D2 Knockout Mice Research Articles

The Validation of a Precursor Lesion of Epithelial Ovarian Cancer in Fancd2-KO Mice.

Ovarian cancer (OC) has the highest mortality rate of all gynaecological malignancies. The asymptomatic nature and limited understanding of early disease hamper research into early-stage OC. Therefore, there is an urgent need for models of early-stage OC to be characterised to improve the understanding of early neoplastic transformations. This study sought to validate a unique mouse model for early OC development. The homozygous Fanconi anaemia complementation group D2 knock-out mice (Fancd2-/-) develop multiple ovarian tumour phenotypes in a sequential manner as they age. Using immunohistochemistry, our group previously identified purported initiating precursor cells, termed 'sex cords', that are hypothesised to progress into epithelial OC in this model. To validate this hypothesis, the sex cords, tubulostromal adenomas and equivalent controls were isolated using laser capture microdissection for downstream multiplexed gene expression analyses using the Genome Lab GeXP Genetic Analysis System. Principal component analysis and unbiased hierarchical clustering of the resultant expression data from approximately 90 OC-related genes determined that cells from the sex cords and late-stage tumours clustered together, confirming the identity of the precursor lesion in this model. This study, therefore, provides a novel model for the investigation of initiating neoplastic events that can accelerate progress in understanding early OC.

Loss of individualized behavioral trajectories in adult neurogenesis-deficient cyclin D2 knockout mice.

There is still limited mechanistic insight into how the interaction of individuals with their environment results in the emergence of individuality in behavior and brain structure. Nevertheless, the idea that personal activity shapes the brain is implicit in strategies for healthy cognitive aging as well as in the idea that individuality is reflected in the brain's connectome. We have shown that even isogenic mice kept in a shared enriched environment (ENR) developed divergent and stable social and exploratory trajectories. As these trajectories-measured as roaming entropy (RE)-positively correlated with adult hippocampal neurogenesis, we hypothesized that a feedback between behavioral activity and adult hippocampal neurogenesis might be a causal factor in brain individualization. We used cyclin D2 knockout mice with constitutively extremely low levels of adult hippocampal neurogenesis and their wild-type littermates. We housed them for 3 months in a novel ENR paradigm, consisting of 70 connected cages equipped with radio frequency identification antennae for longitudinal tracking. Cognitive performance was evaluated in the Morris Water Maze task (MWM). With immunohistochemistry we confirmed that adult neurogenesis correlated with RE in both genotypes and that D2 knockout mice had the expected impaired performance in the reversal phase of the MWM. But whereas the wild-type animals developed stable exploratory trajectories with increasing variance, correlating with adult neurogenesis, this individualizing phenotype was absent in D2 knockout mice. Here the behaviors started out more random and revealed less habituation and low variance. Together, these findings suggest that adult neurogenesis contributes to experience-dependent brain individualization.

Exploring stem cell biology in pituitary tumors and derived organoids.

Pituitary tumorigenesis is highly prevalent and causes major endocrine disorders. Hardly anything is known on the behavior of the local stem cells in this pathology. Here, we explored the stem cells' biology in mouse and human pituitary tumors using transcriptomic, immunophenotyping and organoid approaches. In the prolactinoma-growing pituitary of dopamine receptor D2 knock-out mice, the stem cell population displays an activated state in terms of proliferative activity and distinct cytokine/chemokine phenotype. Organoids derived from the tumorous glands' stem cells recapitulated these aspects of the stem cells' activation nature. Upregulated cytokines, in particular interleukin-6, stimulated the stem cell-derived organoid development and growth process. In human pituitary tumors, cells typified by expression of stemness markers, in particular SOX2 and SOX9, were found present in a wide variety of clinical tumor types, also showing a pronounced proliferative status. Organoids efficiently developed from human tumor samples, displaying a stemness phenotype as well as tumor-specific expression fingerprints. Transcriptomic analysis revealed fading of cytokine pathways at organoid development and passaging, but their reactivation did not prove capable of rescuing early organoid expansion and passageability arrest. Taken together, our study revealed and underscored an activated phenotype of the pituitary-resident stem cells in tumorigenic glands and tumors. Our findings pave the way to defining the functional position of the local stem cells in pituitary tumor pathogenesis, at present barely known. Deeper insight can lead to more efficient and targeted clinical management, currently still not satisfactorily.

Impaired Generation of Transit-Amplifying Progenitors in the Adult Subventricular Zone of Cyclin D2 Knockout Mice.

In the adult brain, new neurons are constitutively derived from postnatal neural stem cells/progenitors located in two neurogenic regions: the subventricular zone (SVZ) of the lateral ventricles (migrating and differentiating into different subtypes of the inhibitory interneurons of the olfactory bulbs), and the subgranular layer of the hippocampal dentate gyrus. Cyclin D2 knockout (cD2-KO) mice exhibit reduced numbers of new hippocampal neurons; however, the proliferation deficiency and the dysregulation of adult neurogenesis in the SVZ required further investigation. In this report, we characterized the differentiation potential of each subpopulation of the SVZ neural precursors in cD2-KO mice. The number of newly generated cells in the SVZs was significantly decreased in cD2-KO mice compared to wild type mice (WT), and was not accompanied by elevated levels of apoptosis. Although the number of B1-type quiescent precursors (B1q) and the overall B1-type activated precursors (B1a) were not affected in the SVZ neurogenic niche, the number of transit-amplifying progenitors (TaPs) was significantly reduced. Additionally, the subpopulations of calbindin D28k and calretinin interneurons were diminished in the olfactory bulbs of cD2-KO mice. Our results suggest that cyclin D2 might be critical for the proliferation of neural precursors and progenitors in the SVZ—the transition of B1a into TaPs and, thereafter, the production of newly generated interneurons in the olfactory bulbs. Untangling regulators that functionally modulate adult neurogenesis provides a basis for the development of regenerative therapies for injuries and neurodegenerative diseases.

Dopaminergic Signaling in the Nucleus Accumbens Modulates Stress-Coping Strategies during Inescapable Stress.

Maladaptation to stress is a critical risk factor in stress-related disorders, such as major depression and post-traumatic stress disorder (PTSD). Dopamine signaling in the nucleus accumbens (NAc) has been shown to modulate behavior by reinforcing learning and evading aversive stimuli, which are important for the survival of animals under environmental challenges such as stress. However, the mechanisms through which dopaminergic transmission responds to stressful events and subsequently regulates its downstream neuronal activity during stress remain unknown. To investigate how dopamine signaling modulates stress-coping behavior, we measured the subsecond fluctuation of extracellular dopamine concentration and pH using fast scanning cyclic voltammetry (FSCV) in the NAc, a postsynaptic target of midbrain dopaminergic neurons, in male mice engaged in a tail suspension test (TST). The results revealed a transient decrease in dopamine concentration and an increase in pH levels when the animals changed behaviors, from being immobile to struggling. Interestingly, optogenetic inhibition of dopamine release in NAc, potentiated the struggling behavior in animals under the TST. We then addressed the causal relationship of such a dopaminergic transmission with behavioral alterations by knocking out both the dopamine receptors, i.e., D1 and D2, in the NAc using viral vector-mediated genome editing. Behavioral analyses revealed that male D1 knock-out mice showed significantly more struggling bouts and longer struggling durations during the TST, while male D2 knock-out mice did not. Our results therefore indicate that D1 dopaminergic signaling in the NAc plays a pivotal role in the modulation of stress-coping behaviors in animals under tail suspension stress.SIGNIFICANCE STATEMENT The tail suspension test (TST) has been widely used as a despair-based behavioral assessment to screen the antidepressant so long. Despite its prevalence in the animal studies, the neural substrate underlying the changes of behavior during the test remains unclear. This study provides an evidence for a role of dopaminergic transmission in the modulation of stress-coping behavior during the TST, a despair test widely used to screen the antidepressants in rodents. Taking into consideration the fact that the dopamine metabolism is upregulated by almost all antidepressants, a part of which acts directly on the dopaminergic transmission, current results would uncover the molecular mechanism through which the dopaminergic signaling mediates antidepressant effect with facilitation of the recovery from the despair-like behavior in the TST.

Dopamine D1 and muscarinic acetylcholine receptors in dorsal striatum are required for high speed running

Dopamine (DA) signaling in the basal ganglia plays important roles in motor control. Motor deficiencies were previously reported in dopamine receptor D1 (D1R) and D2 (D2R) knockout mice. While these results indicate the involvement of DA receptors in motor execution, the null knockout (KO) mouse lacks the specificity necessary to determine when and where in the brain D1R and D2R function in motor execution. To address these questions, we restricted the loss of function temporally and spatially by using D1R conditional knockdown (cKD) mice and mice injected with antagonists against DA receptors directly into the dorsal striatum. In addition, we address the DA and acetylcholine (ACh) balance hypothesis by using antagonists against ACh receptors. We tested the motor ability of the mice with a newly devised task named the accelerating step-wheel. In this task, the maximum running speed was measured in a situation where the wheel rotation speed was gradually accelerated in one trial. We found significant decreases in the maximum running speed of D1R cKD mice and the mice injected with the antagonist against D1R or muscarinic ACh receptor. These results indicated that D1R and muscarinic ACh receptor in the dorsal striatum play pivotal roles in the execution of walking/running.

A Global Loss of Dio2 Leads to Unexpected Changes in Function and Fiber Types of Slow Skeletal Muscle in Male Mice.

The type 2 iodothyronine-deiodinase (D2) enzyme converts T4 to T3, and mice deficient in this enzyme [D2 knockout (D2KO) mice] have decreased T3 derived from T4 in skeletal muscle despite normal circulating T3 levels. Because slow skeletal muscle is particularly susceptible to changes in T3 levels, we expected D2 inactivation to result in more pronounced slow-muscle characteristics in the soleus muscle, mirroring hypothyroidism. However, ex vivo studies of D2KO soleus revealed higher rates of twitch contraction and relaxation and reduced resistance to fatigue. Immunostaining of D2KO soleus showed that these properties were associated with changes in muscle fiber type composition, including a marked increase in the number of fast, glycolytic type IIB fibers. D2KO soleus muscle fibers had a larger cross-sectional area, and this correlated with increased myonuclear accretion in myotubes formed from D2KO skeletal muscle precursor cells differentiated in vitro. Consistent with our functional findings, D2KO soleus gene expression was markedly different from that in hypothyroid wild-type (WT) mice. Comparison of gene expression between euthyroid WT and D2KO mice indicated that PGC-1α, a T3-dependent regulator of slow muscle fiber type, was decreased by ∼50% in D2KO soleus. Disruption of Dio2 in the C2C12 myoblast cell line led to a significant decrease in PGC-1α expression and a faster muscle phenotype upon differentiation. These results indicate that D2 loss leads to significant changes in soleus contractile function and fiber type composition that are inconsistent with local hypothyroidism and suggest that reduced levels of PCG-1α may contribute to the observed phenotypical changes.

Regional brain volume changes following chronic antipsychotic administration are mediated by the dopamine D2 receptor

BackgroundNeuroanatomical alterations are well established in patients suffering from schizophrenia, however the extent to which these changes are attributable to illness, antipsychotic drugs (APDs), or their interaction is unclear. APDs have been extremely effective for treatment of positive symptoms in major psychotic disorders. Their therapeutic effects are mediated, in part, through blockade of D2-like dopamine (DA) receptors, i.e. the D2, D3 and D4 dopamine receptors. Furthermore, the dependency of neuroanatomical change on DA system function and D2-like receptors has yet to be explored. MethodsWe undertook a preclinical longitudinal study to examine the effects of typical (haloperidol (HAL)) and atypical (clozapine (CLZ)) APDs in wild type (WT) and dopamine D2 knockout (D2KO) mice over 9-weeks using structural magnetic resonance imaging (MRI). ResultsChronic typical APD administration in WT mice was associated with reductions in total brain (p = 0.009) and prelimbic area (PL) (p = 0.02) volumes following 9-weeks, and an increase in striatal volume (p = 0.04) after six weeks. These APD-induced changes were not present in D2KOs, where, at baseline, we observed significantly smaller overall brain volume (p < 0.01), thinner cortices (q < 0.05), and enlarged striata (q < 0.05). Stereological assessment revealed increased glial density in PL area of HAL treated wild types. Interestingly, in WT and D2KO mice, chronic CLZ administration caused more limited changes in brain structure. ConclusionsOur results present evidence for the role of D2 DA receptors in structural alterations induced by the administration of the typical APD HAL and that chronic administration of CLZ has a limited influence on brain structure.

Excessive D1 Dopamine Receptor Activation in the Dorsal Striatum Promotes Autistic-Like Behaviors.

The dopamine system has been characterized in motor function, goal-directed behaviors, and rewards. Recent studies recognize various dopamine system genes as being associated with autism spectrum disorder (ASD). However, how dopamine system dysfunction induces ASD pathophysiology remains unknown. In the present study, we demonstrated that mice with increased dopamine functions in the dorsal striatum via the suppression of dopamine transporter expression in substantia nigra neurons or the optogenetic stimulation of the nigro-striatal circuitry exhibited sociability deficits and repetitive behaviors relevant to ASD pathology in animal models, while these behavioral changes were blocked by a D1 receptor antagonist. Pharmacological activation of D1 dopamine receptors in normal mice or the genetic knockout (KO) of D2 dopamine receptors also produced typical autistic-like behaviors. Moreover, the siRNA-mediated inhibition of D2 dopamine receptors in the dorsal striatum was sufficient to replicate autistic-like phenotypes in D2 KO mice. Intervention of D1 dopamine receptor functions or the signaling pathways-related D1 receptors in D2 KO mice produced anti-autistic effects. Together, our results indicate that increased dopamine function in the dorsal striatum promotes autistic-like behaviors and that the dorsal striatum is the neural correlate of ASD core symptoms.

PET radiotracer development for imaging high-affinity state of dopamine D2 and D3 receptors: Binding studies of fluorine-18 labeled aminotetralins in rodents.

Imaging the high-affinity, functional state (HA) of dopamine D2 and D3 receptors has been pursued in PET imaging studies of various brain functions. We report further evaluation of 18 F-5-OH-FPPAT, and the newer 18 F-5-OH-FHXPAT and 18 F-7-OH-FHXPAT. Syntheses of 18 F-5-OH-FHXPAT and 18 F-7-OH-FHXPAT were improved by modifications of our previously reported procedures. Brain slices and brain homogenates from male Sprague-Dawley rats were used with the 3 radiotracers (74-111 kBq/cc). Competition with dopamine (1-100 nM) and Gpp(NH)p (10-50 µM) were carried out to demonstrate binding to dopamine D2 and D3 HA-states and binding kinetics of 18 F-5-OH-FPPAT measured. Ex vivo brain slice autoradiography was carried out on rats administered with 18 F-5-OH-FHXPAT to ascertain HA-state binding. PET/CT imaging in rats and wild type (WT) and D2 knock-out mice were carried out using 18 F-7-OH-FHXPAT (2-37 MBq). Striatum was clearly visualized by the three radiotracers in brain slices and dopamine displaced more than 80% of binding, with dissociation rate in homogenates of 2.2 × 10-2 min-1 for 18 F-5-OH-FPPAT. Treatment with Gpp(NH)p significantly reduced 50-80% striatal binding with faster dissociation rates (5.0 × 10-2 min-1 ), suggesting HA-state binding of 18 F-5-OH-FPPAT and 18 F-5-OH-FHXPAT. Striatal binding of 18 F-5-OH-FHXPAT in ex vivo brain slices were sensitive to Gpp(NH)p, suggesting HA-state binding in vivo. PET binding ratios of 18 F-7-OH-FHXPAT in rat brain were ventral striatum/cerebellum = 2.09 and dorsal striatum/cerebellum = 1.65; similar binding ratios were found in the D2 WT mice. These results suggest that in vivo PET measures of agonists in the brain at least in part reflect binding to the membrane-bound HA-state of the dopamine receptor.

Effects of D1 receptor knockout on fear and reward learning

Dopamine signaling is involved in a variety of neurobiological processes that contribute to learning and memory. D1-like dopamine receptors (including D1 and D5 receptors) are thought to be involved in memory and reward processes, but pharmacological approaches have been limited in their ability to distinguish between D1 and D5 receptors. Here, we examine the effects of a specific knockout of D1 receptors in associative learning tasks involving aversive (shock) or appetitive (cocaine) unconditioned stimuli. We find that D1 knockout mice show similar levels of cued and contextual fear conditioning to WT controls following conditioning protocols involving one, two, or four shocks. D1 knockout mice show increased generalization of fear conditioning and extinction across contexts, revealed as increased freezing to a novel context following conditioning and decreased freezing to an extinguished cue during a contextual renewal test. Further, D1 knockout mice show mild enhancements in extinction following an injection of SKF81297, a D1/D5 receptor agonist, suggesting a role for D5 receptors in extinction enhancements induced by nonspecific pharmacological agonists. Finally, although D1 knockout mice show decreased locomotion induced by cocaine, they are able to form a cocaine-induced conditioned place preference. We discuss these findings in terms of the role of dopamine D1 receptors in general learning and memory processes.

Reversal of an Unconditioned Behavioral Preference for Specific Food Pellets by Intervention of Whisker Sensory Inputs.

Adenylyl cyclase type-5 (AC5) is preferentially expressed in the dorsal striatum. Recently, we reported that AC5 knockout (KO) mice preferred food pellets carrying an olfactory cue produced by AC5 KO mice during food consumption (AC5 KO pellets) over food pellets that had been taken by wildtype (WT) mice. In the present study, we demonstrated that whisker trimming on the right side of the face but not the left in AC5 KO mice blocked the behavioral preference for AC5 KO pellets. Conversely, whisker trimming on the right but not the left in WT mice induced a behavioral preference for AC5 KO pellets. Mice lacking D2 dopamine receptor (D2 KO mice) also showed a behavioral preference for AC5 KO pellets. In D2 mice, whisker trimming on the right side of the face but not the left blocked a behavioral preference for AC5 KO food pellets. AC5 KO mice had increased level of phospho-CaMKIIα in the dorsal striatum, and WT mice with whiskers cut on either side also showed increased p-CaMKIIα level in the dorsal striatum. The siRNA-mediated inhibition of CaMKIIα in the dorsal striatum in either the right or the left hemisphere in AC5 KO mice and D2 KO mice blocked the behavioral preference for AC5 KO pellets. However, behavioral changes induced by this inhibition on each side showed asymmetrical time courses. These results suggest that an unconditioned behavioral preference for specific food pellets can be switched on or off based on the balance of states of neural activity in the dorsal striatum regulated by a signaling pathway centered on AC5 and D2 and the sensory inputs of whiskers from the right side of the face.

Cyclin D1 Promotes Androgen-Dependent DNA Damage Repair in Prostate Cancer Cells.

Therapy resistance and poor outcome in prostate cancer is associated with increased expression of cyclin D1. Androgens promote DNA double-strand break repair to reduce DNA damage, and cyclin D1 was also shown to enhance DNA damage repair (DDR). In this study, we investigated the significance of cyclin D1 in androgen-induced DDR using established prostate cancer cells and prostate tissues from cyclin D1 knockout mice. We demonstrate that endogenous cyclin D1 further diminished the dihydrotestosterone (DHT)-dependent reduction of γH2AX foci in vitro. We also show that cyclin D1 was required for the androgen-dependent DNA damage response both in vitro and in vivo. Furthermore, cyclin D1 was required for androgen-enhanced DDR and radioresistance of prostate cancer cells. Moreover, microarray analysis of primary prostate epithelial cells from cyclin D1-deficient and wild-type mice demonstrated that most of the DHT-dependent gene expression changes are also cyclin D1 dependent. Collectively, our findings suggest that the hormone-mediated recruitment of cyclin D1 to sites of DDR may facilitate the resistance of prostate cancer cells to DNA damage therapies and highlight the need to explore other therapeutic approaches in prostate cancer to prevent or overcome drug resistance.

Efficacy of Hybrid Tetrahydrobenzo[d]thiazole Based Aryl Piperazines D-264 and D-301 at D2 and D3 Receptors

In elucidating the role of pharmacodynamic efficacy at D3 receptors in therapeutic effectiveness of dopamine receptor agonists, the influence of study system must be understood. Here two compounds with D3 over D2 selectivity developed in our earlier work, D-264 and D-301, are compared in dopamine receptor-mediated G-protein activation in striatal regions of wild-type and D2 receptor knockout mice and in CHO cells expressing D2 or D3 receptors. In caudate-putamen of D2 knockout mice, D-301 was ~3-fold more efficacious than D-264 in activating G-proteins as assessed by [(35)S]GTPγS binding; in nucleus accumbens, D-301 stimulated G-protein activation whereas D-264 did not. In contrast, the two ligands exerted similar efficacy in both regions of wild-type mice, suggesting both ligands activate D2 receptors with similar efficacy. In D2 and D3 receptor-expressing CHO cells, D-264 and D-301 appeared to act in the [(35)S]GTPγS assay as full agonists because they produced maximal stimulation equal to dopamine. Competition for [(3)H]spiperone binding was then performed to determine Ki/EC50 ratios as an index of receptor reserve for each ligand. Action of D-301, but not D-264, showed receptor reserve in D3 but not in D2 receptor-expressing cells, whereas dopamine showed receptor reserve in both cell lines. Gαo1 is highly expressed in brain and is important in D2-like receptor-G protein coupling. Transfection of Gαo1 in D3- but not D2-expressing CHO cells led to receptor reserve for D-264 without altering receptor expression levels. D-301 and dopamine exhibited receptor reserve in D3-expressing cells both with and without transfection of Gαo1. Altogether, these results indicate that D-301 has greater intrinsic efficacy to activate D3 receptors than D-264, whereas the two compounds act on D2 receptors with similar intrinsic efficacy. These findings also suggest caution in interpreting Emax values from functional assays in receptor-transfected cell models without accounting for receptor reserve.

Transplanted Dentate Progenitor Cells Show Increased Survival in an Enriched Environment But Do Not Exert a Neurotrophic Effect on Spatial Memory Within 2 Weeks of Engraftment.

Cyclin D2 knockout mice show decreased levels of endogenous dentate neurogenesis. We investigated whether transplanted dentate progenitor cells from wild-type mice respond in vivo to an enriched environment and whether they improve deficient dentate neurogenesis through a neurotrophic effect. Adult cyclin D2 knockout mice were transplanted with passaged adult progenitor cells and kept in an enriched environment or under standard housing conditions in isolation. After 1 week, animals living in an enriched environment underwent water maze testing. Progenitor cells grown on a laminin/poly-d-lysine monolayer expressed Sox2 and nestin and could be differentiated in vitro into neurons and astrocytes. After transplantation into the dentate gyrus, cells preferentially survived along the laminin-rich ependymal lining of the basal cistern or basal membrane of capillaries. A subpopulation of transplanted cells migrated into the interstitial space of the hippocampus and was not associated with laminin. Environmental enrichment led to a significant increase in the survival of transplanted progenitor cells on laminin in the dentate gyrus after 2 weeks. However, animals did not show an enhanced performance in the Morris water maze, and transplantation failed to exert a neurotrophic effect on endogenous neurogenesis after 2 weeks. However, a major limitation of the study is the short-term period of investigation, which may have been insufficient to capture functional effects. In conclusion, initial survival of transplanted neural progenitor cells was dependent on the presence of laminin and was significantly enhanced by environmental enrichment. Further studies are needed to address whether an enriched environment continues to promote graft survival over longer periods of time.

Epileptogenesis following Kainic Acid-Induced Status Epilepticus in Cyclin D2 Knock-Out Mice with Diminished Adult Neurogenesis.

The goal of this study was to determine whether a substantial decrease in adult neurogenesis influences epileptogenesis evoked by the intra-amygdala injection of kainic acid (KA). Cyclin D2 knockout (cD2 KO) mice, which lack adult neurogenesis almost entirely, were used as a model. First, we examined whether status epilepticus (SE) evoked by an intra-amygdala injection of KA induces cell proliferation in cD2 KO mice. On the day after SE, we injected BrdU into mice for 5 days and evaluated the number of DCX- and DCX/BrdU-immunopositive cells 3 days later. In cD2 KO control animals, only a small number of DCX+ cells was observed. The number of DCX+ and DCX/BrdU+ cells/mm of subgranular layer in cD2 KO mice increased significantly following SE (p<0.05). However, the number of newly born cells was very low and was significantly lower than in KA-treated wild type (wt) mice. To evaluate the impact of diminished neurogenesis on epileptogenesis and early epilepsy, we performed video-EEG monitoring of wt and cD2 KO mice for 16 days following SE. The number of animals with seizures did not differ between wt (11 out of 15) and cD2 KO (9 out of 12) mice. The median latency to the first spontaneous seizure was 4 days (range 2 – 10 days) in wt mice and 8 days (range 2 – 16 days) in cD2 KO mice and did not differ significantly between groups. Similarly, no differences were observed in median seizure frequency (wt: 1.23, range 0.1 – 3.4; cD2 KO: 0.57, range 0.1 – 2.0 seizures/day) or median seizure duration (wt: 51 s, range 23 – 103; cD2 KO: 51 s, range 23 – 103). Our results indicate that SE-induced epileptogenesis is not disrupted in mice with markedly reduced adult neurogenesis. However, we cannot exclude the contribution of reduced neurogenesis to the chronic epileptic state.

Pituitary tumors contain a side population with tumor stem cell-associated characteristics.

Pituitary adenomas cause significant endocrine and mass-related morbidity. Little is known about the mechanisms that underlie pituitary tumor pathogenesis. In the present study, we searched for a side population (SP) in pituitary tumors representing cells with high efflux capacity and potentially enriched for tumor stem cells (TSCs). Human pituitary adenomas contain a SP irrespective of hormonal phenotype. This adenoma SP, as well as the purified SP (pSP) that is depleted from endothelial and immune cells, is enriched for cells that express 'tumor stemness' markers and signaling pathways, including epithelial-mesenchymal transition (EMT)-linked factors. Pituitary adenomas were found to contain self-renewing sphere-forming cells, considered to be a property of TSCs. These sphere-initiating cells were recovered in the pSP. Because benign pituitary adenomas do not grow in vitro and have failed to expand in immunodeficient mice, the pituitary tumor cell line AtT20 was further used. We identified a SP in this cell line and found it to be more tumorigenic than the non-SP 'main population'. Of the two EMT regulatory pathways tested, the inhibition of chemokine (C-X-C motif) receptor 4 (CXCR4) signaling reduced EMT-associated cell motility in vitro as well as xenograft tumor growth, whereas the activation of TGFβ had no effect. The human adenoma pSP also showed upregulated expression of the pituitary stem cell marker SOX2. Pituitaries from dopamine receptor D2 knockout (Drd2(-/-)) mice that bear prolactinomas contain more pSP, Sox2(+), and colony-forming cells than WT glands. In conclusion, we detected a SP in pituitary tumors and identified TSC-associated characteristics. The present study adds new elements to the unraveling of pituitary tumor pathogenesis and may lead to the identification of new therapeutic targets.

The selective loss of the type 2 iodothyronine deiodinase in mouse thyrotrophs increases basal TSH but blunts the thyrotropin response to hypothyroidism.

The type 2 iodothyronine deiodinase (D2) is essential for feedback regulation of TSH by T4. We genetically inactivated in vivo D2 in thyrotrophs using a mouse model of Cga-driven cre recombinase. Pituitary D2 activity was reduced 90% in the Cga-cre D2 knockout (KO) mice compared with control Dio2(fl/fl) mice. There was no growth or reproductive phenotype. Basal TSH levels were increased 1.5- to 1.8-fold, but serum T4 and T3 were not different from the controls in adult mice. In hypothyroid adult mice, suppression of TSH by T4, but not T3, was impaired. Despite mild basal TSH elevation, the TSH increase in response to hypothyroidism was 4-fold reduced in the Cga-cre D2KO compared with control mice despite an identical level of pituitary TSH α- and β-subunit mRNAs. In neonatal Cga-cre D2KO mice, TSH was also 2-fold higher than in the controls, but serum T4 was elevated. Despite a constant TSH, serum T4 increased 2-3-fold between postnatal day (P) 5 and P15 in both genotypes. The pituitary, but not cerebrocortical, D2 activity was markedly elevated in P5 mice decreasing towards adult levels by P17. In conclusion, a congenital severe reduction of thyrotroph D2 causes a major impairment of the TSH response to hypothyroidism. This would be deleterious to the compensatory adaptation of the thyroid gland to iodine deficiency.

Abnormal motor phenotype at adult stages in mice lacking type 2 deiodinase.

BackgroundThyroid hormones have a key role in both the developing and adult central nervous system and skeletal muscle. The thyroid gland produces mainly thyroxine (T4) but the intracellular concentrations of 3,5,3′-triiodothyronine (T3; the transcriptionally active hormone) in the central nervous system and skeletal muscle are modulated by the activity of type 2 deiodinase (D2). To date no neurological syndrome has been associated with mutations in the DIO2 gene and previous studies in young and juvenile D2-knockout mice (D2KO) did not find gross neurological alterations, possibly due to compensatory mechanisms.AimThis study aims to analyze the motor phenotype of 3-and-6-month-old D2KO mice to evaluate the role of D2 on the motor system at adult stages in which compensatory mechanisms could have failed.ResultsMotor abilities were explored by validated tests. In the footprint test, D2KO showed an altered global gait pattern (mice walked slower, with shorter strides and with a hindlimb wider base of support than wild-type mice). No differences were detected in the balance beam test. However, a reduced latency to fall was found in the rotarod, coat-hanger and four limb hanging wire tests indicating impairment on coordination and prehensile reflex and a reduction of muscle strength. In histological analyses of cerebellum and skeletal muscle, D2KO mice did not present gross structural abnormalities. Thyroid hormones levels and deiodinases activities were also determined. In D2KO mice, despite euthyroid T3 and high T4 plasma levels, T3 levels were significantly reduced in cerebral cortex (48% reduction) and skeletal muscle (33% reduction), but not in the cerebellum where other deiodinase (type 1) is expressed.ConclusionsThe motor alterations observed in D2KO mice indicate an important role for D2 in T3 availability to maintain motor function and muscle strength. Our results suggest a possible implication of D2 in motor disorders.

Mice with ablated adult brain neurogenesis are not impaired in antidepressant response to chronic fluoxetine

The neurogenesis hypothesis of major depression has two main facets. One states that the illness results from decreased neurogenesis while the other claims that the very functioning of antidepressants depends on increased neurogenesis. In order to verify the latter, we have used cyclin D2 knockout mice (cD2 KO mice), known to have virtually no adult brain neurogenesis, and we demonstrate that these mice successfully respond to chronic fluoxetine. After unpredictable chronic mild stress, mutant mice showed depression-like behavior in forced swim test, which was eliminated with chronic fluoxetine treatment, despite its lack of impact on adult hippocampal neurogenesis in cD2 KO mice. Our results suggest that new neurons are not indispensable for the action of antidepressants such as fluoxetine. Using forced swim test and tail suspension test, we also did not observe depression-like behavior in control cD2 KO mice, which argues against the link between decreased adult brain neurogenesis and major depression.