Biallelic variants in SLC4A10 encoding a sodium-dependent bicarbonate transporter lead to a neurodevelopmental disorder.

PURPOSE: SLC4A10 encodes a plasma membrane-bound transporter, which mediates Na+-dependent HCO3- import, thus mediating net acid extrusion. Slc4a10 knockout mice show collapsed brain ventricles, an increased seizure threshold, mild behavioral abnormalities, impaired vision, and deafness. METHODS: Utilizing exome/genome sequencing in families with undiagnosed neurodevelopmental disorders and international data sharing, 11 patients from 6 independent families with biallelic variants in SLC4A10 were identified. Clinico-radiological and dysmorphology assessments were conducted. A minigene assay, localization studies, intracellular pH recordings, and protein modeling were performed to study the possible functional consequences of the variant alleles. RESULTS: The families harbor 8 segregating ultra-rare biallelic SLC4A10 variants (7 missense and 1 splicing). Phenotypically, patients present with global developmental delay/intellectual disability and central hypotonia, accompanied by variable speech delay, microcephaly, cerebellar ataxia, facial dysmorphism, and infrequently, epilepsy. Neuroimaging features range from some non-specific to distinct neuroradiological findings, including slit ventricles and a peculiar form of bilateral curvilinear nodular heterotopia. In silico analyses showed 6 of 7 missense variants affect evolutionarily conserved residues. Functional analyses supported the pathogenicity of 4 of 7 missense variants. CONCLUSION: We provide evidence that pathogenic biallelic SLC4A10 variants can lead to neurodevelopmental disorders characterized by variable abnormalities of the central nervous system, including altered brain ventricles, thus resembling several features observed in knockout mice.

SLC4A10 mutation causes a neurological disorder associated with impaired GABAergic transmission.

SLC4A10 is a plasma-membrane bound transporter that utilizes the Na+ gradient to drive cellular HCO3- uptake, thus mediating acid extrusion. In the mammalian brain, SLC4A10 is expressed in principal neurons and interneurons, as well as in epithelial cells of the choroid plexus, the organ regulating the production of CSF. Using next generation sequencing on samples from five unrelated families encompassing nine affected individuals, we show that biallelic SLC4A10 loss-of-function variants cause a clinically recognizable neurodevelopmental disorder in humans. The cardinal clinical features of the condition include hypotonia in infancy, delayed psychomotor development across all domains and intellectual impairment. Affected individuals commonly display traits associated with autistic spectrum disorder including anxiety, hyperactivity and stereotyped movements. In two cases isolated episodes of seizures were reported in the first few years of life, and a further affected child displayed bitemporal epileptogenic discharges on EEG without overt clinical seizures. While occipitofrontal circumference was reported to be normal at birth, progressive postnatal microcephaly evolved in 7 out of 10 affected individuals. Neuroradiological features included a relative preservation of brain volume compared to occipitofrontal circumference, characteristic narrow sometimes 'slit-like' lateral ventricles and corpus callosum abnormalities. Slc4a10 -/- mice, deficient for SLC4A10, also display small lateral brain ventricles and mild behavioural abnormalities including delayed habituation and alterations in the two-object novel object recognition task. Collapsed brain ventricles in both Slc4a10-/- mice and affected individuals suggest an important role of SLC4A10 in the production of the CSF. However, it is notable that despite diverse roles of the CSF in the developing and adult brain, the cortex of Slc4a10-/- mice appears grossly intact. Co-staining with synaptic markers revealed that in neurons, SLC4A10 localizes to inhibitory, but not excitatory, presynapses. These findings are supported by our functional studies, which show the release of the inhibitory neurotransmitter GABA is compromised in Slc4a10-/- mice, while the release of the excitatory neurotransmitter glutamate is preserved. Manipulation of intracellular pH partially rescues GABA release. Together our studies define a novel neurodevelopmental disorder associated with biallelic pathogenic variants in SLC4A10 and highlight the importance of further analyses of the consequences of SLC4A10 loss-of-function for brain development, synaptic transmission and network properties.

Heteromeric clusters of ubiquitinated ER-shaping proteins drive ER-phagy.

Membrane-shaping proteins characterized by reticulon homology domains play an important part in the dynamic remodelling of the endoplasmic reticulum (ER). An example of such a protein is FAM134B, which can bind LC3 proteins and mediate the degradation of ER sheets through selective autophagy (ER-phagy)1. Mutations in FAM134B result in a neurodegenerative disorder in humans that mainly affects sensory and autonomic neurons2. Here we report that ARL6IP1, another ER-shaping protein that contains a reticulon homology domain and is associated with sensory loss3, interacts with FAM134B and participates in the formation of heteromeric multi-protein clusters required for ER-phagy. Moreover, ubiquitination of ARL6IP1 promotes this process. Accordingly, disruption of Arl6ip1 in mice causes an expansion of ER sheets in sensory neurons that degenerate over time. Primary cells obtained from Arl6ip1-deficient mice or from patients display incomplete budding of ER membranes and severe impairment of ER-phagy flux. Therefore, we propose that the clustering of ubiquitinated ER-shaping proteins facilitates the dynamic remodelling of the ER during ER-phagy and is important for neuronal maintenance.

Triac Treatment Prevents Neurodevelopmental and Locomotor Impairments in Thyroid Hormone Transporter Mct8/Oatp1c1 Deficient Mice.

Patients with inactive thyroid hormone (TH) transporter MCT8 display intellectual disability due to compromised central TH transport and action. As a therapeutic strategy, application of thyromimetic, MCT8-independent compounds Triac (3,5,3'-triiodothyroacetic acid), and Ditpa (3,5-diiodo-thyropropionic acid) was proposed. Here, we directly compared their thyromimetic potential in Mct8/Oatp1c1 double knock-out mice (Dko) modeling human MCT8 deficiency. Dko mice received either Triac (50 ng/g or 400 ng/g) or Ditpa (400 ng/g or 4000 ng/g) daily during the first three postnatal weeks. Saline-injected Wt and Dko mice served as controls. A second cohort of Dko mice received Triac (400 ng/g) daily between postnatal weeks 3 and 6. Thyromimetic effects were assessed at different postnatal stages by immunofluorescence, ISH, qPCR, electrophysiological recordings, and behavior tests. Triac treatment (400 ng/g) induced normalized myelination, cortical GABAergic interneuron differentiation, electrophysiological parameters, and locomotor performance only when administered during the first three postnatal weeks. Ditpa (4000 ng/g) application to Dko mice during the first three postnatal weeks resulted in normal myelination and cerebellar development but only mildly improved neuronal parameters and locomotor function. Together, Triac is highly-effective and more efficient than Ditpa in promoting CNS maturation and function in Dko mice yet needs to be initiated directly after birth for the most beneficial effects.

GMPPA defects cause a neuromuscular disorder with α-dystroglycan hyperglycosylation.

GDP-mannose-pyrophosphorylase-B (GMPPB) facilitates the generation of GDP-mannose, a sugar donor required for glycosylation. GMPPB defects cause muscle disease due to hypoglycosylation of α-dystroglycan (α-DG). Alpha-DG is part of a protein complex, which links the extracellular matrix with the cytoskeleton, thus stabilizing myofibers. Mutations of the catalytically inactive homolog GMPPA cause alacrima, achalasia, and mental retardation syndrome (AAMR syndrome), which also involves muscle weakness. Here, we showed that Gmppa-KO mice recapitulated cognitive and motor deficits. As structural correlates, we found cortical layering defects, progressive neuron loss, and myopathic alterations. Increased GDP-mannose levels in skeletal muscle and in vitro assays identified GMPPA as an allosteric feedback inhibitor of GMPPB. Thus, its disruption enhanced mannose incorporation into glycoproteins, including α-DG in mice and humans. This increased α-DG turnover and thereby lowered α-DG abundance. In mice, dietary mannose restriction beginning after weaning corrected α-DG hyperglycosylation and abundance, normalized skeletal muscle morphology, and prevented neuron degeneration and the development of motor deficits. Cortical layering and cognitive performance, however, were not improved. We thus identified GMPPA defects as the first congenital disorder of glycosylation characterized by α-DG hyperglycosylation, to our knowledge, and we have unraveled underlying disease mechanisms and identified potential dietary treatment options.

Disruption of KCC2 in Parvalbumin-Positive Interneurons Is Associated With a Decreased Seizure Threshold and a Progressive Loss of Parvalbumin-Positive Interneurons.

GABAA receptors are ligand-gated ion channels, which are predominantly permeable for chloride. The neuronal K-Cl cotransporter KCC2 lowers the intraneuronal chloride concentration and thus plays an important role for GABA signaling. KCC2 loss-of-function is associated with seizures and epilepsy. Here, we show that KCC2 is expressed in the majority of parvalbumin-positive interneurons (PV-INs) of the mouse brain. PV-INs receive excitatory input from principle cells and in turn control principle cell activity by perisomatic inhibition and inhibitory input from other interneurons. Upon Cre-mediated disruption of KCC2 in mice, the polarity of the GABA response of PV-INs changed from hyperpolarization to depolarization for the majority of PV-INs. Reduced excitatory postsynaptic potential-spike (E-S) coupling and increased spontaneous inhibitory postsynaptic current (sIPSC) frequencies further suggest that PV-INs are disinhibited upon disruption of KCC2. In vivo, PV-IN-specific KCC2 knockout mice display a reduced seizure threshold and develop spontaneous sometimes fatal seizures. We further found a time dependent loss of PV-INs, which was preceded by an up-regulation of pro-apoptotic genes upon disruption of KCC2.

Gain-of-function mutation in SCN11A causes itch and affects neurogenic inflammation and muscle function in Scn11a+/L799P mice.

Mutations in the genes encoding for voltage-gated sodium channels cause profound sensory disturbances and other symptoms dependent on the distribution of a particular channel subtype in different organs. Humans with the gain-of-function mutation p.Leu811Pro in SCN11A (encoding for the voltage-gated Nav1.9 channel) exhibit congenital insensitivity to pain, pruritus, self-inflicted injuries, slow healing wounds, muscle weakness, Charcot-like arthropathies, and intestinal dysmotility. As already shown, knock-in mice (Scn11a+/L799P) carrying the orthologous mutation p.Leu799Pro replicate reduced pain sensitivity and show frequent tissue lesions. In the present study we explored whether Scn11a+/L799P mice develop also pruritus, muscle weakness, and changes in gastrointestinal transit time. Furthermore, we analyzed morphological and functional differences in nerves, skeletal muscle, joints and small intestine from Scn11a+/L799P and Scn11a+/+ wild type mice. Compared to Scn11a+/+ mice, Scn11a+/L799P mice showed enhanced scratching bouts before skin lesions developed, indicating pruritus. Scn11a+/L799P mice exhibited reduced grip strength, but no disturbances in motor coordination. Skeletal muscle fiber types and joint architecture were unaltered in Scn11a+/L799P mice. Their gastrointestinal transit time was unaltered. The small intestine from Scn11a+/L799P showed a small shift towards less frequent peristaltic movements. Similar proportions of lumbar dorsal root ganglion neurons from Scn11a+/L799P and Scn11a+/+ mice were calcitonin gene-related peptide (CGRP-) positive, but isolated sciatic nerves from Scn11a+/L799P mice exhibited a significant reduction of the capsaicin-evoked release of CGRP indicating reduced neurogenic inflammation. These data indicate important Nav1.9 channel functions in several organs in both humans and mice. They support the pathophysiological relevance of increased basal activity of Nav1.9 channels for sensory abnormalities (pain and itch) and suggest resulting malfunctions of the motor system and of the gastrointestinal tract. Scn11a+/L799P mice are suitable to investigate the role of Nav1.9, and to explore the pathophysiological changes and mechanisms which develop as a consequence of Nav1.9 hyperactivity.

DNA Methylation-Mediated Modulation of Endocytosis as Potential Mechanism for Synaptic Function Regulation in Murine Inhibitory Cortical Interneurons.

The balance of excitation and inhibition is essential for cortical information processing, relying on the tight orchestration of the underlying subcellular processes. Dynamic transcriptional control by DNA methylation, catalyzed by DNA methyltransferases (DNMTs), and DNA demethylation, achieved by ten-eleven translocation (TET)-dependent mechanisms, is proposed to regulate synaptic function in the adult brain with implications for learning and memory. However, focus so far is laid on excitatory neurons. Given the crucial role of inhibitory cortical interneurons in cortical information processing and in disease, deciphering the cellular and molecular mechanisms of GABAergic transmission is fundamental. The emerging relevance of DNMT and TET-mediated functions for synaptic regulation irrevocably raises the question for the targeted subcellular processes and mechanisms. In this study, we analyzed the role dynamic DNA methylation has in regulating cortical interneuron function. We found that DNMT1 and TET1/TET3 contrarily modulate clathrin-mediated endocytosis. Moreover, we provide evidence that DNMT1 influences synaptic vesicle replenishment and GABAergic transmission, presumably through the DNA methylation-dependent transcriptional control over endocytosis-related genes. The relevance of our findings is supported by human brain sample analysis, pointing to a potential implication of DNA methylation-dependent endocytosis regulation in the pathophysiology of temporal lobe epilepsy, a disease characterized by disturbed synaptic transmission.

Visual deprivation independent shift of ocular dominance induced by cross-modal plasticity.

There is convincing evidence that the deprivation of one sense can lead to adaptive neuronal changes in spared primary sensory cortices. However, the repercussions of late-onset sensory deprivations on functionality of the remaining sensory cortices are poorly understood. Using repeated intrinsic signal imaging we investigated the effects of whisker or auditory deprivation (WD or AD, respectively) on responsiveness of the binocular primary visual cortex (V1) in fully adult mice. The binocular zone of mice is innervated by both eyes, with the contralateral eye always dominating V1 input over ipsilateral eye input, the normal ocular dominance (OD) ratio. Strikingly, we found that 3 days of WD or AD induced a transient shift of OD, which was mediated by a potentiation of V1 input through the ipsilateral eye. This cross-modal effect was accompanied by strengthening of layer 4 synapses in V1, required visual experience through the ipsilateral eye and was mediated by an increase of the excitation/inhibition ratio in V1. Finally, we demonstrate that both WD and AD induced a long-lasting improvement of visual performance. Our data provide evidence that the deprivation of a non-visual sensory modality cross-modally induces experience dependent V1 plasticity and improves visual behavior, even in adult mice.

The Na+/H+ Exchanger Nhe1 Modulates Network Excitability via GABA Release.

Brain functions are extremely sensitive to pH changes because of the pH-dependence of proteins involved in neuronal excitability and synaptic transmission. Here, we show that the Na+/H+ exchanger Nhe1, which uses the Na+ gradient to extrude H+, is expressed at both inhibitory and excitatory presynapses. We disrupted Nhe1 specifically in mice either in Emx1-positive glutamatergic neurons or in parvalbumin-positive cells, mainly GABAergic interneurons. While Nhe1 disruption in excitatory neurons had no effect on overall network excitability, mice with disruption of Nhe1 in parvalbumin-positive neurons displayed epileptic activity. From our electrophysiological analyses in the CA1 of the hippocampus, we conclude that the disruption in parvalbumin-positive neurons impairs the release of GABA-loaded vesicles, but increases the size of GABA quanta. The latter is most likely an indirect pH-dependent effect, as Nhe1 was not expressed in purified synaptic vesicles itself. Conclusively, our data provide first evidence that Nhe1 affects network excitability via modulation of inhibitory interneurons.

Homeostatic plasticity and synaptic scaling in the adult mouse auditory cortex.

It has been demonstrated that sensory deprivation results in homeostatic adjustments recovering neuronal activity of the deprived cortex. For example, deprived vision multiplicatively scales up mEPSC amplitudes in the primary visual cortex, commonly referred to as synaptic scaling. However, whether synaptic scaling also occurs in auditory cortex after auditory deprivation remains elusive. Using periodic intrinsic optical imaging in adult mice, we show that conductive hearing loss (CHL), initially led to a reduction of primary auditory cortex (A1) responsiveness to sounds. However, this was followed by a complete recovery of A1 activity evoked sounds above the threshold for bone conduction, 3 days after CHL. Over the same time course patch-clamp experiments in slices revealed that mEPSC amplitudes in A1 layers 2/3 pyramids scaled up multiplicatively in CHL mice. No recovery of sensory evoked A1 activation was evident in TNFα KO animals, which lack synaptic scaling. Additionally, we could show that the suppressive effect of sounds on visually evoked visual cortex activity completely recovered along with TNFα dependent A1 homeostasis in WT animals. This is the first demonstration of homeostatic multiplicative synaptic scaling in the adult A1. These findings suggest that mild hearing loss massively affects auditory processing in adult A1.

The sorting receptor Rer1 controls Purkinje cell function via voltage gated sodium channels.

Rer1 is a sorting receptor in the early secretory pathway that controls the assembly and the cell surface transport of selected multimeric membrane protein complexes. Mice with a Purkinje cell (PC) specific deletion of Rer1 showed normal polarization and differentiation of PCs and normal development of the cerebellum. However, PC-specific loss of Rer1 led to age-dependent motor deficits in beam walk, ladder climbing and gait. Analysis of brain sections revealed a specific degeneration of PCs in the anterior cerebellar lobe in old animals. Electrophysiological recordings demonstrated severe deficits in spontaneous action potential generation. Measurements of resurgent currents indicated decreased surface densities of voltage-gated sodium channels (Nav), but not changes in individual channels. Analysis of mice with a whole brain Rer1-deletion demonstrated a strong down-regulation of Nav1.6 and 1.1 in the absence of Rer1, whereas protein levels of the related Cav2.1 and of Kv3.3 and 7.2 channels were not affected. The data suggest that Rer1 controls the assembly and transport of Nav1.1 and 1.6, the principal sodium channels responsible for recurrent firing, in PCs.

NKCC1-Mediated GABAergic Signaling Promotes Postnatal Cell Death in Neocortical Cajal-Retzius Cells.

During early development, a substantial proportion of central neurons undergoes programmed cell death. This activity-dependent process is essential for the proper structural and functional development of the brain. To uncover cell type-specific differences in the regulation of neuronal survival versus apoptosis, we studied activity-regulated cell death in Cajal-Retzius neurons (CRNs) and the overall neuronal population in the developing mouse cerebral cortex. CRNs in the upper neocortical layer represent an early-born neuronal population, which is important for cortical development and largely disappears by apoptosis during neonatal stages. In contrast to the overall neuronal population, activity blockade with tetrodotoxin improved survival of CRNs in culture. Activation of GABAA receptors also blocked spontaneous activity and caused overall cell death including apoptosis of CRNs. Blockade of the Na-K-Cl transporter NKCC1 in vitro or its genetic deletion in vivo rescued CRNs from apoptosis. This effect was mediated by blockade of the p75NTR receptor signaling pathway. In summary, we discovered a novel developmental death pathway mediated by NKCC1, via GABAA receptor-mediated membrane depolarization and p75NTR signaling in CRNs. This pathway controls apoptosis of CRNs and may be critically involved in neurodevelopmental disorders such as autism and schizophrenia.

Reduced tonic inhibition after stroke promotes motor performance and epileptic seizures.

Stroke survivors often recover from motor deficits, either spontaneously or with the support of rehabilitative training. Since tonic GABAergic inhibition controls network excitability, it may be involved in recovery. Middle cerebral artery occlusion in rodents reduces tonic GABAergic inhibition in the structurally intact motor cortex (M1). Transcript and protein abundance of the extrasynaptic GABAA-receptor complex α4ß3δ are concurrently reduced (δ-GABAARs). In vivo and in vitro analyses show that stroke-induced glutamate release activates NMDA receptors, thereby reducing KCC2 transporters and down-regulates δ-GABAARs. Functionally, this is associated with improved motor performance on the RotaRod, a test in which mice are forced to move in a similar manner to rehabilitative training sessions. As an adverse side effect, decreased tonic inhibition facilitates post-stroke epileptic seizures. Our data imply that early and sometimes surprisingly fast recovery following stroke is supported by homeostatic, endogenous plasticity of extrasynaptic GABAA receptors.

Disruption of Slc4a10 augments neuronal excitability and modulates synaptic short-term plasticity.

Slc4a10 is a Na(+)-coupled Cl(-)-HCO3 (-) exchanger, which is expressed in principal and inhibitory neurons as well as in choroid plexus epithelial cells of the brain. Slc4a10 knockout (KO) mice have collapsed brain ventricles and display an increased seizure threshold, while heterozygous deletions in man have been associated with idiopathic epilepsy and other neurological symptoms. To further characterize the role of Slc4a10 for network excitability, we compared input-output relations as well as short and long term changes of evoked field potentials in Slc4a10 KO and wildtype (WT) mice. While responses of CA1 pyramidal neurons to stimulation of Schaffer collaterals were increased in Slc4a10 KO mice, evoked field potentials did not differ between genotypes in the stratum radiatum or the neocortical areas analyzed. Paired pulse facilitation was diminished in the hippocampus upon disruption of Slc4a10. In the neocortex paired pulse depression was increased. Though short term plasticity is modulated via Slc4a10, long term potentiation appears independent of Slc4a10. Our data support that Slc4a10 dampens neuronal excitability and thus sheds light on the pathophysiology of SLC4A10 associated pathologies.

Regulation of endoplasmic reticulum turnover by selective autophagy.

The endoplasmic reticulum (ER) is the largest intracellular endomembrane system, enabling protein and lipid synthesis, ion homeostasis, quality control of newly synthesized proteins and organelle communication. Constant ER turnover and modulation is needed to meet different cellular requirements and autophagy has an important role in this process. However, its underlying regulatory mechanisms remain unexplained. Here we show that members of the FAM134 reticulon protein family are ER-resident receptors that bind to autophagy modifiers LC3 and GABARAP, and facilitate ER degradation by autophagy ('ER-phagy'). Downregulation of FAM134B protein in human cells causes an expansion of the ER, while FAM134B overexpression results in ER fragmentation and lysosomal degradation. Mutant FAM134B proteins that cause sensory neuropathy in humans are unable to act as ER-phagy receptors. Consistently, disruption of Fam134b in mice causes expansion of the ER, inhibits ER turnover, sensitizes cells to stress-induced apoptotic cell death and leads to degeneration of sensory neurons. Therefore, selective ER-phagy via FAM134 proteins is indispensable for mammalian cell homeostasis and controls ER morphology and turnover in mice and humans.

ProSAP1 and membrane nanodomain-associated syndapin I promote postsynapse formation and function.

Insights into mechanisms coordinating membrane remodeling, local actin nucleation, and postsynaptic scaffolding during postsynapse formation are important for understanding vertebrate brain function. Gene knockout and RNAi in individual neurons reveal that the F-BAR protein syndapin I is a crucial postsynaptic coordinator in formation of excitatory synapses. Syndapin I deficiency caused significant reductions of synapse and dendritic spine densities. These syndapin I functions reflected direct, SH3 domain-mediated associations and functional interactions with ProSAP1/Shank2. They furthermore required F-BAR domain-mediated membrane binding. Ultra-high-resolution imaging of specifically membrane-associated, endogenous syndapin I at membranes of freeze-fractured neurons revealed that membrane-bound syndapin I preferentially occurred in spines and formed clusters at distinct postsynaptic membrane subareas. Postsynaptic syndapin I deficiency led to reduced frequencies of miniature excitatory postsynaptic currents, i.e., to defects in synaptic transmission phenocopying ProSAP1/Shank2 knockout, and impairments in proper synaptic ProSAP1/Shank2 distribution. Syndapin I-enriched membrane nanodomains thus seem to be important spatial cues and organizing platforms, shaping dendritic membrane areas into synaptic compartments.

A de novo gain-of-function mutation in SCN11A causes loss of pain perception.

The sensation of pain protects the body from serious injury. Using exome sequencing, we identified a specific de novo missense mutation in SCN11A in individuals with the congenital inability to experience pain who suffer from recurrent tissue damage and severe mutilations. Heterozygous knock-in mice carrying the orthologous mutation showed reduced sensitivity to pain and self-inflicted tissue lesions, recapitulating aspects of the human phenotype. SCN11A encodes Nav1.9, a voltage-gated sodium ion channel that is primarily expressed in nociceptors, which function as key relay stations for the electrical transmission of pain signals from the periphery to the central nervous system. Mutant Nav1.9 channels displayed excessive activity at resting voltages, causing sustained depolarization of nociceptors, impaired generation of action potentials and aberrant synaptic transmission. The gain-of-function mechanism that underlies this channelopathy suggests an alternative way to modulate pain perception.

Expression of coagulation factors and their receptors in tumor tissue and coagulation factor upregulation in peripheral blood of patients with cerebral carcinoma metastases.

BACKGROUND: Patients with malignancies often suffer from thrombembolic events that complicate the course of cancer disease and reduce the patients' quality of life or shorten the survival time in severe cases. This phenomenon is also known for patients with primary or secondary brain tumors; but the reasons are not identified. METHODS: We performed a prospective case-controlled study of patients with brain metastases but without any active peripheral tumor site. Blood of patients was collected perioperatively and investigated for coagulation factor activities. Moreover, we analyzed the expression of coagulation factors and their receptors within the tumor material of brain metastases from clear-cell renal cell carcinomas and small-cell carcinomas of the lung. RESULTS: Here, we show that even patients without an active peripheral tumor disease that means without any tumor masses outside the central nervous system after anticancer treatment by surgery, radiation therapy, or chemotherapy but with symptomatic brain metastasis develop an increased systemic activation of multiple coagulation factors. The pro-coagulatory state is expressed preoperatively, but also can be observed in the early postoperative period. Additionally to that, intracerebral metastases of clear-cell renal cell carcinomas and of small-cell carcinomas of the lung express prothrombin, thrombin, factor X, and the protease-activated receptors type 1, 2, 3, and 4. CONCLUSIONS: These observations support the hypothesis of a link between the hemostatic system in the periphery and the malignant tumor disease even when the tumor is an intracerebral metastasis and the affected patient currently is free of a systemically active tumor. The results of this study support the hypothesis that the concerted action of coagulation factors and their receptors within the metastasis tissue itself and the systemic coagulation system could control the malignant behavior of tumor disease and make larger prospective trials mandatory.

Synaptic glutamate release is modulated by the Na+ -driven Cl-/HCO3⁻ exchanger Slc4a8.

On the one hand, neuronal activity can cause changes in pH; on the other hand, changes in pH can modulate neuronal activity. Consequently, the pH of the brain is regulated at various levels. Here we show that steady-state pH and acid extrusion were diminished in cultured hippocampal neurons of mice with a targeted disruption of the Na(+)-driven Cl(-)/HCO(3)(-) exchanger Slc4a8. Because Slc4a8 was found to predominantly localize to presynaptic nerve endings, we hypothesize that Slc4a8 is a key regulator of presynaptic pH. Supporting this hypothesis, spontaneous glutamate release in the CA1 pyramidal layer was reduced but could be rescued by increasing the intracellular pH. The reduced excitability in vitro correlated with an increased seizure threshold in vivo. Together with the altered kinetics of stimulated synaptic vesicle release, these data suggest that Slc4a8 modulates glutamate release in a pH-dependent manner.