Tet1 Isoforms Differentially Regulate Gene Expression, Synaptic Transmission, and Memory in the Mammalian Brain.

The dynamic regulation of DNA methylation in postmitotic neurons is necessary for memory formation and other adaptive behaviors. Ten-eleven translocation 1 (TET1) plays a part in these processes by oxidizing 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), thereby initiating active DNA demethylation. However, attempts to pinpoint its exact role in the nervous system have been hindered by contradictory findings, perhaps due in part, to a recent discovery that two isoforms of the Tet1 gene are differentially expressed from early development into adulthood. Here, we demonstrate that both the shorter transcript (Tet1S ) encoding an N-terminally truncated TET1 protein and a full-length Tet1 (Tet1FL ) transcript encoding canonical TET1 are co-expressed in the adult mouse brain. We show that Tet1S is the predominantly expressed isoform and is highly enriched in neurons, whereas Tet1FL is generally expressed at lower levels and more abundant in glia, suggesting their roles are at least partially cell type-specific. Using viral-mediated, isoform and neuron-specific molecular tools, we find that the individual repression of each transcript leads to the dysregulation of unique gene ensembles and contrasting changes in basal synaptic transmission. In addition, Tet1S repression enhances, while Tet1FL impairs, hippocampal-dependent memory in male mice. Together, our findings demonstrate that each Tet1 isoform serves a distinct role in the mammalian brain.SIGNIFICANCE STATEMENT In the brain, activity-dependent changes in gene expression are required for the formation of long-term memories. DNA methylation plays an essential role in orchestrating these learning-induced transcriptional programs by influencing chromatin accessibility and transcription factor binding. Once thought of as a stable epigenetic mark, DNA methylation is now known to be impermanent and dynamically regulated, driving neuroplasticity in the brain. We found that Tet1, a member of the ten-eleven translocation (TET) family of enzymes that mediates removal of DNA methyl marks, is expressed as two separate isoforms in the adult mouse brain and that each differentially regulates gene expression, synaptic transmission and memory formation. Together, our findings demonstrate that each Tet1 isoform serves a distinct role in the CNS.

[Alterations in ATP-dependence of swelling-activated Cl- current associated with neuroendocrine differentiation of LNCaP human prostate cancer epithelial cells].

Increasing population of malignant, apoptosis resistant neuroendocrine (NE) cells due to differentiation of prostate epithelial/basal cells is a hallmark of advanced, androgen-independent prostate cancer, for which there is no successful therapy. Acquisition of apoptosis resistance involves alterations in the mechanisms of cell volume homeostasis, of which volume-regulate anion channels (VRAC) that carry swelling-activated Cl- current (I(Cl,swell)) represent one of the key determinants. Given that VRAC function is generally known to be ATP-dependent, here we investigated how such dependence may evolve during NE differentiation of LNCaP prostate cancer epithelial cells. In the whole-cell patch-clamp recording mode I(Cl,swell) could be activated in response to hypotonicity-induced cell swelling in control and NE-differentiated (by incubation in membrane-permeable cAMP analogs) LNCaP cells even following total depletion of intracellular ATP using a cocktail of metabolic inhibitors. However, this basal I(Cl,swell) had about 30% higher density and was less inactivating in NE-differentiated cells. Inclusion of 5 mM Mg-ATP in the patch pipette caused I(Cl,swell) augmentation in both cell types. The augmentation in the control cells was more prominent and occurred mostly at the expense of a non-inactivating current component. We conclude that I(Cl,swell) in LNCaP cells consists of a non-inactivating, ATP-dependent and inactivating, ATP-independent components. NE differentiation promotes the increase of non-inactivating component and partial loss of its ATP sensitivity making the whole I(Cl,swell) less ATP-sensitive as well. By largely avoiding the ATP metabolic control I(Cl,swell) may contribute to better control of cell volume under metabolic stress and thus enhance the survival rates of apoptosis-resistant NE cells.

Alterations in the regulatory volume decrease (RVD) and swelling-activated Cl- current associated with neuroendocrine differentiation of prostate cancer epithelial cells.

Neuroendocrine (NE) differentiation of prostate epithelial/basal cells is a hallmark of advanced, androgen-independent prostate cancer, for which there is no successful therapy. Here we report for the first time on alterations in regulatory volume decrease (RVD) and its key determinant, swelling-activated Cl- current (I(Cl,swell)), associated with NE differentiation of androgen-dependent LNCaP prostate cancer epithelial cells. NE-differentiating regimens, namely, chronic cAMP elevation or androgen deprivation, resulted in generally augmented I(Cl,swell) and enhanced RVD. This occurred as a result of both the increased endogenous expression of ClC-3, which is a volume-sensitive Cl- channel involved, as we show, in I(Cl,swell) in LNCaP (lymph-node carcinoma of the prostate) cells and the weaker negative I(Cl,swell) control from Ca2+ entering via store-dependent pathways. The changes in the RVD of NE-differentiated cells generally mimicked those reported for Bcl-2-conferred apoptotic resistance. Our results suggest that strengthening the mechanism that helps to maintain volume constancy may contribute to better survival rates of apoptosis-resistant NE cells.

[Effect of neuroendocrine cell differentiation of the human prostatic neoplasm cell line LNCaP on the characteristics of volume-sensitive chloride content]. / Vplyv neiroendokrynnoho dyferentsiiuvannia klityn raku prostaty liudyny liniï LNCaP na kharakterystyky ob'iemchutlyvoho khlornoho strumu.

By means of patch-clamp technique we examined changes in volume-regulated chloride current (ICl,swell) at neuroendocrine differentiation of androgen-dependent LNCaP prostate cancer cells. In those cells with neuroendocrine differentiation resulted from an increase in the intracellular cAMP, ICl,swell became much faster in response to applying external hypotonic solution and cell swelling. Changes in final rectification and voltage-dependent inactivation were not detected, as compared to the control cells. The differentiation also diminished ICl,swell blockade by Ca2+ transported via store-operated channels (SOC). On the base of our data we suggest that potentiation of the current at neuroendocrine differentiation, at least in part, resulted from a decrease in an inhibitory effect of Ca2+, transported into a cell through SOC, on volume-sensitive chloride current. Accelerated current in those cells might be induced by cytoskeleton rearrangement at the neuron-like growth.

[Effect of intracellular trypsin on characteristics of voltage-dependent inactivation of chloride currents in prostatic cancer cells]. / Vplyv vnutrishn'oklitynnoho vvedennia trypsynu na kharaterystyky potentsialzalezhnoï inaktyvatsiï khlornoho strumu v klitynakh raku prostaty.

By means of the patch-clamp technique we have studied the effects of intracellular applied trypsin, a known modulator of membrane channel function, on the properties of the Cl- current induced by hypotonicity-obliged cell swelling (ICl, swell) in human prostate cancer epithelial cells, LNCaP. Intracellular infusion of 1 mg/ml of trypsin into LNCaP cells via the patch pipette shortened the delay for the onset and the time of development of ICl, swell in response to hypotonicity as well as accelerated the rate of current diminution following the return to isotonic conditions. The maximal density of ICl, swell in the presence of intracellular trypsin was 2-fold higher while the current voltage-dependent inactivation at high depolarizing potentials was virtually eliminated. Intracellular co-application of the trypsin inhibitor together with trypsin abolished all effects of trypsin. We conclude that VRACs share a great degree of functional and structural homology to voltage-gated Na+, K+ and Cl- channels by having intracellular inactivation domain subjected to proteolytic cleavage that function in conformity with "ball-and-chain" inactivation model.