Kosara Smiljanic

Caloric Restriction Suppresses Microglial Activation and Prevents Neuroapoptosis Following Cortical Injury in Rats

Traumatic brain injury (TBI) is a widespread cause of death and a major source of adult disability. Subsequent pathological events occurring in the brain after TBI, referred to as secondary injury, continue to damage surrounding tissue resulting in substantial neuronal loss. One of the hallmarks of the secondary injury process is microglial activation resulting in increased cytokine production. Notwithstanding that recent studies demonstrated that caloric restriction (CR) lasting several months prior to an acute TBI exhibits neuroprotective properties, understanding how exactly CR influences secondary injury is still unclear. The goal of the present study was to examine whether CR (50% of daily food intake for 3 months) alleviates the effects of secondary injury on neuronal loss following cortical stab injury (CSI). To this end, we examined the effects of CR on the microglial activation, tumor necrosis factor-α (TNF-α) and caspase-3 expression in the ipsilateral (injured) cortex of the adult rats during the recovery period (from 2 to 28 days) after injury. Our results demonstrate that CR prior to CSI suppresses microglial activation, induction of TNF-α and caspase-3, as well as neurodegeneration following injury. These results indicate that CR strongly attenuates the effects of secondary injury, thus suggesting that CR may increase the successful outcome following TBI.

Behavioral and biochemical effects of various food-restriction regimens in the rats.

In this paper we describe the effects of six different food restriction (FR) regimens on amphetamine (AMPH)-induced locomotor and nonlocomotor activities in male rats. Changes in serum corticosterone (CORT), insulin and glucose levels were also examined. Each regimen was implemented through different daily food allowance (50%, 25% and 12.5% of the daily food intake, referred to as 50%, 75% and 87.5% FR groups, respectively) and by a specific feeding regimen - either every day (ED) or every other day (EOD). AMPH injection led to a significant increase of locomotor activity in all rats subjected to FR compared to ad libitum fed rats. A significant increase of nonlocomotor activity was observed only in the 75% FR and 87.5% FR groups. The serum CORT levels were significantly elevated and the serum insulin and glucose levels were significantly decreased in all of the FR groups in comparison to the AL rats. The results presented in this paper suggest that the ED regimens produced changes in motor activity and biochemical parameters, which were more-or-less dependent on the degree of FR. In contrast, the EOD regimens induced very similar changes irrespective of the degree of FR degree. Our data support the possible mechanistic roles of CORT and insulin in the effect of FR on locomotor activity, since the most pronounced increase of serum CORT and more pronounced decrease in serum insulin concentration was observed in the groups that also exhibited the highest locomotor activities.

Loss of Cathepsin B and L Leads to Lysosomal Dysfunction, NPC-Like Cholesterol Sequestration and Accumulation of the Key Alzheimer's Proteins.

Proper function of lysosomes is particularly important in neurons, as they cannot dilute accumulated toxic molecules and aggregates by cell division. Thus, impairment of lysosomal function plays an important role in neuronal degeneration and in the pathogenesis of numerous neurodegenerative diseases. In this work we analyzed how inhibition and/or loss of the major lysosomal proteases, the cysteine cathepsins B and L (CtsB/L), affects lysosomal function, cholesterol metabolism and degradation of the key Alzheimers disease (AD) proteins. Here, we show that cysteine CtsB/L, and not the aspartyl cathepsin D (CtsD), represent a major lysosomal protease(s) that control lysosomal function, intracellular cholesterol trafficking and AD-like amyloidogenic features. Intriguingly, accumulation of free cholesterol in late endosomes/lysosomes upon CtsB/L inhibition resembled a phenotype characteristic for the rare neurodegenerative disorder Niemann-Pick type C (NPC). CtsB/L inhibition and not the inhibition of CtsD led to lysosomal impairment assessed by decreased degradation of EGF receptor, enhanced LysoTracker staining and accumulation of several lysosomal proteins LC3II, NPC1 and NPC2. By measuring the levels of NPC1 and ABCA1, the two major cholesterol efflux proteins, we showed that CtsB/L inhibition or genetic depletion caused accumulation of the NPC1 in lysosomes and downregulation of ABCA1 protein levels and its expression. Furthermore, we revealed that CtsB/L are involved in degradation of the key Alzheimer’s proteins: amyloid-β peptides (Aβ) and C-terminal fragments of the amyloid precursor protein (APP) and in degradation of β-secretase (BACE1). Our results imply CtsB/L as major regulators of lysosomal function and demonstrate that CtsB/L may play an important role in intracellular cholesterol trafficking and in degradation of the key AD proteins. Our findings implicate that enhancing the activity or levels of CtsB/L could provide a promising and a common strategy for maintaining lysosomal function and for preventing and/or treating neurodegenerative diseases.

Induction of TNF-α signaling cascade in neonatal rat brain during propofol anesthesia

Propofol anesthesia can trigger pro‐ and anti‐apoptotic signaling pathways in the rat brain. In our previous work, we demonstrated that propofol causes widespread apoptotic neurodegeneration in 7‐postnatal‐day‐old (PND7) but not in PND14 rat neurons. The mechanism responsible for these opposing outcomes is unknown, apparently linked to the specific stage of brain development. The present study aims to elucidate the anti‐apoptotic process that is activated in the cortex and thalamus of PND14 Wistar rats during the first 48 h after the onset of propofol anesthesia. We showed that the expression of tumor necrosis factor‐α (TNF‐α) and several components of its pathway, TNFR1 and caspase‐8, was significantly increased in the cortex and thalamus. Nuclear factor kappa B (NF‐κB) p65 was downregulated in the cortex and upregulated in the thalamus. The expression of c‐Fos was upregulated only in the cortex, showing opposed profile compared to NF‐κB p65. Double immunofluorescence staining revealed the colocalization of NF‐κB p65 with neuronal marker (NeuN), but with predominantly cytoplasmic localization. Finally, X‐linked inhibitor of apoptosis protein (XIAP) was upregulated in both examined structures. Immunohistochemical staining with Iba‐1 revealed that the treatment did not induce changes in microglial morphology. Our results (i) reveal that the simultaneous activation of pro‐ and anti‐apoptotic signaling occurs after propofol anesthesia, and (ii) pinpoint the potential neuroprotective role of XIAP in anesthesia‐induced neurotoxicity.

Neonatal propofol anesthesia modifies activity-dependent processes and induces transient hyperlocomotor response to d-amphetamine during adolescence in rats.

This study examined the influence of propofol anesthesia on the expression of activity‐regulated molecules (BDNF and c‐Fos) and synaptic plasticity markers (synaptophysin, GAP‐43, drebrin) in the frontal cortex and thalamus of 7‐day‐old (P7) rats. Although these brain regions are the main targets of anesthetic action, they are contained in the cortico–striato–thalamo–cortical feedback loops, involved in naturally occurring and drug‐induced psychoses. Therefore, functional integrity of these loops was examined in adolescent and adult rats through d‐amphetamine‐induced hyperactivity. Propofol treatment (25 mg/kg) decreased exon‐specific and total BDNF mRNA expression in the frontal cortex and thalamus, in a time‐dependent manner. BDNF protein level was increased in the frontal cortex and decreased in the thalamus, which was accompanied by the change of phospho‐TrkB expression. Similarly to BDNF, the expression of c‐Fos was decreased in the frontal cortex while it was changed only at the protein level in the thalamus. Synaptic plasticity markers changed in a time‐ and region‐specific manner, indicating increased synaptogenesis in the frontal cortex and synapse elimination in the thalamus in P7 rats after the propofol anesthesia exposure. These early molecular changes were followed by time‐related, increased motor reaction to d‐amphetamine in adolescent, but not in adult rats. Our study revealed that exposure of immature brain to propofol anesthesia during the critical phase of development provoked immediate changes in activity‐dependent processes and synaptic adjustment, influencing brain capacity to integrate later developmental events and resulting in temporary altered response to acute psychotropic stimulation during adolescence.

Expression profiles of cholesterol metabolism-related genes are altered during development of experimental autoimmune encephalomyelitis in the rat spinal cord

Increased evidence suggests that dysregulation of cholesterol metabolism may be a key event contributing to progression of multiple sclerosis (MS). Using an experimental autoimmune encephalomyelitis (EAE) model of MS we revealed specific changes in the mRNA and protein expression of key molecules involved in the maintaining of cholesterol homeostasis in the rat spinal cord: 3-hydroxy-3-methylglutaryl-coenzyme-A reductase (HMGCR), apolipoprotein E (ApoE) and cholesterol 24-hydroxylase (CYP46A1) during the course of disease. The presence of myelin lipid debris was seen only at the peak of EAE in demyelination loci being efficiently removed during the recovery period. Since CYP46A1 is responsible for removal of cholesterol excess, we performed a detailed profiling of CYP46A1 expression and revealed regional and temporal specificities in its distribution. Double immunofluorescence staining demonstrated CYP46A1 localization with neurons, infiltrated macrophages, microglia and astrocytes in the areas of demyelination, suggesting that these cells play a role in cholesterol turnover in EAE. We propose that alterations in the regulation of cholesterol metabolism at the onset and peak of EAE may add to the progression of disease, while during the recovery period may have beneficial effects contributing to the regeneration of myelin sheath and restoration of neuronal function.

Brain molecular changes and behavioral alterations induced by propofol anesthesia exposure in peripubertal rats

Propofol is commonly used in modern anesthesiology. Some findings suggest that it is highly addictive.

Long-term dietary restriction differentially affects the expression of BDNF and its receptors in the cortex and hippocampus of middle-aged and aged male rats

Dietary restriction (DR) exerts significant beneficial effects in terms of aging and age-related diseases in many organisms including humans. The present study aimed to examine the influence of long-term DR on the BDNF system at the transcriptional and translational levels in the cortex and hippocampus of middle-aged (12-month-old) and aged (24-month-old) male Wistar rats. The obtained results revealed that the DR upregulated the expression of exon-specific BDNF transcripts in both regions, followed by elevated levels of mBDNF only in the cortex in middle-aged animals. In aged animals, DR modulated BDNF protein levels by increasing proBDNF and by declining mBDNF levels. Additionally, elevated levels of the full-length TrkB accompanied by a decreased level of the less-glycosylated TrkB protein were observed in middle-aged rats following DR, while in aged rats, DR amplified only the expression of the less-glycosylated form of TrkB. The levels of phosphorylated TrkBY816 were stable during aging regardless of feeding. Reduced levels of p75NTR were detected in both regions of middle-aged DR-fed animals, while a significant increase was measured in the cortex of aged DR-fed rats. These findings shed additional light on DR as a modulator of BDNF system revealing its disparate effects in middle-aged and aged animals. Given the importance of the proBDNF/BDNF circuit-level expression in different brain functions and various aspects of behavior, it is necessary to further elucidate the optimal duration of the applied dietary regimen with regard to the animal age in order to achieve its most favorable effects.

Effects of Different Dietary Protocols on General Activity and Frailty of Male Wistar Rats During Aging

Dietary restriction (DR) is an important experimental paradigm for lifespan and healthspan extension, but its specific contribution regarding the type, onset, and duration are still debatable. This study was designed to examine the impact of different dietary protocols by assessing the behavioral changes during aging. We exposed male Wistar rats of various age to ad libitum (AL) or DR (60 per cent of AL daily intake) feeding regimens with different onsets. The impact of DR on locomotor activity, memory, and learning was examined in 12-, 18-, and 24-month-old treated animals and controls using open field and Y-maze tests. We have also evaluated the effects of different DRs through the quantification of animal frailty, using behavioral data to create the frailty score. Our results indicated that DR improves general animal activity and spatial memory and decreases frailty with the effect being highly dependent on DR duration and onset. Notably, life-long restriction started at young age had the most profound effect. In contrast, shorter duration and later onset of restricted diet had significantly lower or no impact on animals behavior and frailty. This study signifies the importance of DR starting point and duration as critical determinants of DR effects on healthspan.

Long-term intermittent feeding restores impaired GR signaling in the hippocampus of aged rat.

Diminished glucocorticoid signaling is associated with an age-related decline in hippocampal functioning. In this study we demonstrate the effect of intermittent, every other day (EOD) feeding on the glucocorticoid hormone/glucocorticoid receptor (GR) system in the hippocampus of middle-aged (18-month-old) and aged (24-month-old) Wistar rats. In aged ad libitum-fed rats, a decrease in the level of total GR and GR phosphorylated at Ser(232) (pGR) was detected. Conversely, aged rats subjected to EOD feeding, starting from 6 months of age, showed an increase in GR and pGR levels and a higher content of hippocampal corticosterone. Furthermore, prominent nuclear staining of pGR was observed in CA1 pyramidal and DG granule neurons of aged EOD-fed rats. These changes were accompanied by increased Sgk-1 and decreased GFAP transcription, pointing to upregulated transcriptional activity of GR. EOD feeding also induced an increase in the expression of the mineralocorticoid receptor. Our results reveal that intermittent feeding restores impaired GR signaling in the hippocampus of aged animals by inducing rather than by stabilizing GR signaling during aging.