👤 Attila D Kovács

Fasting triggers complex physiological and neuroimmune adaptations, yet its impact on hypothalamic microglia and the underlying regulatory role of glucocorticoids remains incompletely understood. The present study focused on fasting-induced systemic changes and cellular adaptations seen in the hypothalamus where components of metabolic- hormonal- and immune regulations are integrated. Adult male microglia reporter (CX3CR1 Overnight fasting resulted in a decrease in energy expenditure and respiratory exchange ratio (RER) indicating conservation of energy and a metabolic shift towards utilization of fatty acids as alternative energy source. Fasting increased hypothalamic expression of orexigenic neuropeptides and mRNA levels of Pdk4, Glut1, and Mct2 genes, in line with metabolic compensation. Upregulation of hypothalamic Crh and increased plasma concentration of corticosterone indicated sustained activation of the HPA axis. Importantly, fasting promoted an anti-inflammatory milieu in the hypothalamus characterized by elevated Il-4, Il-10 and IkBα genes without significant activation of pro-inflammatory cytokines (e.g., Il-1β, Il-6, Tnfα). Morphological analysis revealed region-specific changes in microglia number and branching complexity, particularly in hypothalamic regions directly exposed to blood-borne signals. Functional profiling showed increased microglial expression of IkBα and decreased pIkBα, indicating suppressed NFkB signaling. Adrenalectomy (1 week) and acute pharmacological inhibition of corticosterone synthesis (methyrapone) revealed that fasting-induced anti-inflammatory and metabolic gene expression, as well as microglial plasticity were largely glucocorticoid dependent. Hypothalamic expression of fasting-related neuropeptides (Npy, Agrp) and genes, related to the metabolic shift (Pdk4, Glut-1, Mct2, Angptl4) as well as some immune-related genes (Il10, Iba1) was dependent on presence of the adrenal gland or fasting-induced elevation of corticosterone. These findings highlight short term fasting as a potent modulator of hypothalamic immune-metabolic crosstalk and reveal critical role of adrenal glucocorticoids in orchestrating microglial responses to energetic challenges. The results have potential implications for therapeutic interventions targeting metabolic and inflammatory disorders. Show less

Obesity is a global health challenge that can lead to various complications, such as metabolic syndrome, diabetes mellitus, and cardiovascular diseases. Heat shock proteins are evolutionarily conserved chaperones that help maintain cellular protein homeostasis. Their expression is dysregulated in various chronic diseases, including diabetes mellitus and hyperlipidemia, and they also regulate inflammatory processes. Therefore, the present study aimed to investigate the effects of a small heat shock protein, HSPB1, on the comorbidities and complications of obesity in a transgenic mouse model. Male and female human apolipoprotein B-100 (APOB) transgenic mice fed with a high-fat diet (HFD) from months 3-10 of age were used as a model of metabolic syndrome (MetS). To study whether HSPB1 influences the development of MetS, APOB animals were crossed with HSPB1-overexpressing mice. Age and sex-matched wild-type and human HSPB1-overexpressing mice were used as controls. Changes in cardiac morphology and function were assessed by transthoracic echocardiography at month 9. At month 10, serum triglyceride and cholesterol concentrations were determined by enzymatic colorimetric assays. Pathological changes in the liver were studied on hematoxylin-eosin-stained sections. Expression levels of genes involved in inflammation and metabolism were measured by quantitative real-time polymerase chain reaction in the liver, left ventricle, and visceral white adipose tissue (vWAT). The body weight and serum LDL-cholesterol levels were significantly higher in the APOB animals than in the wild-type mice in both sexes. Notably, HSPB1 overexpression further increased weight gain in female APOB animals. Conversely, in APOB males, HSPB1 overexpression decreased LDL-cholesterol levels without significantly affecting body weight. Furthermore, in APOB females, HSPB1 overexpression elevated Fgf-21 expression in the vWAT, restored Lpl levels, and reduced the expression of several cytokines in the liver. APOB males developed left ventricular hypertrophy (LVH) with diastolic dysfunction. HSPB1 overexpression induced LVH without cardiac dysfunction in the wild-type animals. Both sexes of APOB animals developed MetS. APOB males presented LVH with preserved ejection fraction (EF); however, APOB females showed enlarged left ventricular end-systolic volume (LVESV). In APOB animals, HSPB1 overexpression exerted a sex-dependent influence on obesity-related alterations, including weight gain, hypercholesterolemia, and hepatic and vWAT gene expression. Show less

Batten disease is a group of mostly pediatric neurodegenerative lysosomal storage disorders caused by mutations in the CLN1-14 genes. We have recently shown that acidified drinking water attenuated neuropathological changes and improved motor function in the Cln1 Show less

Psoriatic patients have considerably higher odds of being obese compared with the general population; however, the exact pathophysiological link between psoriasis and obesity needs to be elucidated. To investigate the association of psoriasis with established obesity-related gene variants, we conducted a population-based case-control study including 3541 subjects (574 psoriasis cases and 2967 controls from the general Hungarian population). Genotyping of 20 SNPs at ADIPOQ, BDNF, FTO, GNPDA2, LEPR, MC4R, NEGR1, NPY, PPARG, TMEM18, and UCP2 were determined, and differences in genotype and allele distributions were investigated. Multiple logistic regression analyses were implemented. Analysis revealed an association between the G allele of the rs1137101 polymorphism (LEPR gene) and obesity risk (OR: 3.30 (1.45; 7.50), Our results suggest that in psoriatic patients, there are prominent differences in the causes of obesity that should be accounted for, including not only environmental factors but also patient characteristics, such as the time of disease onset as well as genetic factors. Show less

Activation of the type I angiotensin receptor (AT1-R) in vascular smooth muscle cells (VSMCs) plays a crucial role in the regulation of blood pressure; however, it is also responsible for the development of pathological conditions such as vascular remodeling, hypertension and atherosclerosis. Stimulation of the VSMC by angiotensin II (AngII) promotes a broad variety of biological effects, including gene expression changes. In this paper, we have taken an integrated approach in which an analysis of AngII-induced gene expression changes has been combined with the use of small-molecule inhibitors and lentiviral-based gene silencing, to characterize the mechanism of signal transduction in response to AngII stimulation in primary rat VSMCs. We carried out Affymetrix GeneChip experiments to analyze the effects of AngII stimulation on gene expression; several genes, including Show less

CLN3 Batten disease (CLN3 disease) is a pediatric lysosomal storage disorder that presents with progressive blindness, motor and cognitive decline, seizures, and premature death. CLN3 disease results from mutations in CLN3 with the most prevalent mutation, a 966 bp deletion spanning exons 7-8, affecting ~ 75% of patients. Mouse models with complete Cln3 deletion or Cln3 Show less

Among Neuronal Ceroid Lipofuscinoses (NCLs), which are childhood fatal neurodegenerative disorders, the juvenile onset form (JNCL) is the most common. JNCL is caused by recessive mutations in the CLN3 gene. CLN3 encodes a lysosomal/endosomal transmembrane protein but its precise function is not completely known. We have previously reported that in baby hamster kidney (BHK) cells stably expressing myc-tagged human CLN3 (myc-CLN3), hyperosmotic conditions drastically increased myc-CLN3 mRNA and protein expression. In the present study, we analyzed the consequences of hyperosmolarity, and increased CLN3 expression on cathepsin D (CTSD) activity and prosaposin processing using BHK cells transiently or stably expressing myc-CLN3. We found that hyperosmolarity increased lysotracker staining of lysosomes, and elevated the levels of myc-CLN3 and lysosome-associated membrane protein-1 (LAMP1). Hyperosmolarity, independently of the expression level of myc-CLN3, decreased the levels of PSAP and saposin D, which are protein cofactors in sphingolipid metabolism. The lysosomal enzyme cathepsin D (CTSD) mediates the proteolytic cleavage of PSAP precursor into saposins A-D. Myc-CLN3 colocalized with CTSD and activity of CTSD decreased as myc-CLN3 expression increased, and clearly decreased under hyperosmotic conditions. Nevertheless, levels of CTSD measured by Western blotting were not altered under any studied condition. Our results suggest a direct involvement of CLN3 in the regulation of CTSD activity. J. Cell. Biochem. 118: 3883-3890, 2017. © 2017 Wiley Periodicals, Inc. Show less

Mutations of the CLN3 gene lead to juvenile neuronal ceroid lipofuscinosis (JNCL), an autosomal recessive lysosomal storage disorder that causes progressive neurodegeneration in children and adolescents. There is evidence of immune system involvement in pathology that has been only minimally investigated. We characterized bone marrow stem cell-derived antigen presenting cells (APCs), peritoneal macrophages, and leukocytes from spleen and blood, harvested from the Cln3(-/-) mouse model of JNCL. We detected dramatically elevated CD11c surface levels and increased total CD11c protein in Cln3(-/-) cell samples compared to wild type. This phenotype was specific to APCs and also to a loss of CLN3, as surface levels did not differ from wild type in other leukocyte subtypes nor in cells from two other NCL mouse models. Subcellularly, CD11c was localized to lipid rafts, indicating that perturbation of surface levels is attributable to derangement of raft dynamics, which has previously been shown in Cln3 mutant cells. Interrogation of APC function revealed that Cln3(-/-) cells have increased adhesiveness to CD11c ligands as well as an abnormal secretory pattern that closely mimics what has been previously reported for Cln3 mutant microglia. Our results show that CLN3 deficiency alters APCs, which can be a major contributor to the autoimmune response in JNCL. Show less

Mutations in the CLN3 gene cause a fatal neurodegenerative disorder, juvenile CLN3 disease. Exploring the cause of the motor coordination deficit in the Cln3(-/-) mouse model of the disease we have previously found that attenuation of AMPA receptor activity in 1-month-old Cln3(-/-) mice significantly improves their motor coordination [20]. To elucidate the mechanism of the abnormally increased AMPA receptor function in Cln3(-/-) mice, we examined the surface expression of AMPA receptors using surface cross-linking in brain slices from 1-month-old wild type (WT) and Cln3(-/-) mice. In surface cross-linked brain samples, Western blotting for AMPA receptor subunits revealed significantly increased surface levels of GluA1 and GluA2 in the cerebellum, and of GluA2 in the cortex and striatum of Cln3(-/-) mice as compared to WT mice. Expression levels of the GluA4 subunit were similar in the cerebellum of WT and Cln3(-/-) mice. While intracellular GluA1 levels in the WT and Cln3(-/-) cerebellum or cortex were similar, the intracellular expression of GluA1 in the Cln3(-/-) striatum was decreased to 56% of the WT level. Our results show a prominent increase in AMPA receptor surface expression in the brain of Cln3(-/-) mice and suggest that CLN3 is involved in the regulation of AMPA receptor surface expression. Show less

Mutations in the CLN3 gene cause a fatal neurodegenerative disorder: juvenile CLN3 disease, also known as juvenile Batten disease. The two most commonly utilized mouse models of juvenile CLN3 disease are Cln3-knockout (Cln3(-/-)) and Cln3(Δex7/8)-knock-in mice, the latter mimicking the most frequent disease-causing human mutation. To determine which mouse model has the most pronounced neurological phenotypes that can be used as outcome measures for therapeutic studies, we compared the exploratory activity, motor function and depressive-like behavior of 1-, 3- and 6-month-old Cln3(-/-) and Cln3(Δex7/8)-knock-in mice on two different genetic backgrounds (129S6/SvEv and C57BL/6J). Although, in many cases, the behavior of Cln3(-/-) and Cln3(Δex7/8) mice was similar, we found genetic-background-, gender- and age-dependent differences between the two mouse models. We also observed large differences in the behavior of the 129S6/SvEv and C57BL/6J wild-type strains, which highlights the strong influence that genetic background can have on phenotype. Based on our results, Cln3(-/-) male mice on the 129S6/SvEv genetic background are the most appropriate candidates for therapeutic studies. They exhibit motor deficits at 1 and 6 months of age in the vertical pole test, and they were the only mice to show impaired motor coordination in the rotarod test at both 3 and 6 months. Cln3(-/-) males on the C57BL/6J background and Cln3(Δex7/8) males on the 129S6/SvEv background also provide good outcome measures for therapeutic interventions. Cln3(-/-) (C57BL/6J) males had serious difficulties in climbing down (at 1 and 6 months) and turning downward on (at 1, 3 and 6 months) the vertical pole, whereas Cln3(Δex7/8) (129S6/SvEv) males climbed down the vertical pole drastically slower than wild-type males at 3 and 6 months of age. Our study demonstrates the importance of testing mouse models on different genetic backgrounds and comparing males and females in order to find the most appropriate disease model for therapeutic studies. Show less

The fatal, primarily childhood neurodegenerative disorders, neuronal ceroid lipofuscinoses (NCLs), are currently associated with mutations in 13 genes. The protein products of these genes (CLN1 to CLN14) differ in their function and their intracellular localization. NCL-associated proteins have been localized mostly in lysosomes (CLN1, CLN2, CLN3, CLN5, CLN7, CLN10, CLN12 and CLN13) but also in the Endoplasmic Reticulum (CLN6 and CLN8), or in the cytosol associated to vesicular membranes (CLN4 and CLN14). Some of them such as CLN1 (palmitoyl protein thioesterase 1), CLN2 (tripeptidyl-peptidase 1), CLN5, CLN10 (cathepsin D), and CLN13 (cathepsin F), are lysosomal soluble proteins; others like CLN3, CLN7, and CLN12, have been proposed to be lysosomal transmembrane proteins. In this review, we give our views and attempt to summarize the proposed and confirmed functions of each NCL protein and describe and discuss research results published since the last review on NCL proteins. This article is part of a Special Issue entitled: "Current Research on the Neuronal Ceroid Lipofuscinoses (Batten Disease)". Show less

Juvenile CLN3 disease (formerly known as juvenile neuronal ceroid lipofuscinosis) is a fatal childhood neurodegenerative disorder caused by mutations in the CLN3 gene. CLN3 encodes a putative lysosomal transmembrane protein with unknown function. Previous cell culture studies using CLN3-overexpressing vectors and/or anti-CLN3 antibodies with questionable specificity have also localized CLN3 in cellular structures other than lysosomes. Osmoregulation of the mouse Cln3 mRNA level in kidney cells was recently reported. To clarify the subcellular localization of the CLN3 protein and to investigate if human CLN3 expression and localization is affected by osmotic changes we generated a stably transfected BHK (baby hamster kidney) cell line that expresses a moderate level of myc-tagged human CLN3 under the control of the human ubiquitin C promoter. Hyperosmolarity (800 mOsm), achieved by either NaCl/urea or sucrose, dramatically increased the mRNA and protein levels of CLN3 as determined by quantitative real-time PCR and Western blotting. Under isotonic conditions (300 mOsm), human CLN3 was found in a punctate vesicular pattern surrounding the nucleus with prominent Golgi and lysosomal localizations. CLN3-positive early endosomes, late endosomes and cholesterol/sphingolipid-enriched plasma membrane microdomain caveolae were also observed. Increasing the osmolarity of the culture medium to 800 mOsm extended CLN3 distribution away from the perinuclear region and enhanced the lysosomal localization of CLN3. Our results reveal that CLN3 has multiple subcellular localizations within the cell, which, together with its expression, prominently change following osmotic stress. These data suggest that CLN3 is involved in the response and adaptation to cellular stress. Show less

Currently there is no treatment for juvenile Batten disease, a fatal childhood neurodegenerative disorder caused by mutations in the CLN3 gene. The Cln3-knockout (Cln3(Δex1-6)) mouse model recapitulates several features of the human disorder. Cln3(Δex1-6) mice, similarly to juvenile Batten disease patients, have a motor coordination deficit detectable as early as postnatal day 14. Previous studies demonstrated that acute attenuation of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA)-type glutamate receptor activity by the non-competitive AMPA antagonist, EGIS-8332, in both 1- and 6-7-month-old Cln3(Δex1-6) mice results in improvement in motor coordination. Here we show that acute inhibition of N-methyl-D-aspartate (NMDA)-type glutamate receptors by memantine (1 and 5 mg/kg i.p.) had no effect on the impaired motor coordination of one-month-old Cln3(Δex1-6) mice. At a later stage of the disease, in 6-7-month-old Cln3(Δex1-6) mice, memantine induced a delayed but extended (8 days) improvement of motor skills similarly to that observed previously with EGIS-8332 treatment. An age-dependent therapeutic effect of memantine implies that the pathomechanism in juvenile Batten disease changes during disease progression. In contrast to acute treatment, repeated administration of memantine or EGIS-8332 (1 mg/kg, once a week for 4 weeks) to 6-month-old Cln3(Δex1-6) mice had no beneficial effect on motor coordination. Moreover, repeated treatments did not impact microglial activation or the survival of vulnerable neuron populations. Memantine did not affect astrocytosis in the cortex. EGIS-8332, however, decreased astrocytic activation in the somatosensory barrelfield cortex. Acute inhibition of NMDA receptors can induce a prolonged therapeutic effect, identifying NMDA receptors as a new therapeutic target for juvenile Batten disease. Show less

The juvenile onset form of neuronal ceroid lipofuscinoses (JNCL) is a recessively inherited lysosomal storage disorder characterized by progressive neurodegeneration. JNCL results from mutations in the CLN3 gene that encodes a lysosomal membrane protein with unknown function. Utilizing a Cln3-knock-out mouse model of JNCL that was created on the 129S6/SvEv genetic background, we have previously demonstrated that CLN3-deficient cerebellar granule cells (CGCs) have a selectively increased sensitivity to AMPA-type glutamate receptor-mediated toxicity. Our recent findings that CGCs from 129S6/SvEv and C57BL/6J wild type (WT) mice have significant differences in glutamate receptor expression and in excitotoxic vulnerability indicated that the genetic background possibly have a strong influence on how glutamate receptor function is dysregulated in CLN3-deficient neurons. Indeed, here we show that in the Cln3(Δex7/8)-knock-in mouse model, that is on the C57BL/6J genetic background, mimics the most frequent mutation observed in JNCL patients and considered a null mutant, the sensitivity of CGCs to both AMPA- and NMDA-type glutamate receptor overactivations is altered. Cultured wild type and Cln3(Δex7/8) CGCs were equally sensitive to AMPA toxicity after 2 or 3 weeks in vitro, whereas the subunit-selective AMPA receptor agonist, CPW-399, induced significantly more cell death in mature, 3-week-old Cln3(Δex7/8) cultures. NMDA receptor-mediated toxicity changed during in vitro development: Cln3(Δex7/8) CGCs were less sensitive to high concentration of NMDA after 2 weeks in culture but became more vulnerable than their WT counterparts after 3 weeks in vitro. Abnormally altered glutamate receptor function in the cerebellum may result in motor deficits, and we confirmed that 7-week-old Cln3(Δex7/8) mice, similarly to Cln3-knock-out mice, have a motor coordination deficit as measured by an accelerating rotarod. Our results demonstrate altered glutamate receptor function in Cln3(Δex7/8) neurons and suggest that both AMPA and NMDA receptors are potential therapeutic targets in JNCL. Show less

Mutations in the CLN3 gene cause juvenile Batten disease, a fatal pediatric neurodegenerative disorder. The Cln3-knockout (Cln3(Δex1-6)) mouse model of the disease displays many pathological characteristics of the human disorder including a deficit in motor coordination. We have previously found that attenuation of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA)-type glutamate receptor activity in one-month-old Cln3(Δex1-6) mice resulted in an immediate improvement of their motor skills. Here we show that at a later stage of the disease, in 6-7-month-old Cln3(Δex1-6) mice, acute inhibition of AMPA receptors by a single intraperitoneal injection (1mg/kg) of the non-competitive AMPA antagonist, EGIS-8332, does not have an immediate effect. Instead, it induces a delayed but prolonged improvement of motor skills. Four days after the injection of the AMPA antagonist, Cln3(Δex1-6) mice reached the same motor skill level as their wild type (WT) counterparts, an improvement that persisted for an additional four days. EGIS-8332 was rapidly eliminated from the brain as measured by HPLC-MS/MS. Histological analysis performed 8 days after the drug administration revealed that EGIS-8332 did not have any impact upon glial activation or the survival of vulnerable neuron populations in 7-month-old Cln3(Δex1-6) mice. We propose that temporary inhibition of AMPA receptors can induce a prolonged correction of the pre-existing abnormal glutamatergic neurotransmission in vivo for juvenile Batten disease. Show less

Juvenile Batten disease, caused by mutations in the CLN3 gene, is a fatal, incurable neurodegenerative disorder in children. The Cln3-loss-of-function (Cln3(Deltaex1-6)) mouse model of the disease exhibits many characteristic pathological features of the human disorder including a deficit in motor skills. Our recent findings [Kovács, A.D., Weimer, J.M., Pearce, D.A., 2006. Selectively increased sensitivity of cerebellar granule cells to AMPA receptor-mediated excitotoxicity in a mouse model of Batten disease. Neurobiol. Dis. 22, 575-585] suggested that the neurological deficit in the Cln3(Deltaex1-6) mouse model of the disease might result from an abnormally increased AMPA receptor activity in the cerebellum. Therefore, we tested if administration of low doses of an AMPA receptor antagonist, that attenuate AMPA receptor function but avoid a toxic, complete blockade of the receptor, have beneficial effects in Cln3(Deltaex1-6) mice. Here we show that attenuation of AMPA receptor activity by a single intraperitoneal injection of the non-competitive AMPA antagonist, EGIS-8332 (1 mg/kg), significantly improves the motor skills of Cln3(Deltaex1-6) mice. Our results provide a new, promising therapeutic approach for juvenile Batten disease. Show less

Batten disease, a lysosomal storage disorder, is caused by mutations in the CLN3 gene. The Cln3-knockout (Cln3-/-) mouse model of the disease exhibits many characteristic pathological features of the human disorder. Here, we show that Cln3-/- mice, similarly to Batten disease patients, have a deficit in cerebellar motor coordination. To explore the possible cellular cause of this functional impairment, we compared the vulnerability of wild type (WT) and Cln3-/- cerebellar granule cell cultures to different toxic insults. We have found that cultured Cln3-/- cerebellar granule cells are selectively more vulnerable to AMPA-type glutamate receptor-mediated toxicity than their WT counterparts. This selective sensitivity was also observed in organotypic cerebellar slice cultures. Our results suggest that lack of the CLN3 protein has a significant influence on the function of AMPA receptors in cerebellar granule neurons, and that AMPA receptor dysregulation may be a major contributor to the cerebellar dysfunction in Batten disease. Show less