New germline BRCA2 gene variant in the Tuvinian Mongol breast cancer patients.

To date, there are a limited number of reports on inherited gene mutations associated with breast cancer (BC) among Mongoloid indigenous people in Russia. The present study aimed at identifying the BC-associated genes in 26 Russian Mongoloid BC patients (Buryats, Tuvinians and others). The median age of the patients at the time of breast cancer diagnosis was 41 years (range 25-51 years). Genomic DNA isolated from blood samples was used to prepare libraries using a capture-based target enrichment kit (Hereditary Cancer Solution™, SOPHiA GENETICS, Switzerland) covering 27 genes (ATM, APC, BARD1, BRCA1, BRCA2, BRIP1, CDH1, CHEK2, EPCAM, FAM175A, MLH1, MRE11A, MSH2, MSH6, MUTYH, NBN, PALB2, PIK3CA, PMS2, PMS2CL, PTEN, RAD50, RAD51C, RAD51D, STK11, TP53 and XRCC2). Next-generation sequencing (NGS) was performed on an Illumina NextSeq 500 System (Illumina, USA). In our study, we found 1 Indel and 11 SNPs that passed filters during variant calling. We identified a highly pathogenic germline rs483353122 (c.8208_8209insAG, p.Leu2737Serfs*2) in the BRCA2 gene in six unrelated Tuvinian Mongol BC patients. We also identified a likely damaging germline rs35352891 in the MUTYH gene (c.1118C>T, p.Ala373Val) in one Buryat Mongol BC patient. Other SNPs were classified as variants of uncertain significance. To the best of our knowledge, this report is the first to describe the highly pathogenic variant in the BRCA2 gene (rs483353122) and the likely damaging germline variant in the MUTYH gene (rs35352891) in Russian Mongoloid BC patients with young-onset and/or bilateral and/or familial BC. Further studies are therefore necessary to evaluate the contributions of novel sequence variants to hereditary BC.

Deletion of ecto-5'-nucleotidase (CD73) reveals direct action potential-dependent adenosine release.

Purinergic signaling is a highly complex system of extracellular communication involved in many physiological and pathological functions in the mammalian brain. Its complexity stems from the multitude of purine receptor subtypes and endogenous purine receptor ligands (including ATP, ADP, UTP, UDP, and adenosine). Potentially all of these ligands could be directly released, and some could also arise from extracellular metabolism. A widely held consensus is that, except under pathological conditions, extracellular adenosine arises only from ectoATPase-mediated metabolism of previously released ATP. Here, we have used mice that lack the CD73 gene (encoding ecto-5'-nucleotidase that converts AMP to adenosine) to test whether action potential-dependent adenosine release in the cerebellum depends on prior ATP release. Surprisingly, we have uncovered two parallel pathways of adenosine release: one that is indirect via glutamate receptor-dependent release of ATP and a second of equal amplitude that has no dependence on prior release of ATP and thus represents the direct release of adenosine. This component of adenosine release is blocked by bafilomycin and modulated by mGlu4 receptor activation, strongly supporting adenosine release by exocytosis from parallel fibers. Our findings are a major step in understanding the mechanisms of adenosine release and are likely to have implications for all aspects of physiology where adenosine plays a key modulatory role.

Receptor-mediated modulation of activity-dependent adenosine release in rat cerebellum.

Although the neuromodulator adenosine plays an important role in many central nervous system physiological and pathological processes, the properties and mechanisms of extracellular adenosine production are still unclear. In previous work, we determined that two forms of adenosine release can be evoked in the molecular layer of the cerebellum: one independent of ionotropic glutamate receptor activation (evoked by a train of stimuli) and one mainly dependent on the activation of ionotropic glutamate receptors (evoked by a single stimulus in 4-aminopyridine). Here we have investigated how these different forms of adenosine release are modulated by metabotropic receptors (A(1), GABA(B) and mGlu4). Although both types of adenosine release are inhibited by the activation of metabotropic receptors, single stimulus-evoked release was much more potently inhibited suggesting differential coupling between receptors and adenosine release mechanisms. Metabotropic receptor antagonists revealed that endogenous A(1) receptor activation plays the major role in controlling adenosine release and determine the relationship between stimulus strength and adenosine release. The major mechanism of modulation is through control of ionotropic glutamate receptor activation with block of metabotropic receptors inducing glutamate receptor-dependent adenosine release. In contrast to metabotropic receptor agonists, which inhibit adenylyl cyclase, activation of adenylyl cyclase (with forskolin) increased both glutamate receptor-dependent and independent adenosine release. This is the first time that the control of adenosine release by endogenous modulators has been studied and like classical neurotransmitters, adenosine release is controlled by an interplay of presynaptic modulators. This article is part of a Special Issue entitled 'Post-Traumatic Stress Disorder'.

L-Dopa activates histaminergic neurons.

L-Dopa is the most effective treatment of early and advanced stages of Parkinson's disease (PD), but its chronic use leads to loss of efficiency and dyskinesia. This is delayed by lower dosage at early stages, made possible by additional treatment with histamine antagonists. We present here evidence that histaminergic tuberomamillary nucleus (TMN) neurons, involved in the control of wakefulness, are excited under L-Dopa (EC50 15 µM), express Dopa decarboxylase and show dopamine immunoreactivity. Dopaergic excitation was investigated with patch-clamp recordings from brain slices combined with single-cell RT-PCR analysis of dopamine receptor expression. In addition to the excitatory dopamine 1 (D1)-like receptors, TMN neurons express D2-like receptors, which are coupled through phospholipase C (PLC) to transient receptor potential canonical (TRPC) channels and the Na+/Ca2+ exchanger. D2 receptor activation enhances firing frequency, histamine release in freely moving rats (microdialysis) and wakefulness (EEG recordings). In histamine deficient mice the wake-promoting action of the D2 receptor agonist quinpirole (1 mg kg⁻¹, I.P.) is missing. Thus the histamine neurons can, subsequent to L-Dopa uptake, co-release dopamine and histamine from their widely projecting axons. Taking into consideration the high density of histaminergic fibres and the histamine H3 receptor heteromerization either with D1 or with D2 receptors in the striatum, this study predicts new avenues for PD therapy.

The dynamics of single spike-evoked adenosine release in the cerebellum.

The purine adenosine is a potent neuromodulator in the brain, with roles in a number of diverse physiological and pathological processes. Modulators such as adenosine are difficult to study as once released they have a diffuse action (which can affect many neurones) and, unlike classical neurotransmitters, have no inotropic receptors. Thus rapid postsynaptic currents (PSCs) mediated by adenosine (equivalent to mPSCs) are not available for study. As a result the mechanisms and properties of adenosine release still remain relatively unclear. We have studied adenosine release evoked by stimulating the parallel fibres in the cerebellum. Using adenosine biosensors combined with deconvolution analysis and mathematical modelling, we have characterised the release dynamics and diffusion of adenosine in unprecedented detail. By partially blocking K+ channels, we were able to release adenosine in response to a single stimulus rather than a train of stimuli. This allowed reliable sub-second release of reproducible quantities of adenosine with stereotypic concentration waveforms that agreed well with predictions of a mathematical model of purine diffusion. We found no evidence for ATP release and thus suggest that adenosine is directly released in response to parallel fibre firing and does not arise from extracellular ATP metabolism. Adenosine release events showed novel short-term dynamics, including facilitated release with paired stimuli at millisecond stimulation intervals but depletion-recovery dynamics with paired stimuli delivered over minute time scales. These results demonstrate rich dynamics for adenosine release that are placed, for the first time, on a quantitative footing and show strong similarity with vesicular exocytosis.

Excitation of histaminergic tuberomamillary neurons by thyrotropin-releasing hormone.

The histaminergic tuberomamillary nucleus (TMN) controls arousal and attention, and the firing of TMN neurons is state-dependent, active during waking, silent during sleep. Thyrotropin-releasing hormone (TRH) promotes arousal and combats sleepiness associated with narcolepsy. Single-cell reverse-transcription-PCR demonstrated variable expression of the two known TRH receptors in the majority of TMN neurons. TRH increased the firing rate of most (ca 70%) TMN neurons. This excitation was abolished by the TRH receptor antagonist chlordiazepoxide (CDZ; 50 mum). In the presence of tetrodotoxin (TTX), TRH depolarized TMN neurons without obvious change of their input resistance. This effect reversed at the potential typical for nonselective cation channels. The potassium channel blockers barium and cesium did not influence the TRH-induced depolarization. TRH effects were antagonized by inhibitors of the Na(+)/Ca(2+) exchanger, KB-R7943 and benzamil. The frequency of GABAergic spontaneous IPSCs was either increased (TTX-insensitive) or decreased [TTX-sensitive spontaneous IPSCs (sIPSCs)] by TRH, indicating a heterogeneous modulation of GABAergic inputs by TRH. Facilitation but not depression of sIPSC frequency by TRH was missing in the presence of the kappa-opioid receptor antagonist nor-binaltorphimine. Montirelin (TRH analog, 1 mg/kg, i.p.) induced waking in wild-type mice but not in histidine decarboxylase knock-out mice lacking histamine. Inhibition of histamine synthesis by (S)-alpha-fluoromethylhistidine blocked the arousal effect of montirelin in wild-type mice. We conclude that direct receptor-mediated excitation of rodent TMN neurons by TRH demands activation of nonselective cation channels as well as electrogenic Na(+)/Ca(2+) exchange. Our findings indicate a key role of the brain histamine system in TRH-induced arousal.

P2Y receptor-mediated excitation in the posterior hypothalamus.

Histaminergic neurons located in the posterior hypothalamus (tuberomamillary nucleus, TMN) project widely through the whole brain controlling arousal and attention. They are tonically active during wakefulness but cease firing during sleep. As a homeostatic theory of sleep involves ATP depletion and adenosine accumulation in the brain, we investigated the role of ATP and its analogues as well as adenosine on neuronal activity in the TMN. We show increased firing of rat TMN neurons by ATP, ADP, UTP and 2meSATP, indicating activation of receptors belonging to the P2Y family. Adenosine affected neither membrane potential nor firing of these cells. Single-cell reverse transcriptase-polymerase chain reaction revealed that P2Y1 and P2Y4 are prevailing receptors in TMN neurons. P2Y1 receptor mRNA was detected with a higher frequency in 2-week-old than in 4-week-old rats; in accordance, 2meSATP was more potent than ATP. Semi-quantitative real-time polymerase chain reaction revealed a developmental downregulation of mRNA levels for P2Y1 and P2Y4 receptors. Immunocytochemistry demonstrated neuronal and glial localization of the P2Y1 receptor protein. Network activity measured with multielectrode arrays in primary cultures made from the posterior hypothalamus was enhanced by UTP and 2meSATP (P2Y4 and P2Y1 agonists, respectively). ATP caused an inhibition of firing, which was reversed in the presence of suramin or gabazine [gamma-aminobutyric acid (GABA)A receptor antagonist], indicating that GABAergic neurons are preferentially activated by ATP in this network. Excitation of the wake-active TMN neurons by nucleotides and the lack of adenosine action may be important factors in sleep-wake regulation.