Phenylalanine hydroxylase contributes to serotonin synthesis in mice.

Serotonin is an important signaling molecule in the periphery and in the brain. The hydroxylation of tryptophan is the first and rate-limiting step of its synthesis. In most vertebrates, two enzymes have been described to catalyze this step, tryptophan hydroxylase (TPH) 1 and 2, with expression localized to peripheral and neuronal cells, respectively. However, animals lacking both TPH isoforms still exhibit about 10% of normal serotonin levels in the blood demanding an additional source of the monoamine. In this study, we provide evidence by the gain and loss of function approaches in in vitro and in vivo systems, including stable-isotope tracing in mice, that phenylalanine hydroxylase (PAH) is a third TPH in mammals. PAH contributes to serotonin levels in the blood, and may be important as a local source of serotonin in organs in which no other TPHs are expressed, such as liver and kidney.

Reducing l-lactate release from hippocampal astrocytes by intracellular oxidation increases novelty induced activity in mice.

Astrocytes are in control of metabolic homeostasis in the brain and support and modulate neuronal function in various ways. Astrocyte-derived l-lactate (lactate) is thought to play a dual role as a metabolic and a signaling molecule in inter-cellular communication. The biological significance of lactate release from astrocytes is poorly understood, largely because the tools to manipulate lactate levels in vivo are limited. We therefore developed new viral vectors for astrocyte-specific expression of a mammalianized version of lactate oxidase (LOx) from Aerococcus viridans. LOx expression in astrocytes in vitro reduced their intracellular lactate levels as well as the release of lactate to the extracellular space. Selective expression of LOx in astrocytes of the dorsal hippocampus in mice resulted in increased locomotor activity in response to novel stimuli. Our findings suggest that a localized decreased intracellular lactate pool in hippocampal astrocytes could contribute to greater responsiveness to environmental novelty. We expect that use of this molecular tool to chronically limit astrocytic lactate release will significantly facilitate future studies into the roles and mechanisms of intercellular lactate communication in the brain.

Alterations in BDNF Protein Concentrations in the Hippocampus do not Explain the Pro-Neurogenic Effect of Citalopram on Adult Neurogenesis.

INTRODUCTION: Brain-derived neurotrophic factor (BDNF) has been implicated in the pro-neurogenic effect of selective serotonin reuptake inhibitors. In this study, we used Tph2 -/- mice lacking brain serotonin to dissect the interplay between BDNF and the serotonin system in mediating the effects of antidepressant pharmacotherapy on adult neurogenesis in the hippocampus. METHODS: Besides citalopram (CIT), we tested tianeptine (TIA), an antidepressant whose mechanism of action is not well understood. Specifically, we examined cell survival and endogenous concentrations of BDNF following daily injection of the drugs. RESULTS: Twenty-one days of CIT, but not of TIA, led to a significant increase in the survival of newly generated cells in the dentate gyrus of wild-type mice, without a significant effect on BDNF protein levels by either treatment. In Tph2 -/- mice, adult neurogenesis was consistently increased. Furthermore, Tph2 -/- mice showed increased BDNF protein levels, which were not affected by TIA but were significantly reduced by CIT. DISCUSSION: We conclude that the effects of CIT on adult neurogenesis are not explained by changes in BDNF protein concentrations in the hippocampus.

Priming of LTP in amygdala and hippocampus by prior paired pulse facilitation paradigm in mice lacking brain serotonin.

This study focuses on analyzing long-term potentiation (LTP) changes in the lateral nucleus of the amygdala (LA) and in the CA1 region of the hippocampus in slices derived from mice deficient in tryptophan hydroxylase 2 (TPH2-/- ), the rate-limiting enzyme for 5-HT synthesis in the brain. We found a reduced LTP in both brain structures in TPH2-/- mice. However, we found no changes in the magnitude of LTP in TPH2-/- mice compared to wildtype mice when it was preceded by a paired pulse protocol. Whereas the magnitude of long-term depression (LTD) did not differ between wildtype and TPH2-/- mice, priming synapses by LTD-induction facilitated subsequent CA1-LTP in wildtype mice to a greater extent than in TPH2-/- mice. In the LA we found no differences between the genotypes in this protocol of metaplasticity. These data show that, unlike exogenous 5-HT application, lack of 5-HT in the brain impairs cellular mechanisms responsible for induction of LTP. It is supposed that suppression of LTP observed in TPH2-/- mice might be compensated by mechanisms of metaplasticity induced by paired pulse stimulation or low frequency stimulation before the induction of LTP.

Depletion of angiotensin-converting enzyme 2 reduces brain serotonin and impairs the running-induced neurogenic response.

Physical exercise induces cell proliferation in the adult hippocampus in rodents. Serotonin (5-HT) and angiotensin (Ang) II are important mediators of the pro-mitotic effect of physical activity. Here, we examine precursor cells in the adult brain of mice lacking angiotensin-converting enzyme (ACE) 2, and explore the effect of an acute running stimulus on neurogenesis. ACE2 metabolizes Ang II to Ang-(1-7) and is essential for the intestinal uptake of tryptophan (Trp), the 5-HT precursor. In ACE2-deficient mice, we observed a decrease in brain 5-HT levels and no increase in the number of BrdU-positive cells following exercise. Targeting the Ang II/AT1 axis by blocking the receptor, or experimentally increasing Trp/5-HT levels in the brain of ACE2-deficient mice, did not rescue the running-induced effect. Furthermore, mice lacking the Ang-(1-7) receptor, Mas, presented a normal neurogenic response to exercise. Our results identify ACE2 as a novel factor required for exercise-dependent modulation of adult neurogenesis and essential for 5-HT metabolism.

Glio- and neuro-protection by prosaposin is mediated by orphan G-protein coupled receptors GPR37L1 and GPR37.

Discovery of neuroprotective pathways is one of the major priorities for neuroscience. Astrocytes are natural neuroprotectors and it is likely that brain resilience can be enhanced by mobilizing their protective potential. Among G-protein coupled receptors expressed by astrocytes, two highly related receptors, GPR37L1 and GPR37, are of particular interest. Previous studies suggested that these receptors are activated by a peptide Saposin C and its neuroactive fragments (prosaptide TX14(A)), which were demonstrated to be neuroprotective in various animal models by several groups. However, pairing of Saposin C or prosaptides with GPR37L1/GPR37 has been challenged and presently GPR37L1/GPR37 have regained their orphan status. Here, we demonstrate that in their natural habitat, astrocytes, these receptors mediate a range of effects of TX14(A), including protection from oxidative stress. The Saposin C/GPR37L1/GPR37 pathway is also involved in the neuroprotective effect of astrocytes on neurons subjected to oxidative stress. The action of TX14(A) is at least partially mediated by Gi-proteins and the cAMP-PKA axis. On the other hand, when recombinant GPR37L1 or GPR37 are expressed in HEK293 cells, they are not functional and do not respond to TX14(A), which explains unsuccessful attempts to confirm the ligand-receptor pairing. Therefore, this study identifies GPR37L1/GPR37 as the receptors for TX14(A), and, by extension of Saposin C, and paves the way for the development of neuroprotective therapeutics acting via these receptors.

Increased brain-derived neurotrophic factor (BDNF) protein concentrations in mice lacking brain serotonin.

The interplay between BDNF signaling and the serotonergic system remains incompletely understood. Using a highly sensitive enzyme-linked immunosorbent assay, we studied BDNF concentrations in hippocampus and cortex of two mouse models of altered serotonin signaling: tryptophan hydroxylase (Tph)2-deficient (Tph2 (-/-)) mice lacking brain serotonin and serotonin transporter (SERT)-deficient (SERT(-/-)) mice lacking serotonin re-uptake. Surprisingly, hippocampal BDNF was significantly elevated in Tph2 (-/-) mice, whereas no significant changes were observed in SERT(-/-) mice. Furthermore, BDNF levels were increased in the prefrontal cortex of Tph2 (-/-) but not of SERT(-/-) mice. Our results emphasize the interaction between serotonin signaling and BDNF. Complete lack of brain serotonin induces BDNF expression.

Serotonin is required for exercise-induced adult hippocampal neurogenesis.

Voluntary wheel running has long been known to induce precursor cell proliferation in adult hippocampal neurogenesis in rodents. However, mechanisms that couple activity with the promitotic effect are not yet fully understood. Using tryptophan hydroxylase (TPH) 2 deficient (Tph2-deficient) mice that lack brain serotonin, we explored the relationship between serotonin signaling and exercise-induced neurogenesis. Surprisingly, Tph2-deficient mice exhibit normal baseline hippocampal neurogenesis but impaired activity-induced proliferation. Our data demonstrate that the proproliferative effect of running requires the release of central serotonin in young-adult and aged mice. Lack of brain serotonin further results in alterations at the stage of Sox2-positive precursor cells, suggesting physiological adaptations to changes in serotonin supply to maintain homeostasis in the neurogenic niche. We conclude that serotonin plays a direct and acute regulatory role in activity-dependent hippocampal neurogenesis. The understanding of exercise-induced neurogenesis might offer preventive but also therapeutic opportunities in depression and age-related cognitive decline.

miR-483-5p offsets functional and behavioural effects of stress in male mice through synapse-targeted repression of Pgap2 in the basolateral amygdala.

Severe psychological trauma triggers genetic, biochemical and morphological changes in amygdala neurons, which underpin the development of stress-induced behavioural abnormalities, such as high levels of anxiety. miRNAs are small, non-coding RNA fragments that orchestrate complex neuronal responses by simultaneous transcriptional/translational repression of multiple target genes. Here we show that miR-483-5p in the amygdala of male mice counterbalances the structural, functional and behavioural consequences of stress to promote a reduction in anxiety-like behaviour. Upon stress, miR-483-5p is upregulated in the synaptic compartment of amygdala neurons and directly represses three stress-associated genes: Pgap2, Gpx3 and Macf1. Upregulation of miR-483-5p leads to selective contraction of distal parts of the dendritic arbour and conversion of immature filopodia into mature, mushroom-like dendritic spines. Consistent with its role in reducing the stress response, upregulation of miR-483-5p in the basolateral amygdala produces a reduction in anxiety-like behaviour. Stress-induced neuromorphological and behavioural effects of miR-483-5p can be recapitulated by shRNA mediated suppression of Pgap2 and prevented by simultaneous overexpression of miR-483-5p-resistant Pgap2. Our results demonstrate that miR-483-5p is sufficient to confer a reduction in anxiety-like behaviour and point to miR-483-5p-mediated repression of Pgap2 as a critical cellular event offsetting the functional and behavioural consequences of psychological stress.

Growth retardation and altered autonomic control in mice lacking brain serotonin.

Serotonin synthesis in mammals is initiated by 2 distinct tryptophan hydroxylases (TPH), TPH1 and TPH2. By genetically ablating TPH2, we created mice (Tph2(-/-)) that lack serotonin in the central nervous system. Surprisingly, these mice can be born and survive until adulthood. However, depletion of serotonin signaling in the brain leads to growth retardation and 50% lethality in the first 4 weeks of postnatal life. Telemetric monitoring revealed more extended daytime sleep, suppressed respiration, altered body temperature control, and decreased blood pressure (BP) and heart rate (HR) during nighttime in Tph2(-/-) mice. Moreover, Tph2(-/-) females, despite being fertile and producing milk, exhibit impaired maternal care leading to poor survival of their pups. These data confirm that the majority of central serotonin is generated by TPH2. TPH2-derived serotonin is involved in the regulation of behavior and autonomic pathways but is not essential for adult life.

Tph2 Gene Expression Defines Ethanol Drinking Behavior in Mice.

Indirect evidence supports a link between disrupted serotonin (5-hydroxytryptamine; 5-HT) signaling in the brain and addictive behaviors. However, the effects of hyposerotonergia on ethanol drinking behavior are contradictory. In this study, mice deficient in tryptophan hydroxylase 2 (Tph2-/-), the rate-limiting enzyme of 5-HT synthesis in the brain, were used to assess the role of central 5-HT in alcohol drinking behavior. Life-long 5-HT depletion in these mice led to an increased ethanol consumption in comparison to wild-type animals in a two-bottle choice test. Water consumption was increased in naïve 5-HT-depleted mice. However, exposure of Tph2-/- animals to ethanol resulted in the normalization of water intake to the level of wild-type mice. Tph2 deficiency in mice did not interfere with ethanol-evoked antidepressant response in the forced swim test. Gene expression analysis in wild-type animals revealed no change in Tph2 expression in the brain of mice consuming ethanol compared to control mice drinking water. However, within the alcohol-drinking group, inter-individual differences in chronic ethanol intake correlated with Tph2 transcript levels. Taken together, central 5-HT is an important modulator of drinking behavior in mice but is not required for the antidepressant effects of ethanol.

Expression of Microbial Enzymes in Mammalian Astrocytes to Modulate Lactate Release.

Astrocytes support and modulate neuronal activity through the release of L-lactate. The suggested roles of astrocytic lactate in the brain encompass an expanding range of vital functions, including central control of respiration and cardiovascular performance, learning, memory, executive behaviour and regulation of mood. Studying the effects of astrocytic lactate requires tools that limit the release of lactate selectively from astrocytes. Here, we report the validation in vitro of novel molecular constructs derived from enzymes originally found in bacteria, that when expressed in astrocytes, interfere with lactate handling. When lactate 2-monooxygenase derived from M. smegmatis was specifically expressed in astrocytes, it reduced intracellular lactate pools as well as lactate release upon stimulation. D-lactate dehydrogenase derived from L. bulgaricus diverts pyruvate towards D-lactate production and release by astrocytes, which may affect signalling properties of lactate in the brain. Together with lactate oxidase, which we have previously described, this set of transgenic tools can be employed to better understand astrocytic lactate release and its role in the regulation of neuronal activity in different behavioural contexts.

Tryptophan hydroxylase as novel target for the treatment of depressive disorders.

Serotonin (5-HT) is a monoamine implicated in a variety of physiological processes that functions either as a neurotransmitter or as a peripheral hormone. Pharmacological and genetic studies in humans and experimental animals have shown that 5-HT is important for the pathophysiology of depressive disorders. The 5-HT system is thus already a main target for the therapy of these diseases. The peripheral and cerebral biosynthesis of 5-HT is initiated by two distinct tryptophan hydroxylases: TPH1 and TPH2. This duality of the serotonergic system and the existence of a brain-specific TPH isoform provide a promising new target for pharmacological intervention with higher selectivity and specificity and, therefore, possibly with reduced side effects and increased efficiency. This paper summarizes the data which support TPH2 as novel drug target and discusses strategies for its pharmacological exploitation.

Targeted Manipulation of Brain Serotonin: RNAi-Mediated Knockdown of Tryptophan Hydroxylase 2 in Rats.

Tryptophan hydroxylase (TPH) is the rate-limiting enzyme in the biosynthesis of the biogenic monoamine serotonin (5-hydroxytryptamine, 5-HT). Two existing TPH isoforms are responsible for the generation of two distinct serotonergic systems in vertebrates. TPH1, predominantly expressed in the gastrointestinal tract and pineal gland, mediates 5-HT biosynthesis in non-neuronal tissues, while TPH2, mainly found in the raphe nuclei of the brain stem, is accountable for the production of 5-HT in the brain. Neuronal 5-HT is a key regulator of mood and behavior and its deficiency has been implicated in a variety of neuropsychiatric disorders, e.g., depression and anxiety. To gain further insights into the complexity of central 5-HT modulations of physiological and pathophysiological processes, a new transgenic rat model, allowing an inducible gene knockdown of Tph2, was established based on doxycycline-inducible shRNA-expression. Biochemical phenotyping revealed a functional knockdown of Tph2 mRNA expression following oral doxycycline administration, with subsequent reductions in the corresponding levels of TPH2 enzyme expression and activity. Transgenic rats showed also significantly decreased tissue levels of 5-HT and its degradation product 5-Hydroxyindoleacetic acid (5-HIAA) in the raphe nuclei, hippocampus, hypothalamus, and cortex, while peripheral 5-HT concentrations in the blood remained unchanged. In summary, this novel transgenic rat model allows inducible manipulation of 5-HT biosynthesis specifically in the brain and may help to elucidate the role of 5-HT in the pathophysiology of affective disorders.

Rodents and humans are able to detect the odour of L-Lactate.

L-Lactate (LL) is an essential cellular metabolite which can be used to generate energy. In addition, accumulating evidence suggests that LL is used for inter-cellular signalling. Some LL-sensitive receptors have been identified but we recently proposed that there may be yet another unknown G-protein coupled receptor (GPCR) sensitive to LL in the brain. Olfactory receptors (ORs) represent the largest family of GPCRs and some of them are expressed outside the olfactory system, including brain, making them interesting candidates for non-olfactory LL signalling. One of the "ectopically" expressed ORs, Olfr78 in mice (Olr59 in rats and OR51E2 in humans), reportedly can be activated by LL. This implies that both rodents and humans should be able to detect the LL odour. Surprisingly, this has never been demonstrated. Here we show that mice can detect the odour of LL in odour detection and habituation-dishabituation tasks, and discriminate it from peppermint and vanilla odours. Behaviour of the Olfr78 null mice and wildtype mice in odour detection task was not different, indicating that rodents are equipped with more than one LL-sensitive OR. Rats were also able to use the smell of LL as a cue in an odour-reward associative learning task. When presented to humans, more than 90% of participants detected a smell of LL in solution. Interestingly, LL was perceived differently than acetate or propionate-LL was preferentially reported as a pleasant sweet scent while acetate and propionate were perceived as repulsive sour/acid smells. Subjective perception of LL smell was different in men and women. Taken together, our data demonstrate that both rodents and humans are able to detect the odour of LL. Moreover, in mice, LL perception is not purely mediated by Olfr78. Discovery of further LL-sensitive OR might shed the light on their contribution to LL signalling in the body.

Effects of ACE2 deficiency on physical performance and physiological adaptations of cardiac and skeletal muscle to exercise.

The renin-angiotensin system (RAS) is related to physiological adaptations induced by exercise. Angiotensin-converting enzyme (ACE) 2 is a major regulator of the RAS in tissues, as it metabolizes angiotensin (Ang) II to Ang-(1-7). The aim of this study was to determine the effects of ACE2 deficiency on physical performance and physiological adaptations induced by voluntary running. Physical performance, body composition and plasma angiotensin levels, as well as tissue morphology and gene expression of RAS components in the left ventricle (LV) and skeletal muscle (gastrocnemius), were evaluated in ACE2-deficient (ACE2(-/y)) and wild-type (ACE2(+/y)) mice after 6 weeks of voluntary wheel running. ACE2(-/y) mice run less than ACE2(+/y) mice (19±4.7 vs. 26±12.6 revolutions per day × 100, P<0.01). The ACE2(+/y) group presented a lower fat mass (15±1.1%) and higher muscle mass (76.6±1.6%) after 6 weeks of voluntary running compared with the sedentary control group (fat mass: 18.3±2.1%; muscle mass: 72.7±2.2). However, no change in body composition was observed in ACE2(-/y) mice after exercise. Heart and skeletal muscle hypertrophy was observed only in trained ACE2(+/y) mice. Besides a small decrease in Ang I in ACE2(-/y) mice, plasma levels of angiotensin peptides remained unchanged by exercise or ACE2 deficiency. In the LV of trained animals, AT2 gene expression was higher in ACE2(+/y) compared with ACE2(-/y) mice. ACE2 deficiency leads to an increase in AT1 gene expression in skeletal muscle. ACE expression in soleus was increased in all exercised groups. ACE2 deficiency affects physical performance and impairs cardiac and skeletal muscle adaptations to exercise.

Is L-lactate a novel signaling molecule in the brain?

In the brain, L-lactate is produced by both neurons and astrocytes. There is no doubt that neurons use L-lactate as a supplementary fuel although the importance of this energy source is disputed. Irrespective of its caloric value, L-lactate might also have a signaling role in the brain. Here, we review several current hypotheses of L-lactate mediated signaling. Some proposed mechanisms require L-lactate entry into the neurons leading to a shift in ATP/ADP ratio or redox state. Others postulate interaction with either known receptor HCA1 (GPR81) or a novel, yet unidentified receptor. We argue that the sensitivity of any such mechanism has to match the concentration range of extracellular L-lactate, which is less than ~1.5 mmol/L under physiologic conditions. From that point of view, some of the proposed mechanisms require supraphysiologic levels of L-lactate and could be engaged during ischemia or seizures when L-lactate concentration rises dramatically. Currently, we do not know whether L-lactate production in the brain occurs in microdomains, which might create higher than average local concentrations. Nevertheless, it is clear that in the brain, as in the peripheral tissues, L-lactate is not only used as a source of energy but also acts as a signaling molecule.

Reduced isolation-induced pup ultrasonic communication in mouse pups lacking brain serotonin.

BACKGROUND: Serotonin (5-hydroxytryptamine, 5-HT) is a key modulatory neurotransmitter in the mammalian central nervous system (CNS) that plays an important role as a developmental signal. Several lines of evidence associate altered 5-HT signaling with psychopathology in humans, particularly neurodevelopmental disorders such as autism spectrum disorders (ASD). ASD are characterized by persistent social and communication deficits along with stereotyped and repetitive patterns of behavior, with all symptoms emerging early during development. METHODS: Here, we employed a mouse model devoid of brain 5-HT due to the lack of the gene encoding tryptophan hydroxylase 2 (Tph2), the initial and rate-limiting enzyme of 5-HT synthesis in the CNS. Tph2 null mutant (Tph2 (-/-) ) mice show normal prenatal development; however, they display for yet unknown reasons severe growth retardation during the first postnatal weeks. We investigated, therefore, whether Tph2 (-/-) mice display deficits in isolation-induced ultrasonic vocalizations (USV) as pups during early life. Isolation-induced USV are the most commonly studied behavioral measure to assess developmental delays and communication deficits in rodent models for ASD, particularly as they serve an important communicative function in coordinating mother-pup interactions. RESULTS: Tph2 (-/-) mouse pups displayed a clear deficit in the emission of isolation-induced USV, as compared to heterozygous and wildtype littermates, exactly during growth retardation onset, including reduced call numbers and deficits in call clustering and temporal organization. CONCLUSIONS: The ultrasonic communication impairment displayed by Tph2 (-/-) mouse pups is likely to result in a deficient mother-infant interaction, presumably contributing to their growth retardation phenotype, and represents a prominent feature relevant to ASD.

Life without brain serotonin: reevaluation of serotonin function with mice deficient in brain serotonin synthesis.

Tryptophan hydroxylase (TPH) is a rate limiting enzyme in the synthesis of serotonin (5-HT), a monoamine which works as an autacoid in the periphery and as a neurotransmitter in the central nervous system. In 2003 we have discovered the existence of a second Tph gene, which is expressed exclusively in the brain, and, therefore, is responsible for the 5-HT synthesis in the central nervous system. In the following years several research groups have independently generated Tph2-deficient mice. In this review we will summarize the data gained from the existing mouse models with constitutive or conditional deletion of the Tph2 gene, focusing on biochemical, developmental, and behavioral consequences of Tph2-deficiency.