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Mouse specific expression of the novel splice variant of Pcp2/L7

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Barski J. J. , Przybyla M. , Nowacka-Chmielewska M.

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nr Suppl.1

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INTRODUCTION: Pcp2/L7 is a member of the GoLoco protein family with a very cell‑specific expression in cerebellar Purkinje cells and retinal bipolar neurons. Its precise functional role remains still unclear. Sparse studies indicated its possible role as a guanine nucleotide dissociation inhibitor or guanine nucleotide exchange factor. Studies on genomic structure of pcp2 gene revealed some alternative splice variants expressed in Purkinje cells and retinal bipolar neurons. AIM(S): Here we attempted to shed some light on the conservation of a novel pcp2 splice variant in closely related laboratory rodents: mouse, rat and hamster. METHOD(S): PCR: Both splice variants were amplified with the primers L7sense and L7anti, which yielded two reaction products of 371 and 312 bp. The novel splice variant including exon 3B was detected using the primers L7sense (as above) and L73Aanti, for a product size of 274 bp. Quantitative real‑time PCR (qPCR) analysis: To validate the results obtained by RT-PCR, we conducted an additional qPCR experiment to investigate the expression of Pcp2 transcripts with greater accuracy. The region of interest was amplified from cDNA resulting in a product size of 90 bp.Relative gene expression levels were calculated with the 2‑ΔΔCt method. RESULTS:In our approach we were able to confirm expression of the novel longer transcript in mouse, however PCR amplification on cDNA from rat and hamster did not reveal the long splice variant with additional exon 3B*. CONCLUSIONS: Obtained data indicate, that the novel splicing variant of pcp2 is mouse‑specific and is expressed only in Purkinje cells and retinal bipolar neurons in this species. In course of the evolution it appeared probably in that species as result of a spontaneous mutation. Herewith we suggest a very specific and not known yet function of pcp2 in the mouse eye and/or Purkinje cells. FINANCIAL SUPPORT: Research supported by the Ministry of Science and Higher Education core grant #KNW-2-011/D/4/N.

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Ketogenic diets based on fat of either animal or plant origin have different effects on the abundance of autophagic vesicles in mouse brain

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Liskiewicz D. , Liskiewicz A. , Nowacka-Chmielewska M. , Jablonska K. , Grabowski M. , Student S. , Slugocka A. , Przybyla M. , Barski J. J. , Malecki A.

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nr Suppl.1

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INTRODUCTION: Autophagy is a cellular recycling mechanism essential for maintenance of cell homeostasis and viability, especially during stress conditions; hence, autophagy is involved in a number of physiological and pathological processes. Autophagy is thought to be involved in anti‑aging and neuroprotective effects of caloric restriction, Sirtuin 1 activation, inhibition of insulin/ insulin-like growth factor signaling, and administration of rapamycin, resveratrol, and metformin. The ketogenic diet mimics the biochemical actions of fasting and exerts many physiological and cellular responses similar to those evoked by intermittent energy restriction. Despite this, the relationship between nutritional ketosis and autophagy has been a largely unexplored field. AIM(S): The aim of this study was to verify the hypothesis that ketogenic diets affect the process of autophagosome formation in the hippocampus and/or cerebral cortex. METHOD(S): 9-week-old male mice were fed with one of two differently composed ketogenic chows – based on the fat of either animal or plant origin (KA, KP respectively) or with standard rodent chow (SD) – for 6 subsequent weeks. Western blotting, (LC3, p62), QRT‑PCR (LC3A, LC3B, p62), and confocal microscopy (LC3 puncta) were employed to monitor autophagy in hippocampal and cerebrocortical samples. RESULTS: Western blot results revealed increased levels of LC3 II protein – a marker of autophagosomes – in the hippocampus and frontal cortex of mice treated with the ketogenic diet. This observation was confirmed by the evaluation of a number of LC3 puncta with immunofluorescence microscopy. The size of this effect was dependent on the composition of the diet. CONCLUSIONS: This study reports, for the first time, an upregulation of autophagosome synthesis in the brain of animals fed with the ketogenic diet. Our results make a significant contribution to the understanding of the mechanisms of ketogenic diet action. FINANCIAL SUPPORT: This research is supported by the National Science Center grant no. 2017/01/X/ NZ3/00984.

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Global proteomic analysis of brain changes induced by 'lifestyle' modifications

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Nowacka-Chmielewska M. , Liskiewicz D. , Liskiewicz A. , Marczak L. , Wojakowska A. , Przybyla M. , Grabowski M. , Jablonska K. , Barski J. J. , Malecki A.

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nr Suppl.1

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In the pathogenesis of central nervous system (CNS) disorders, an increasingly important role is attributed recently to unhealthy lifestyle, which consists primarily of a high caloric diet (i.e., western), chronic exposure to stress, and lack of physical activity. However, the mechanisms responsible for energy metabolism impairment induced by unhealthy lifestyles compromising CNS functions are poorly understood. Research on the effects of physical activity on the CNS is especially important, because it may result in the development of new methods of therapy inspired by natural protective mechanisms. In our study we employed a new and rarely used approach – a forced running wheel. The lack of electrical stimulus in the aforementioned system successfully makes a breakthrough in the study of animal physical activity. Physiological and behavioral responses of the organism to stress are closely related to sex. Epidemiological studies indicate that women are more vulnerable to the adverse effects of stress and despite that, most of the experimental studies are conveyed on male animals. The investigations were carried on female rats. The main goal of our study was to verify the hypothesis that regular exercise may reduce the disturbances induced by lifestyle modifications, like western diet and/or stress exposure. Adult female rats were fed with the prepared chow reproducing the human western diet and/or subjected to a stress induced by social instability. This stress protocol is characterized by a low degree of invasiveness. To evaluate if regular physical activity may reduce the adverse effects caused by diet and stress, female rats were additionally subjected to the procedure of forced physical activity. A proteomic analysis was conducted on samples obtained from the frontal cortex – a region that plays an important role in cognitive processes as well as is involved in the mechanisms engaged in the response to stress.

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Anxiety-like behavior of laboratory animals on a ketogenic diet

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Przybyla M. , Liskiewicz D. , Nowacka-Chmielewska M. , Liskiewicz A. , Grajoszek A. , Grabowski M. , Jablonska K. , Barski J. J.

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nr Suppl.1

EN

INTRODUCTION: In the recent years, more attention is attributed to the impact of diet on central nervous system function. An increasing number of diseases, including neurological disorders, results from inadequate dietary habits. Diet, however, can affect the function of brain and mental processes in a negative as well as a beneficial way. The diet with well‑documented neuroprotective effects is a high-fat and low-carbohydrate ketogenic diet (KD). METHOD(S): We investigated two type of KD, one of them based on animal (KDA), while the other on vegetable fats (KDB). Both diets were applied to two groups of laboratory animals: mice (129S2/SvPasCrl) and rats (Long‑Evans Rat, Crl: LE) for 6 weeks. CONCLUSIONS: Interestingly, preliminary data indicate a unique anxiogenic action of the KD but only in mice.