, mutations in the NPC2 or NPC1 gene result in pathophysiological accumulation of cholesterol and sphingolipids in LE/LY. The vast majority of Niemann-Pick type C disease patients, 95%, suffer from a mutation of NPC1, and only 5% display a mutation of NPC2. The biochemical phenotype of NP-C1 and NP-C2 appears to be indistinguishable, and both diseases share several commonalities in the clinical manifestation. Studies of the pathological mechanisms underlying NP-C2 are mostly based on NP-C2 animal models and NP-C2 patient-derived fibroblasts. Recently, we established induced pluripotent stem cells (iPSCs), derived from a donor carrying the NPC2 mutations c.58G>T/c.140G>T. Here, we present a profile of pathophysiological in vitro features, shared by NP-C1 and NP-C2, of neural differentiated cells obtained from the patient specific iPSCs. Profiling comprised a determination of the NPC2 protein level, detection of cholesterol accumulation by filipin staining, analysis of oxidative stress, and determination of autophagy. As expected, the NPC2-deficient cells displayed a significantly reduced amount of NPC2 protein, and, accordingly, we observed a significantly increased amount of cholesterol. Most notably, NPC2-deficient cells displayed only a slight increase of reactive oxygen species (ROS), suggesting that they do not suffer from oxidative stress and express catalase at a high level. As a site note, comparable NPC1-deficient cells suffer from a lack of catalase and display an increased level of ROS. In summary, this cell line provides a valuable tool to gain deeper understanding, not only of the pathogenic mechanism of NP-C2, but also of NP-C1.     

Glucosylceramide Mimics: Highly Potent GCase Inhibitors and Selective Pharmacological Chaperones for Mutations Associated with Types 1 and 2 Gaucher Disease

A series of iminoxylitol derivatives carrying a C‐linked di‐O‐acyl or di‐O‐alkyl glyceryl substituent were prepared and characterized. All of these compounds, which were designed as glucosylceramide (GlcCer) mimics, were nanomolar inhibitors of lysosomal β‐glucosidase (glucocerebrosidase, GCase). Two of these pseudoglycolipids were further evaluated for their ability to enhance the activity of mutant GCase in human Gaucher cells. Although the di‐O‐hexyl ether was surprisingly devoid of chaperoning activity on both N370S and L444P GCases, the di‐O‐decanoyl ester was a potent chaperone of the L444P hydrolase, capable of increasing the residual activity of the enzyme by a factor of two at a very low concentration (50 nM ); such a significant effect on the L444P mutation in human fibroblasts has not yet been observed. In heat‐stress studies, the diether was found to be much more effective in stabilizing the wild‐type enzyme than the diester. Four representative pseudoglycolipids were also assayed as inhibitors of GlcCer synthase, because such compounds could find use in the substrate reduction therapy approach to treat lysosomal storage diseases, but these compounds revealed only moderate activity. As efficient pharmacological chaperones, new structures such as the di‐C₁₀‐ester constitute leads for the development of therapeutic agents for types 2 and 3 Gaucher disease, the most severe neuronopathic forms of this lysosomal disease.     

Melatonin as a Signaling Molecule for Metabolism Regulation in Response to Hypoxia in the Crab Neohelice granulata

Melatonin has been identified in a variety of crustacean species, but its function is not as well understood as in vertebrates. The present study investigates whether melatonin has an effect on crustacean hyperglycemic hormone (CHH) gene expression, oxygen consumption (VO₂) and circulating glucose and lactate levels, in response to different dissolved-oxygen concentrations, in the crab Neohelice granulata, as well as whether these possible effects are eyestalk- or receptor-dependent. Melatonin decreased CHH expression in crabs exposed for 45 min to 6 (2, 200 or 20,000 pmol·crab⁻¹) or 2 mgO₂·L⁻¹ (200 pmol·crab⁻¹). Since luzindole (200 nmol·crab⁻¹) did not significantly (p > 0.05) alter the melatonin effect, its action does not seem to be mediated by vertebrate-typical MT1 and MT2 receptors. Melatonin (200 pmol·crab⁻¹) increased the levels of glucose and lactate in crabs exposed to 6 mgO₂·L⁻¹, and luzindole (200 nmol·crab⁻¹) decreased this effect, indicating that melatonin receptors are involved in hyperglycemia and lactemia. Melatonin showed no effect on VO₂. Interestingly, in vitro incubation of eyestalk ganglia for 45 min at 0.7 mgO₂·L⁻¹ significantly (p < 0.05) increased melatonin production in this organ. In addition, injections of melatonin significantly increased the levels of circulating melatonin in crabs exposed for 45 min to 6 (200 or 20,000 pmol·crab⁻¹), 2 (200 and 20,000 pmol·crab⁻¹) and 0.7 (200 or 20,000 pmol·crab⁻¹) mgO₂·L⁻¹. Therefore, melatonin seems to have an effect on the metabolism of N. granulata. This molecule inhibited the gene expression of CHH and caused an eyestalk- and receptor-dependent hyperglycemia, which suggests that melatonin may have a signaling role in metabolic regulation in this crab.     

Xanthine Oxidoreductase-Mediated Superoxide Production Is Not Involved in the Age-Related Pathologies in Sod1-Deficient Mice

Reactive oxygen species (ROS) metabolism is regulated by the oxygen-mediated enzyme reaction and antioxidant mechanism within cells under physiological conditions. Xanthine oxidoreductase (XOR) exhibits two inter-convertible forms (xanthine oxidase (XO) and xanthine dehydrogenase (XDH)), depending on the substrates. XO uses oxygen as a substrate and generates superoxide (O₂^•−) in the catalytic pathway of hypoxanthine. We previously showed that superoxide dismutase 1 (SOD1) loss induced various aging-like pathologies via oxidative damage due to the accumulation of O₂^•− in mice. However, the pathological contribution of XO-derived O₂^•− production to aging-like tissue damage induced by SOD1 loss remains unclear. To investigate the pathological significance of O₂^•− derived from XOR in Sod1^−/− mice, we generated Sod1-null and XO-type- or XDH-type-knock-in (KI) double-mutant mice. Neither XO-type- nor XDH-type KI mutants altered aging-like phenotypes, such as anemia, fatty liver, muscle atrophy, and bone loss, in Sod1^−/− mice. Furthermore, allopurinol, an XO inhibitor, or apocynin, a nicotinamide adenine dinucleotide phosphate oxidase (NOX) inhibitor, failed to improve aging-like tissue degeneration and ROS accumulation in Sod1^−/− mice. These results showed that XOR-mediated O₂^•− production is relatively uninvolved in the age-related pathologies in Sod1^−/− mice.     

Genetic Deletion of NOD1 Prevents Cardiac Ca2+ Mishandling Induced by Experimental Chronic Kidney Disease

Risk of cardiovascular disease (CVD) increases considerably as renal function declines in chronic kidney disease (CKD). Nucleotide-binding oligomerization domain-containing protein 1 (NOD1) has emerged as a novel innate immune receptor involved in both CVD and CKD. Following activation, NOD1 undergoes a conformational change that allows the activation of the receptor-interacting serine/threonine protein kinase 2 (RIP2), promoting an inflammatory response. We evaluated whether the genetic deficiency of Nod1 or Rip2 in mice could prevent cardiac Ca²⁺ mishandling induced by sixth nephrectomy (Nx), a model of CKD. We examined intracellular Ca²⁺ dynamics in cardiomyocytes from Wild-type (Wt), Nod1^−/− and Rip2^−/− sham-operated or nephrectomized mice. Compared with Wt cardiomyocytes, Wt-Nx cells showed an impairment in the properties and kinetics of the intracellular Ca²⁺ transients, a reduction in both cell shortening and sarcoplasmic reticulum Ca²⁺ load, together with an increase in diastolic Ca²⁺ leak. Cardiomyocytes from Nod1^−/−-Nx and Rip2^−/−-Nx mice showed a significant amelioration in Ca²⁺ mishandling without modifying the kidney impairment induced by Nx. In conclusion, Nod1 and Rip2 deficiency prevents the intracellular Ca²⁺ mishandling induced by experimental CKD, unveiling new innate immune targets for the development of innovative therapeutic strategies to reduce cardiac complications in patients with CKD.     

The Antihypertensive Drug Telmisartan Protects Oligodendrocytes from Cholesterol Accumulation and Promotes Differentiation by a PPAR-γ-Mediated Mechanism

Our previous studies have demonstrated that specific peroxisome proliferator-activated receptor-γ (PPAR-γ) agonists play a fundamental role in oligodendrocyte progenitor (OP) differentiation, protecting them against oxidative and inflammatory damage. The antihypertensive drug Telmisartan (TLM) was shown to act as a PPAR-γ modulator. This study investigates the TLM effect on OP differentiation and validates its capability to restore damage in a pharmacological model of Niemann-Pick type C (NPC) disease through a PPAR-γ-mediated mechanism. For the first time in purified OPs, we demonstrate that TLM-induced PPAR-γ activation downregulates the type 1 angiotensin II receptor (AT1), the level of which naturally decreases during differentiation. Like other PPAR-γ agonists, we show that TLM promotes peroxisomal proliferation and promotes OP differentiation. Furthermore, TLM can offset the OP maturation arrest induced by a lysosomal cholesterol transport inhibitor (U18666A), which reproduces an NPC1-like phenotype. In the NPC1 model, TLM also reduces cholesterol accumulation within peroxisomal and lysosomal compartments and the contacts between lysosomes and peroxisomes, revealing that TLM can regulate intracellular cholesterol transport, crucial for myelin formation. Altogether, these data indicate a new potential use of TLM in hypomyelination pathologies such as NPC1, underlining the possible repositioning of the drug already used in other pathologies.     

The Dipeptidyl Peptidase-4 Inhibitor Sitagliptin Protects against Dyslipidemia-Related Kidney Injury in Apolipoprotein E Knockout Mice

The goal of this study was to investigate the possible protective effects of sitagliptin against dyslipidemia-related kidney injury in apolipoprotein E knockout (apoE^−/−) mice. Eight-week-old male apoE^−/− mice were randomized to receive either a high fat diet (HFD, apoE^−/− group) or HFD mixed with sitagliptin (sita + apoE^−/− group) for 16 weeks. A control group of age- and gender-matched C57BL/6J mice were fed a HFD. The apoE^−/− group exhibited increases in body weight and serum lipid levels in addition to high-density lipoprotein, and increases in 24-h urinary 8-hydroxy-2-deoxyguanosine and albuminuria excretion. Decreased insulin sensitivity was also observed in the apoE^−/− group. These mice additionally contained enlargements of the glomerular mesangial matrix area, lipid deposition area, and renal interstitium collagen area. The apoE^−/− group also demonstrated down-regulation of phosphorylated AMP-activated protein kinase (AMPK), increases in renal mRNA expression of transforming growth factor-beta 1 (TGF-β1) and fibronectin (FN), and increased protein expression of Akt, TGF-β1, FN and p38/ERK mitogen-activated protein kinase (MAPK). Sitagliptin treatment successfully ameliorated all the deleterious effects of dyslipidemia tested. To our knowledge, this is the first time that sitagliptin has been shown to reverse the renal dysfunction and structural damage induced by dyslipidemia in apoE^−/− mice. Our results suggest that the renoprotective mechanism of sitagliptin may be due to a reduction in Akt levels, a restoration of AMPK activity, and inhibition of TGF-β1, FN, and p38/ERK MAPK signaling pathways.