Arenguline lähenemine emotsionaalse käitumisega seotud geenide Wfs1 ja Lsampʼi funktsiooni uurimisel

Väitekirja elektrooniline versioon ei sisalda publikatsioonePsühhiaatriliste häirete kujunemises mängib olulist rolli aju areng. Geenid, mis osalevad emotsioonidega seotud ajupiirkondade arengus ja funktsioneerimises, on olulised psühhiaatriliste häirete seisukohalt. Wfs1 ja Lsamp geenid osalevad hirmu- ja ärevuskäitumise regulatsioonis. Wfs1 (Wolframi sündroom 1), nagu nimigi ütleb, on seotud samanimelise haruldase sündroomiga, mille sümptomid on diabetes insipidus, diabetes mellitus, nägemisnärvi kärbumine ja sensorineuraalne kurtus, sageli kaasnevad ka psühhiaatrilised häired. Wfs1 valk mängib olulist rolli insuliini sekretsioonis ja pankrease β-rakkude ellujäämise tagamises, selle funktsioonist ajus on vähem teada. Lsamp (limbilise süsteemiga seotud membraanivalk) osaleb neuriitide väljakasvu, aksonite sihtmärgini jõudmise ja sünaptogeneesi reguleerimises. Lsamp geenil on kaks evolutsiooniliselt konserveerunud alternatiivset esimest eksonit koos eraldi promootoritega, sellise struktuuri funktsionaalne tähtsus on teadmata. Nii Wfs1 kui Lsamp geeni puhul on näidatud kindlate alleelide seotust meeleolu- ja ärevushäirete, skisofreenia ja suitsidaalsusega. Enda doktoritöös uurisin Wfs1 ja Lsamp`i funktsiooni arengulisest vaatevinklist. Selgus, et Wfs1 ekspressioon on evolutsiooniliselt konserveerunud dopamiinergilist sisendit saavates ajupiirkondades, ning et Wfs1 suhtes puudulike hiirte hipokampuses on D1-tüüpi dopamiini retseptorite hulk suurenenud. Arengu käigus ekspresseerus Wfs1 ajutiselt paljudes ajupiirkondades; see laialdane ekspressioon polnud seotud arengulise endoplasmaatilise retiikulumi stressi vastuse regulatsiooniga, mis on üks Wfs1 põhilisi funktsioone täiskasvanud organismis. Lsamp`i puhul näitasime, et selle alternatiivsed promootorid on aktiivsed erinevate funktsioonidega ajupiirkondades: 1a-promootor on põhiline limbilises süsteemis, samas kui 1b-promootori aktiivsus piiritleb sensoorseid juhteteid. Lsamp`i promootorite aktiivsus hiirte hipokampuses, ventraalses striatumis ja temporaalsagaras korreleerus sotsiaalse- ja ärevusega seotud käitumise näitajatega. Saadud tulemused aitavad paremini mõista Wfs1 ja Lsamp`i rolli psühhiaatriliste häirete väljakujunemises ning näitavad suunda edasisteks uuringuteks.Genes that are involved in the development and functioning of the emotional circuits of the brain are highly susceptible targets in psychiatric diseases. Wfs1 and Lsamp, two genes studied in the present thesis, are both involved in the regulation of anxiety- and fear-related behaviour in the adult brain. Wfs1 (Wolfram syndrome 1) is a causative gene for Wolfram syndrome, a rare genetic disorder characterized by diabetes insipidus, diabetes mellitus, optic atrophy and sensorineural deafness. Often, the core symptoms are accompanied by psychiatric manifestations. Wfs1 is important for the survival and functioning of pancreatic β-cells, its roles in the nervous system are not well understood. Lsamp (Limbic system associated membrane protein) is involved in regulating neurite outgrowth, axon targeting and synaptogenesis in the limbic system. Lsamp has two alternative first exons with separate promoters, the role of this conserved gene structure is unknown. Allelic variants of both, Wfs1 and Lsamp, are associated with depression, anxiety disorders, bipolar disorder, schizophrenia and suicidality. We took advantage of the neurodevelopmental approach to study the functions of Wfs1 and Lsamp in the brain. Wfs1 showed evolutionarily conserved expression in the dopaminoceptive brain regions. Relating to this, Wfs1-deficient mice had increased number of D1-type dopamine receptor ligand binding sites in the hippocampi compared to wild-type mice. During the development, Wfs1 was transiently expressed in many brain regions. The widespread expression of Wfs1 in early postnatal mouse brain was not involved in the regulation of developmental endoplasmic reticulum stress, which has previously been shown to be one of the main functions of Wfs1 in the adult organism. The activity pattern of the two alternative promoters of Lsamp was complementary in the mouse brain: 1a promoter was prevailing in the limbic-related structures, while the activity of 1b promoter mainly delineated sensory pathways. The activity of Lsamp promoters in the hippocampus, ventral striatum and temporal lobe correlated with measures of anxiety and social behaviour of mice. The present results help to understand the role of Wfs1 and Lsamp in the context of psychiatric diseases and point direction for further research

GDNF Overexpression from the Native Locus Reveals its Role in the Nigrostriatal Dopaminergic System Function

Peer reviewe

Alternative Promoter Use Governs the Expression of IgLON Cell Adhesion Molecules in Histogenetic Fields of the Embryonic Mouse Brain

The members of the IgLON superfamily of cell adhesion molecules facilitate fundamental cellular communication during brain development, maintain functional brain circuitry, and are associated with several neuropsychiatric disorders such as depression, autism, schizophrenia, and intellectual disabilities. Usage of alternative promoter-specific 1a and 1b mRNA isoforms in Lsamp, Opcml, Ntm, and the single promoter of Negr1 in the mouse and human brain has been previously described. To determine the precise spatiotemporal expression dynamics of Lsamp, Opcml, Ntm isoforms, and Negr1, in the developing brain, we generated isoform-specific RNA probes and carried out in situ hybridization in the developing (embryonic, E10.5, E11.5, 13.5, 17; postnatal, P0) and adult mouse brains. We show that promoter-specific expression of IgLONs is established early during pallial development (at E10.5), where it remains throughout its differentiation through adulthood. In the diencephalon, midbrain, and hindbrain, strong expression patterns are initiated a few days later and begin fading after birth, being only faintly expressed during adulthood. Thus, the expression of specific IgLONs in the developing brain may provide the means for regionally specific functionality as well as for specific regional vulnerabilities. The current study will therefore improve the understanding of how IgLON genes are implicated in the development of neuropsychiatric disorders

Long-term spatial memory and reversal learning ability in the Morris water maze is not altered in <i>Trib3</i>-deficient mice.

<p>Data are means ± SEM from littermate <i>Trib3</i>^−/− (n = 16) and <i>Trib3</i>^+/+ (n = 10) mice. (A) Escape latencies during four days of hidden-platform training performed at four trials per day. (B) Pool quadrant occupancy in a probe trial performed 24 h after the completion of training. The submerged platform was removed from the pool and the swim trajectory of mice was monitored for 1 min. The pool quadrant that previously contained the platform is designated the target quadrant, and the time spent in each quadrant of the pool is presented as percent of the total search time. (C) Reversal training escape latencies during two days of training performed at four trials per day. For reversal training, the hidden platform was repositioned to the pool quadrant opposite of the initial platform location. (D) Results of a probe trial performed 24 h after reversal training. The probe trial was carried out as in B. The reversal target denotes the pool quadrant that contained the platform during the reversal trainings trials. (E) Swimming speed at different stages of the experiment. The mean speed ± SEM from 60-second swimming sessions with no platform are shown. The habituation session was performed one day before the start of training. For <i>Trib3</i>^+/+, n = 5 per sex, and for <i>Trib3</i>^−/−, n = 8 per sex.</p

Rejection of amino acid-imbalanced diet in mice is not influenced by the deletion of <i>Trib3</i>.

<p>(<b>A</b>) Consumption of a diet lacking the essential amino acid leucine (Leu−), compared to the consumption of a corresponding nutritionally complete control diet (Leu+). Adult <i>Trib3</i>^+/+ and <i>Trib3</i>^−/− (n = 7 per genotype) mice were trained with food deprivation during the dark phase, and, during the light phase, food intake was measured for each animal at the indicated time-points by weighing the remaining food. For each animal, the intake of Leu− diet was compared to that of the Leu+ diet, and the average difference in the consumption of the Leu− diet relative to the Leu+ diet is expressed in percent ± SEM for each genotype. (<b>B</b>) Body weight and (<b>C</b>) body weight loss due to overnight food deprivation do not differ between <i>Trib3</i>-deficient and wild type adult mice. For B and C, four-month-old group-housed <i>Trib3</i>^+/+ (n = 5 for both males and females) and <i>Trib3</i>^−/− (n = 8 for both males and females) mice were maintained on the Leu+ diet <i>ad libitum</i> for two weeks to determine their diet-habituated body weight, followed by a single iteration of overnight fasting. The data in B and C are presented as the group means ± SEM.</p

<i>Trib3</i> is upregulated in the mouse anterior piriform cortex in response to leucine-deficient diet.

<p>Adult wild type mice consumed either a diet lacking leucine (Leu−; n = 5) or a corresponding control diet containing leucine (Leu+; n = 5), and, after 6 h of feeding, <i>Trib3</i> expression in the indicated brain regions was quantified by RT-qPCR. The results are presented as the mean ± SEM, and expressed relative to the level of <i>Trib3</i> mRNA in the anterior piriform cortex of the control diet (Leu+) group. *<i>P</i><0.05 comparing leucine-starved and control diet-fed groups.</p

Binding of D₁/D₅ specific ligand [³H]SCH23390 to hippocampal membranes of wt and <i>Wfs1</i> knockout mice.

<p>Comparison of specific binding of radioligand [³H]SCH23390 to hippocampal membranes of wt and <i>Wfs1</i> knockout mice. (A) Binding curve of [³H]SCH23390 binding to pooled samples of wt (triangle) and <i>Wfs1</i> knockout (circle) mice. The membrane suspensions (3 mg/well) were incubated with different concentrations of [³H]SCH23390 for 60 min and bound radioactivity was measured. Data are presented as mean ± SEM from experiments (n = 3) performed in duplicates. (B) The level of [³H]SCH23390 binding sites of individual wt and <i>Wfs1</i> knockout mice determined in hippocampal membrane suspensions (6.7 mg/ml.) incubated with 4 nM radioligand. Data presented as mean ± SEM of all the mice tested. *P < 0.05. Data of individual mice are presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0172825#pone.0172825.s006" target="_blank">S1 Table</a>.</p

Distribution of Wfs1, D₁, and D₅ proteins in selected regions of the adult mouse brain.

<p>Distribution of Wfs1, D1, and D5 proteins in selected regions of the adult mouse brain. Immunohistochemistry on coronal sections. The detected proteins are indicated above. In the cortex Wfs1, D1, and D5 are all present in layer I, upper part of layer II/III, and in layer V (images show somatosensory cortex; A-C); in the hippocampus Wfs1, D1, and D5 are simultaneously present in pyr and slm of CA1 (D-F); Wfs1, D1 and D5 are all present in Rt and Sth; in amygdala Wfs1 is strongly present in CeA, the lateral edge of BL, and medial and cortical nuclei (G-I), whereas D1 and D5 show only weak signal in CeA and BL (G-I); in the dorsolateral thalamus Wfs1, D1, and D5 are delineating dLG and VPM, note that strongly Wfs1-positive fibers are present in fi, but no D1 or D5 is seen there (J-L), in the substantia nigra Wfs1 has similar distribution with D1, but not with D5 (M-O). For abbreviations, see list. Scale bar is 100 μm in A-C, 1 mm in D-L, 500 μm in M-O.</p