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Here we present a deep learning-based image analysis platform (DLAP), tailored to autonomously quantify cell numbers, and fluorescence signals within cellular compartments, derived from RNAscope or immunohistochemistry. We utilised DLAP to analyse subtypes of tyrosine hydroxylase (TH)-positive dopaminergic midbrain neurons in mouse and human brain-sections. These neurons modulate complex behaviour, and are differentially affected in Parkinson's and other diseases. DLAP allows the analysis of large cell numbers, and facilitates the identification of small cellular subpopulations. Using DLAP, we identified a small subpopulation of TH-positive neurons (~5%), mainly located in the very lateral Substantia nigra (SN), that was immunofluorescence-negative for the plasmalemmal dopamine transporter (DAT), with ~40% smaller cell bodies. These neurons were negative for aldehyde dehydrogenase 1A1, with a lower co-expression rate for dopamine-D2-autoreceptors, but a ~7-fold higher likelihood of calbindin-d28k co-expression (~70%). These results have important implications, as DAT is crucial for dopamine signalling, and is commonly used as a marker for dopaminergic SN neurons. A deep learning-based image analysis platform quantifies cell numbers and mRNA or protein derived fluorescence signals, and is used to identify a unique population of dopamine transporter-negative dopaminergic neurons in the lateral Substantia nigra. [ABSTRACT FROM AUTHOR]

Queuosine (Q) is a modified nucleoside at the wobble position of specific tRNAs. In mammals, queuosinylation is facilitated by queuine uptake from the gut microbiota and is introduced into tRNA by the QTRT1‐QTRT2 enzyme complex. By establishing a Qtrt1 knockout mouse model, we discovered that the loss of Q‐tRNA leads to learning and memory deficits. Ribo‐Seq analysis in the hippocampus of Qtrt1‐deficient mice revealed not only stalling of ribosomes on Q‐decoded codons, but also a global imbalance in translation elongation speed between codons that engage in weak and strong interactions with their cognate anticodons. While Q‐dependent molecular and behavioral phenotypes were identified in both sexes, female mice were affected more severely than males. Proteomics analysis confirmed deregulation of synaptogenesis and neuronal morphology. Together, our findings provide a link between tRNA modification and brain functions and reveal an unexpected role of protein synthesis in sex‐dependent cognitive performance. Synopsis: Queuosine (Q) is a modified nucleoside introduced in specific tRNAs by a complex comprising queuine tRNA ribosyltransferase 1 (QTRT1). Loss of Q‐tRNA modification by the knockout of Qtrt1 gene in mice (Q1) leads to an imbalance in codon‐biased protein translation speed, resulting in alterations of hippocampal architecture and sex‐dependent reduction in learning and memory formation.Q‐tRNA levels vary considerably between murine tissues, with the brain showing a high queuosinylation level.Loss of Q results in learning and memory deficits, with females being more affected than male mice.Q1 mice exhibit altered neuronal cytoarchitecture in the hippocampus.Loss of Q leads to ribosome stalling on Q‐decoded codons and alteration of overall protein translation homeostasis.Translational stress and increased eIF2α phosphorylation impair neuronal cognitive functions in Q1 female mice. [ABSTRACT FROM AUTHOR]

Mutations in the human kinesin family member 5A (KIF5A) gene were recently identified as a genetic cause of amyotrophic lateral sclerosis (ALS). Several KIF5A ALS variants cause exon 27 skipping and are predicted to produce motor proteins with an altered C‐terminal tail (referred to as ΔExon27). However, the underlying pathogenic mechanism is still unknown. Here, we confirm the expression of KIF5A mutant proteins in patient iPSC‐derived motor neurons. We perform a comprehensive analysis of ΔExon27 at the single‐molecule, cellular, and organism levels. Our results show that ΔExon27 is prone to form cytoplasmic aggregates and is neurotoxic. The mutation relieves motor autoinhibition and increases motor self‐association, leading to drastically enhanced processivity on microtubules. Finally, ectopic expression of ΔExon27 in Drosophila melanogaster causes wing defects, motor impairment, paralysis, and premature death. Our results suggest gain‐of‐function as an underlying disease mechanism in KIF5A‐associated ALS. Synopsis: The ALS‐associated KIF5A ΔExon27 mutation drives the disease via a gain‐of‐function mechanism caused by constitutive activation and increased motor association and aggregation of mutant KIF5A. KIF5A ΔExon27 is aggregation‐prone and is neurotoxic.KIF5A ΔExon27 displays relieved autoinhibition, increased motor self‐association, and enhanced processivity on microtubules.ALS patient iPSC‐derived motor neurons express mutant KIF5A proteins.Ectopic expression of KIF5A ΔExon27 in Drosophila leads to abnormal wings, motor deficits, paralysis, and premature death. [ABSTRACT FROM AUTHOR]

Multiple pathomechanisms triggered by mutant Huntingtin (mHTT) underlie progressive degeneration of dopaminoceptive striatal neurons in Huntington's disease (HD). The primary cilium is a membrane compartment that functions as a hub for various pathways that are dysregulated in HD, for example, dopamine (DA) receptor transmission and the mechanistic target of rapamycin (mTOR) pathway. The roles of primary cilia (PC) for the maintenance of striatal neurons and in HD progression remain unknown. Here, we investigated PC defects in vulnerable striatal neurons in a progressive model of HD, the mHTT-expressing knock-in zQ175 mice. We found that PC length is affected in striatal but not in cortical neurons, in association with the accumulation of mHTT. To explore the role of PC, we generated conditional mutant mice lacking IFT88, a component of the anterograde intraflagellar transport-B complex lacking PC in dopaminoceptive neurons. This mutation preserved the expression of the dopamine 1 receptor (D1R), and the survival of striatal neurons, but resulted in a mild increase of DA metabolites in the striatum, suggesting an imbalance of ciliary DA receptor transmission. Conditional loss of PC in zQ175 mice did not trigger astrogliosis, however, mTOR signaling was more active and resulted in a more pronounced accumulation of nuclear inclusions containing mHTT. Further studies will be required of aged mice to determine the role of aberrant ciliary function in more advanced stages of HD. [ABSTRACT FROM AUTHOR]

Ribosome biogenesis, including the RNA polymerase 1 (Pol1)-mediated transcription of rRNA, is regulated by the pro-epileptogenic mTOR pathway. Therefore, hippocampal Pol1 activity was examined in mouse models of epilepsy including kainic acid- and pilocarpine-induced status epilepticus (SE) as well as a single seizure in response to pentylenetetrazole (PTZ). Elevated 47S pre-rRNA levels were present acutely after induction of SE suggesting activation of Pol1. Conversely, after a single seizure, 47S pre-rRNA was transiently downregulated with increased levels of unprocessed 18S rRNA precursors in the cornu Ammonis (CA) region. At least in the dentate gyrus (DG), the pilocarpine SE-mediated transient activation of Pol1 did not translate into long-term changes of pre-rRNA levels or total ribosome content. Unaltered hippocampal ribosome content was also found after a 20-day PTZ kindling paradigm with increasing pro-convulsive effects of low dose PTZ that was injected every other day. However, after selectively deleting the essential Pol1 co-activator, transcription initiation factor-1A (Tif1a/Rrn3) from excitatory neurons, PTZ kindling was impaired while DG total ribosome content was moderately reduced and no major neurodegeneration was observed throughout the hippocampus. Therefore, Pol1 activity of excitatory neurons is required for PTZ kindling. As seizures affect ribosome biogenesis without long-term effects on the total ribosome content, such a requirement may be associated with a need to produce specialized ribosomes that promote pro-epileptic plasticity. [ABSTRACT FROM AUTHOR]

Amyotrophic lateral sclerosis (ALS) and frontotemporal degeneration (FTD) are neurodegenerative diseases with distinct clinical appearance. However, both share as major genetic risk factor a C9orf72 locus intronic hexanucleotide expansion. The pathogenic pathways associated with the expansion-dependent neuronal toxicity are still poorly understood. Recent efforts to identify common threads of neuronal dysfunction have pointed towards deficits of ribosomal RNA (rRNA) biogenesis and loss of nucleolar integrity, a condition known as nucleolar stress that is an emerging shared feature among neurodegenerative diseases. Intriguingly, the C9orf72 mutation in ALS-FTD interferes with the function of the nucleolus by transcripts and dipeptide repeats (DPRs) produced by the hexanucleotide expansion. Experimental discrepancies have given rise to different hypotheses with regard to the connection of C9orf72 and nucleolar activity. In this review, we present and discuss emerging concepts concerning the impact of C9orf72 expansion on nucleolar biology. Moreover, we discuss the “nucleolar stress hypothesis,” according to which nucleolar malfunction accompanies, exacerbates, or potentially triggers a degenerative phenotype. Upcoming awareness of the involvement of nucleolar stress in C9orf72 ALS-FTD could shed light into its pathogenesis, enabling potential treatment options aimed at shielding an “Achilles’ heel” of neurons. [ABSTRACT FROM AUTHOR]

Retarded growth and neurodegeneration are hallmarks of the premature aging disease Cockayne syndrome (CS). Cockayne syndrome proteins take part in the key step of ribosomal biogenesis, transcription of RNA polymerase I. Here, we identify a mechanism originating from a disturbed RNA polymerase I transcription that impacts translational fidelity of the ribosomes and consequently produces misfolded proteins. In cells from CS patients, the misfolded proteins are oxidized by the elevated reactive oxygen species (ROS) and provoke an unfolded protein response that represses RNA polymerase I transcription. This pathomechanism can be disrupted by the addition of pharmacological chaperones, suggesting a treatment strategy for CS. Additionally, this loss of proteostasis was not observed in mouse models of CS. • Cockayne syndrome cells show reduced translation fidelity • Elevated oxidative stress and misfolded proteins activate the unfolded protein response • The unfolded protein response suppresses Pol I activity in CS cells • Chemical chaperones alleviate ER stress and restore Pol activity in CS cells Cockayne syndrome is a devastating childhood progeria. Here, Alupei et al. show that cells from CS patients have reduced translation accuracy and elevated ROS, leading to generation of unstable proteins and activation of ER stress. Reducing ER stress by chemical chaperones in these cells rescues RNA polymerase I activity and protein synthesis. [ABSTRACT FROM AUTHOR]

In preclinical studies, endothelin receptor A (ETA) antagonists (ETAi) attenuated the progression of heart failure (HF). However, clinical HF trials failed to demonstrate beneficial effects of ETAi. These conflicting data may be explained by the possibility that established HF drugs such as adrenergic receptor blockers interfered with the mechanism of ETAi action in clinical trials. Here we report that mice lacking ETA only in sympathetic neurons (SN-KO) showed less adverse structural remodeling and cardiac dysfunction in response to pathological pressure overload induced by transverse aortic constriction (TAC). In contrast, mice lacking ETA only in cardiomyocytes (CM-KO) were not protected. TAC led to a disturbed sympathetic nerve function as measured by cardiac norepinephrine (NE) tissue levels and [124l]-metaiodobenzylguani-dine-PET, which was prevented in SN-KO. In a rat model of HF, ETAi improved cardiac and sympathetic nerve function. In cocultures of cardiomyocytes (CMs) and sympathetic neurons (SNs), endothelin-1 (ET1) led to a massive NE release and exaggerated CM hypertrophy compared with CM monocultures. ETA-deficient CMs gained a hypertrophic response through wild-type SNs, but ETA-deficient SNs failed to mediate exaggerated CM hypertrophy. Furthermore, ET1 mediated its effects indirectly via NE in CM-SN cocultures through adrenergic receptors and histone deacetylases, resulting in activation of the prohypertrophic transcription factor myocyte enhancer factor 2. In conclusion, sympathetic ETA amplifies ET1 effects on CMs through adrenergic signaling pathways. Thus, anti-adrenergic therapies may blunt potentially beneficial effects of ETAi. Taken together, this may indicate that patients with β blocker intolerance or disturbed sympathetic nerve function could be evaluated for a potential benefit from ETAi. [ABSTRACT FROM AUTHOR]

Phasic increases in dopamine (DA) are involved in the detection and selection of relevant sensory stimuli. The DAergic and cholinergic system dynamically interact to gate and potentiate sensory inputs to striatum. Striatal cholinergic interneurons (CINs) respond to relevant sensory stimuli with an initial burst, a firing pause, or a late burst, or a combination of these three components. CIN responses coincide with phasic firing of DAergic neurons in vivo. In particular, the late burst of CINs codes for the anticipated reward. To examine whether DAergic midbrain afferents can evoke the different CIN responses, we recorded from adult olfactory tubercle slices in the mouse ventral striatum. Olfactory inputs to striatal projection neurons were gated by the cholinergic tone. Phasic optogenetic activation of DAergic terminals evoked combinations of initial bursts, pauses, and late bursts in subsets of CINs by distinct receptor pathways. Glutamate release from midbrain afferents evoked an NMDAR-dependent initial burst followed by an afterhyperpolarization-induced pause. Phasic release of DA itself evoked acute changes in CIN firing. In particular, in CINs without an initial burst, phasic DA release evoked a pause through D2-type DA receptor activation. Independently, phasic DA activated a slow depolarizing conductance and the late burst through a D1-type DA receptor pathway. In summary, DAergic neurons elicit transient subsecond firing responses in CINs by sequential activation of NMDA, D2-type, and D1-type receptors. This fast control of striatal cholinergic tone by phasic DA provides a novel dynamic link of two transmitter systems central to the detection and selection of relevant stimuli. [ABSTRACT FROM AUTHOR]

It is suggested that striatal cAMP responsive element binding protein (CREB) regulates sensitivity to psychostimulants. To test the cell-specificity of this hypothesis we examined the effects of a dominant-negative CREB protein variant expressed in dopamine receptor D1 (D1R) neurons on cocaine-induced behaviors. A transgenic mouse strain was generated by pronuclear injection of a BAC-derived transgene harboring the A-CREB sequence under the control of the D1R gene promoter. Compared to wild-type, drug-naive mutants showed moderate alterations in gene expression, especially a reduction in basal levels of activity-regulated transcripts such as Arc and Egr2. The behavioral responses to cocaine were elevated in mutant mice. Locomotor activity after acute treatment, psychomotor sensitization after intermittent drug injections and the conditioned locomotion after saline treatment were increased compared to wild-type littermates. Transgenic mice had significantly higher cocaine conditioned place preference, displayed normal extinction of the conditioned preference, but showed an augmented cocaine-seeking response following priming-induced reinstatement. This enhanced cocaine-seeking response was associated with increased levels of activity-regulated transcripts and prodynorphin. The primary reinforcing effects of cocaine were not altered in the mutant mice as they did not differ from wild-type in cocaine self-administration under a fixed ratio schedule at the training dose. Collectively, our data indicate that expression of a dominant-negative CREB variant exclusively in neurons expressing D1R is sufficient to recapitulate the previously reported behavioral phenotypes associated with virally expressed dominant-negative CREB. [ABSTRACT FROM AUTHOR]

Although the main antioxidant therapies failed in the past, these emerging pathways and mechanisms might help to better understand disease mechanisms triggered by oxidative stress. Oxidative stress is typically reported in neurodegenerative diseases. Oxidative stress may directly or indirectly predispose neurons to death as a consequence of mitochondrial dysfunction, altered proteostasis, physiological neurotransmitter metabolism, inflammation, or the deregulation of antioxidant pathways. [Extracted from the article]

Nucleoli are the sites where synthesis of rRNA and ribosomal assembly take place. Along with these 'traditional' roles, the nucleolus controls cellular physiology and homeostasis. The cellular and molecular alterations associated with impaired nucleolar activity ('nucleolar stress') have just started to be systematically explored in the nervous system taking advantage of newly available animal models lacking rRNA synthesis in specific neurons. These studies showed that nucleolar function is necessary for neuronal survival and that its modality of action differs between and within cell types. Nucleolar function is also crucial in pathology as it controls mitochondrial activity and critical stress signaling pathways mimicking hallmarks of human neurodegenerative diseases. This mini-review will focus on the modes of action of nucleolar stress and discuss how the manipulation of nucleolar activity might underscore novel strategies to extend neuronal function and survival. [ABSTRACT FROM AUTHOR]

Neuroendocrine chromaffin cells exist in both intra- and extra-adrenal locations; the organ of Zuckerkandl ( OZ) constitutes the largest accumulation of extra-adrenal chromaffin tissue in mammals. The OZ disappears postnatally by modes that are still enigmatic but can be maintained by treatment with glucocorticoids ( GC). Whether the response to GC reflects a pharmacological or a physiological role of GC has not been clarified. Using mice with a conditional deletion of the GC-receptor ( GR) gene restricted to cells expressing the dopamine β-hydroxylase ( DBH) gene [ GRfl/fl; DBHCre abbreviated ( GRDBHCre)], we now present the first evidence for a physiological role of GC signalling in the postnatal maintenance of the OZ: postnatal losses of OZ chromaffin cells in GRDBHCre mice are doubled compared to wild-type littermates. We find that postnatal cell loss in the OZ starts at birth and is accompanied by autophagy. Electron microscopy reveals autophagic vacuoles and autophagolysosomes in chromaffin cells. Autophagy in OZ extra-adrenal chromaffin cells is confirmed by showing accumulation of p62 protein, which occurs, when autophagy is blocked by deleting the Atg5 gene ( Atg5 DBHCre mice). Cathepsin- D, a lysosomal marker, is expressed in cells that surround chromaffin cells and are positive for the macrophage marker BM8. Macrophages are relatively more abundant in mice lacking the GR, indicating more robust elimination of degenerating chromaffin cells in GRDBHCre mice than in wild-type littermates. In summary, our results indicate that extra-adrenal chromaffin cells in the OZ show signs of autophagy, which accompany their postnatal numerical decline, a process that is controlled by GR signalling. [ABSTRACT FROM AUTHOR]

The brain's noradrenergic system is involved in the development of behaviours induced by drugs of abuse, e.g. dependence and withdrawal, and also reward or psychomotor effects. To investigate how noradrenergic system activity is controlled in the context associated with drug-induced behaviours, we generated a Cre/loxP mouse model in which the essential glutamate NMDA receptor subunit NR1 is ablated in cells expressing dopamine β-hydroxylase (Dbh). As a result, the noradrenergic cells in NR1DbhCre mice lack the NMDA receptor-dependent component of excitatory post-synaptic currents. The mutant mice displayed no obvious behavioural alterations, had unchanged noradrenaline content and mild increase in dopamine levels in the nucleus accumbens. Interestingly, NR1DbhCre animals did not develop morphine-induced psychomotor sensitization. However, when the morphine injections were preceded by treatment with RX821002, an antagonist of α2-adrenergic receptors, the development of sensitization was restored. Conversely, pretreatment with clonidine, an agonist of α2-adrenergic receptors, blocked development of sensitization in wild-type mice. We also found that while the development of tolerance to morphine was normal in mutant mice, withdrawal symptoms were attenuated. These data reveal that NMDA receptors on noradrenergic neurons regulate development of opiate dependence and psychomotor sensitization, by controlling drug-induced noradrenaline signalling. [ABSTRACT FROM PUBLISHER]

Parkinson's disease (PD) is a progressive age-related movement disorder that results primarily from the selective loss of midbrain dopaminergic (DA) neurons. Symptoms of PD can be induced by genetic mutations or by DA neuron-specific toxins. A specific ablation of an essential factor controlling ribosomal RNA transcription, TifIa, in adult mouse DA neurons represses mTOR signaling and leads to progressive neurodegeneration and PD-like phenotype. Using an inducible Cre system in adult mice, we show here that the specific ablation of Pten in adult mouse DA neurons leads to activation of mTOR pathway and is neuroprotective in genetic (TifIa deletion) and neurotoxin-induced (MPTP or 6OHDA) mouse models of PD. Adult mice with DA neuron-specific Pten deletion exhibit elevated expression of tyrosine hydroxylase, a rate-limiting enzyme in the dopamine biosynthesis pathway, associated with increased striatal dopamine content, and increased mRNA levels of Foxa2, Pitx3, En1, Nurr1, and Lmx1b--the essential factors for maintaining physiological functions of adult DA neurons. Pten deletion attenuates the loss of tyrosine hydroxylase-positive cells after 6OHDA treatment, restores striatal dopamine in TifIa-knockout and MPTP-treated mice, and rescues locomotor impairments caused by TifIa loss. Inhibition of Pten-dependent functions in adult DA neurons may represent a promising PD therapy. [ABSTRACT FROM AUTHOR]

Acute striatal lesions increase proliferation in the subventricular zone (SVZ) and induce migration of SVZ neuroblasts to the striatum. However, the potential of these cells to replace acutely degenerated neurons is controversial. The possible contribution of parenchymal progenitors to striatal lesion-induced neurogenesis has been poorly explored. Here, we present a detailed investigation of neurogenesis in the striatum of a mouse model showing slow progressive neurodegeneration of striatal neurons, the Creb1Camkcre4Crem-/- mutant mice (CBCM). By using BrdU time course analyses, intraventricular injections of a cell tracker and 3D reconstructions we showed that neurodegeneration in CBCM mice stimulates the migration of SVZ neuroblasts to the striatum without altering SVZ proliferation. SVZ-neuroblasts migrate as chains through the callosal striatal border and then enter within the striatal parenchyma as individual cells. In addition, a population of clustered neuroblasts showing high turnover rates were observed in the mutant striatum that had not migrated from the SVZ. Clustered neuroblasts might originate within the striatum itself because they are specifically associated with parenchymal proliferating cells showing features of intermediate neuronal progenitors such as clustering, expression of EGF receptor and multiple glial (SOX2, SOX9, BLBP) and neuronal (Dlx, Sp8, and to some extent DCX) markers. Newborn striatal neurons had a short lifespan and did not replace projection neurons nor expressed sets of transcription factors involved in their specification. The differentiation failure of endogenous neuroblasts likely occurred cell autonomously because transplanted wild type embryonic precursors correctly differentiated into striatal projection neurons. Thus, we propose that under progressive degeneration, neither SVZ derived nor intra-striatal generated neurons have the potential to differentiate into striatal projection neurons. [ABSTRACT FROM AUTHOR]

The nucleolus represents an essential stress sensor for the cell. However, the molecular consequences of nucleolar damage and their possible link with neurodegenerative diseases remain to be elucidated. Here, we show that nucleolar damage is present in both genders in Parkinson's disease (PD) and in the pharmacologicalPDmodel induced by the neurotoxin 1,2,3,6-tetrahydro-1-methyl-4-phenylpyridine hydrochloride (MPTP). Mouse mutants with nucleolar disruption restricted to dopaminergic (DA) neurons show phenotypic alterations that resemble PD, such as progressive and differential loss of DA neurons and locomotor abnormalities. At the molecular level, nucleolar disruption results in increased p53 levels and downregulation of mammalian target of rapamycin (mTOR) activity, leading to mitochondrial dysfunction and increased oxidative stress, similar to PD. In turn, increased oxidative stress induced by MPTP causes mTOR and ribosomal RNA synthesis inhibition. Collectively, these observations suggest that the interplay between nucleolar dysfunction and increased oxidative stress, involving p53 and mTOR signaling, may constitute a destructive axis in experimental and sporadic PD. [ABSTRACT FROM AUTHOR]

J. Neurochem. (2010) 115, 563-573. Repeated exposure to opiates leads to cellular and molecular changes and behavioral alterations reflecting a state of dependence. In noradrenergic neurons, cyclic AMP (cAMP)-dependent pathways are activated during opiate withdrawal, but their contribution to the activity of locus coeruleus noradrenergic neurons and behavioral manifestations remains controversial. Here, we test whether the cAMP-dependent transcription factors cAMP responsive element binding protein (CREB) and cAMP-responsive element modulator (CREM) in noradrenergic neurons control the cellular markers and the physical signs of morphine withdrawal in mice. Using the Cre/loxP system we ablated the Creb1 gene in noradrenergic neurons. To avoid adaptive effects because of compensatory up-regulation of CREM, we crossed the conditional Creb1 mutant mice with a Crem−/− line. We found that the enhanced expression of tyrosine hydroxylase normally observed during withdrawal was attenuated in CREB/CREM mutants. Moreover, the withdrawal-associated cellular hyperactivity and c-fos expression was blunted. In contrast, naloxone-precipitated withdrawal signs, such as jumping, paw tremor, tremor and mastication were preserved. We conclude by a specific genetic approach that the withdrawal-associated hyperexcitability of noradrenergic neurons depends on CREB/CREM activity in these neurons, but does not mediate several behavioral signs of morphine withdrawal. [ABSTRACT FROM AUTHOR]

The transcription factor Gata3 is essential for the development of sympathetic neurons and adrenal chromaffin cells. As Gata3 expression is maintained up to the adult stage, we addressed its function in differentiated sympathoadrenal cells at embryonic and adult stages by conditional Gata3 elimination. Inactivation of Gata3 in embryonic DBH-expressing neurons elicits a strong reduction in neuron numbers due to apoptotic cell death and reduced proliferation. No selective effect on noradrenergic gene expression (TH and DBH) was observed. Interestingly, Gata3 elimination in DBH-expressing neurons of adult animals also results in a virtually complete loss of sympathetic neurons. In the Gata3-deficient population, the expression of anti-apoptotic genes (Bcl-2, Bcl-xL, and NFκB) is diminished, whereas the expression of pro-apoptotic genes (Bik, Bok, and Bmf) was increased. The expression of noradrenergic genes (TH and DBH) is not affected. These results demonstrate that Gata3 is continuously required for maintaining survival but not differentiation in the sympathetic neuron lineage up to mature neurons of adult animals. [ABSTRACT FROM AUTHOR]

An introduction is presented in which the editor discusses various articles within the issue on topics including transcriptional deregulation in Huntington's disease, transcription factors associated with development of dopaminergic neurons, and mechanisms of neuronal self-defense based on rRNA.

The family of CREB (cAMP response element-binding protein) transcription factors are involved in a variety of biological processes including the development and plasticity of the nervous system. In the maturing and adult brain, CREB genes are required for activity dependent processes, including synaptogenesis, refinement of connections and long-term potentiation. Here, we use CREB1NescreCREM-/- (cAMP-responsive element modulator) mutants to investigate the role of these genes in stimulus-independent patterns of neural activity at early stages. We show that lack of CREB/CREM genes specifically in neural tissue leads to increased synaptogenesis and to a dramatic increase in the levels of spontaneous network activity at embryonic stages. Thus, the functions of CREB/CREM genes in neural activity differ in distinct periods of neural development. [ABSTRACT FROM AUTHOR]

Transcription of rRNA genes is essential for maintaining nucleolar integrity, a hallmark for the healthy state and proliferation rate of a cell. Inhibition of rRNA synthesis leads to disintegration of the nucleolus, elevated levels of p53, and induction of cell suicide, identifying the nucleolus as a critical stress sensor. Whether deregulation of rRNA synthesis is causally involved in neurodegeneration by promoting cell death and/or by inhibiting cellular growth has however not been addressed. The transcription factor TIF-IA plays a central role in mammalian rRNA synthesis, regulating the transcriptional activity of RNA polymerase I. To investigate the consequences of nucleolar perturbation in the nervous system, we have chosen to specifically ablate the gene encoding the transcription factor TIF-IA in two different contexts: neural progenitors and hippocampal neurons. Here, we show that ablation of TIF-IA leads to impaired nucleolar activity and results in increased levels of the proapoptotic transcription factor p53 in both neural progenitors and hippocampal neurons but induces rapid apoptosis only in neural progenitors. Nondividing cells of the adult hippocampus are more refractory to loss of rRNA transcription and face a protracted degeneration. Our study provides an unexploited strategy to initiate neurodegeneration based on perturbation of nucleolar function and underscores a novel perspective to study the cellular and molecular changes involved in the neurodegenerative processes. [ABSTRACT FROM AUTHOR]

Neuronal activity regulates neurogenesis and neuronal differentiation in the mammalian brain. The commencement of neurotransmitter expression establishes the neuronal phenotype and enables the formation of functional connectivity between neurons. In addition, release of neurotransmitters from differentiating neurons may modulate the behaviour of neural precursors. Here, we show that neuronal activity regulates γ-aminobutyric acid (GABA) expression in neurons generated from stem cells of the striatum and adult subventricular zone (SVZ). Differentiating neurons display spontaneous Ca2+ events, which are voltage-gated calcium channel (VGCC) dependent. Depolarization increases both the frequency of Ca2+ transients and the amount of Ca2+ influx in differentiating neurons. We show that depolarization-dependent GABA expression is regulated by the amplitude and not by the frequency of Ca2+ influx. Brief activation of VGCCs leads to Ca2+ influx that in turn promotes a rapid expression of GABA. Depolarization-dependent GABA expression does not require changes in gene expression. Instead, it involves cAMP-dependent protein kinase (PKA) and Ca2+ and phospholipid-dependent protein kinase (PKC) signalling. Activity increases the number of glutamic acid decarboxylase (GAD) 65-immunoreactive neurons in a PKA-dependent manner, without altering the expression of GAD 65, suggesting that depolarization promotes recruitment of GAD 65 by a post-translational mechanism. In line with this, depolarization does not permanently increase the expression of GABA in neurons derived from neural stem cells of the embryonic striatum, cortex and adult SVZ. Thus, neuronal activity does not merely accelerate neuronal differentiation but it may alter the mechanism of GABA synthesis in newly generated neurons. [ABSTRACT FROM AUTHOR]

The dopamine transporter is an essential component of the dopaminergic synapse. It is located in the presynaptic neurons and regulates extracellular dopamine levels. We generated a transgenic mouse line expressing the Cre recombinase under the control of the regulatory elements of the dopamine transporter gene, for investigations of gene function in dopaminergic neurons. The codon-improved Cre recombinase ( iCre) gene was inserted into the dopamine transporter gene on a bacterial artificial chromosome. The pattern of expression of the bacterial artificial chromosome–dopamine transporter–iCre transgene was similar to that of the endogenous dopamine transporter gene, as shown by immunohistochemistry. Recombinase activity was further studied in mice carrying both the bacterial artificial chromosome–dopamine transporter–iCre transgene and a construct expressing the β-galactosidase gene after Cre-mediated recombination. In situ studies showed that β-galactosidase (5-bromo-4-chloroindol-3-yl β-d-galactoside staining) and the dopamine transporter (immunofluorescence) had identical distributions in the ventral midbrain. We used this animal model to study the distribution of dopamine transporter gene expression in hypothalamic nuclei in detail. The expression profile of tyrosine hydroxylase (an enzyme required for dopamine synthesis) was broader than that of β-galactosidase in A12 to A15. Thus, only a fraction of neurons synthesizing dopamine expressed the dopamine transporter gene. The bacterial artificial chromosome–dopamine transporter–iCre transgenic line is a unique tool for targeting Cre/ loxP-mediated DNA recombination to dopamine neurons for studies of gene function or for labeling living cells, following the crossing of these mice with transgenic Cre reporter lines producing fluorescent proteins. [ABSTRACT FROM AUTHOR]

Background: Dopamine-activated signaling regulates locomotor and emotional responses and alterations in dopamine-signaling are responsible of several psychomotor disorders. In order to identify specific functions of these pathways, the Cre/loxP system has been used. Here, we describe the generation and the characterization of a transgenic mouse line expressing the Cre recombinase in dopaminoceptive neurons. To this purpose, we used as expression vector a 140 kb yeast artificial chromosome (YAC) containing the dopamine D1 receptor gene (Drd1a). Results: In the chosen line, D1Cre, the spatio-temporal pattern of Cre expression closely recapitulated that of the endogenous Drd1a gene, as assessed by immunohistological approaches in embryonic and adult stages. Efficiency of recombination was confirmed by crossing D1Cre with three different loxP lines (Creb/loxP, CaMKIVloxP and GRloxP) and with the R26R reporter. In the three loxP lines studied, recombination was restricted to the area of Cre expression. Conclusion: In view of the patterns of recombination restricted to the major dopaminoceptive regions as seen in the context of the CREB, CaMKIV and GR mutations, the D1Cre line will be a useful tool to dissect the contributions of specific genes to biological processes involving dopamine signaling. [ABSTRACT FROM AUTHOR]

The transcription factor cAMP response element-binding protein (CREB) is involved in multiple aspects of neuronal development and plasticity. Here, we demonstrate that CREB regulates specific phases of adult neurogenesis in the subventricular zone/olfactory bulb (SVZ/OB) system. Combining immunohistochemistry with bromodeoxyuridine treatments, cell tracer injections, cell transplants, and quantitative analyses, we show that although CREB is expressed by the SVZ neuroblasts throughout the neurogenic process, its phosphorylation is transient and parallels neuronal differentiation, increasing during the late phase of tangential migration and decreasing after dendrite elongation and spine formation. In vitro, inhibition of CREB function impairs morphological differentiation of SVZ-derived neuroblasts. Transgenic mice lacking CREB, in a null CREM genetic background, show reduced survival of newborn neurons in the OB. This finding is further supported by peripheral afferent denervation experiments resulting in downregulation of CREB phosphorylation in neuroblasts, the survival of which appears heavily impaired. Together, these findings provide evidence that CREB regulates differentiation and survival of newborn neurons in the OB. [ABSTRACT FROM AUTHOR]

Primary cilia (PC) are microtubule-based protrusions of the cell membrane transducing molecular signals during brain development. Here, we report that PC are required for maintenance of Substantia nigra (SN) dopaminergic (DA) neurons highly vulnerable in Parkinson's disease (PD). Targeted blockage of ciliogenesis in differentiated DA neurons impaired striato-nigral integrity in adult mice. The relative number of SN DA neurons displaying a typical auto-inhibition of spontaneous activity in response to dopamine was elevated under control metabolic conditions, but not under metabolic stress. Strikingly, in the absence of PC, the remaining SN DA neurons were less vulnerable to the PD neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridin (MPTP). Our data indicate conserved PC-dependent neuroadaptive responses to DA lesions in the striatum. Moreover, PC control the integrity and dopamine response of a subtype of SN DA neurons. These results reinforce the critical role of PC as sensors of metabolic stress in PD and other disorders of the dopamine system. [ABSTRACT FROM AUTHOR]

Neurodegenerative diseases pose a major health problem for developed countries. Stress, which induces oxidation in the brain, has been identified as the main risk factor for these disorders. We have developed an antioxidant-enriched drink and have examined its protective properties against acute oxidative stress. We found that addition of red grape polyphenols and MecobalActive® to grape juice did not provoke changes in juice organoleptic characteristics, and that the pasteurization process did not greatly affect the levels of flavonoids and vitamin B12. Out of all combinations, grape juice with red grape polyphenols was selected by expert judges (28.6% selected it as their first choice). In vivo, oral administration of grape juice supplemented with red grape polyphenols exerted an antioxidant effect in the brain of stressed mice reducing two-fold the expression of genes involved in inflammation and oxidation mechanisms and increasing three-fold the expression of genes related to protection against oxidative stress. In addition, we found that this drink augmented antioxidant enzyme activity (17.8 vs. 8.2 nmol/mg), and prevented lipid peroxidation in the brain (49.7 vs. 96.5 nmol/mg). Therefore, we propose supporting the use of this drink by the general population as a new and global strategy for the prevention of neurodegeneration. [ABSTRACT FROM AUTHOR]

Intracellular accumulation of α-synuclein (α-syn) and formation of Lewy bodies are neuropathological characteristics of Parkinson's disease (PD) and related α-synucleinopathies. Oligomerization and spreading of α-syn from neuron to neuron have been suggested as key events contributing to the progression of PD. To directly visualize and characterize α-syn oligomerization and spreading in vivo , we generated two independent conditional transgenic mouse models based on α-syn protein complementation assays using neuron-specifically expressed split Gaussia luciferase or split Venus yellow fluorescent protein (YFP). These transgenic mice allow direct assessment of the quantity and subcellular distribution of α-syn oligomers in vivo. Using these mouse models, we demonstrate an age-dependent accumulation of a specific subtype of α-syn oligomers. We provide in vivo evidence that, although α-syn is found throughout neurons, α-syn oligomerization takes place at the presynapse. Furthermore, our mouse models provide strong evidence for a transsynaptic cell-to-cell transfer of de novo generated α-syn oligomers in vivo. • Inducible split protein mouse models to quantify α-synuclein oligomerization in vivo • Aging promotes accumulation of size-defined α-synuclein oligomers • α-synuclein oligomerization takes place at the presynapse • Cell-to-cell transmission of α-synuclein is demonstrated in an intact mouse brain Kiechle et al. present two transgenic mouse models that allow direct quantitative and spatial detection of α-synuclein (α-syn) oligomers in vivo. They demonstrate α-syn aggregation at the synapse, age-dependent accumulation of 8–16-mer α-syn oligomer species, and trans-cellular oligomer spreading from forebrain to hindbrain neurons. [ABSTRACT FROM AUTHOR]

Parkinson's disease (PD) is the second most common neurodegenerative disorder and is characterized by motor deficits such as tremor, rigidity and bradykinesia. These symptoms are directly caused by the loss of dopaminergic neurons. However, a wealth of clinical evidence indicates that the dopaminergic system is not the only system affected in PD. Postmortem studies of brains from PD patients have revealed the degeneration of noradrenergic neurons in the locus coeruleus (LC) to the same or even greater extent than that observed in the dopaminergic neurons of substantia nigra (SN) and ventral tegmental area (VTA). Moreover, studies performed on rodent models suggest that enhancement of noradrenergic transmission may attenuate the PD-like phenotype induced by MPTP administration, a neurotoxin-based PD model. The aim of this study was to investigate whether chronic treatment with either of two compounds targeting the noradrenergic system (reboxetine or atipamezole) possess the ability to reduce the progression of a PD-like phenotype in a novel mouse model of progressive dopaminergic neurodegeneration induced by the genetic inhibition of rRNA synthesis in dopaminergic neurons, mimicking a PD-like phenotype. The results showed that reboxetine improved the parkinsonian phenotype associated with delayed progression of SN/VTA dopaminergic neurodegeneration and higher dopamine content in the striatum. Moreover, the alpha1-adrenergic agonist phenylephrine enhanced survival of TH+ neurons in primary cell cultures, supporting the putative neuroprotective effects of noradrenergic stimulation. Our results provide new insights regarding the possible influence of the noradrenergic system on dopaminergic neuron survival and strongly support the hypothesis regarding the neuroprotective role of noradrenaline. [ABSTRACT FROM AUTHOR]

A correction to the article "CREB activity in dopamine D1 receptor expressing neurons regulates cocaine-induced behavioral effects" that was published in the 2014 issue is presented.

The aim of this study was to investigate whether conditional inactivation of the glucocorticoid receptors (GRs) in noradrenergic neurons affects animal behavior in mice. Selective ablation of GRs in the noradrenergic system was achieved using the Cre/loxP approach. We crossed transgenic mice expressing the Cre recombinase under the dopamine beta-hydroxylase (DBH) promoter with animals harboring the floxed GR gene. The resulting GRDBHCre mutant mice exhibited no alterations in terms of normal cage behavior, weight gain, spatial memory or spontaneous locomotor activity, regardless of gender. To assess depressive- and anxiety-like behaviors we performed the Tail Suspension Test and the Light-Dark Box Test. While male mutant animals did not show any alternations in both tests, female GRDBHCre mutants displayed depressive- and anxiety-like behavior. Additionally, male GRDBHCre mice were exposed to chronic restraint stress but still exhibited immobility times and anxiety statuses similar to those of non-stressed animals while stressed control mice clearly revealed depressive- and anxiety-like phenotype. Thus, in males the effects of the mutation were precipitated only after chronic restraint stress procedure. Our data reveal a possible gender-dependent role of GRs in the noradrenergic system in anxiety- and depressive-like behavior in mice. [ABSTRACT FROM AUTHOR]

The cyclic-AMP response element-binding (CREB) protein family of transcription factors plays a crucial role in supporting the survival of neurons. However, a cell-autonomous role has not been addressed in vivo. To investigate the cell-specific role of CREB, we used as a model developing sympathetic neurons, whose survival in vitro is dependent on CREB activity. We generated mice lacking CREB in noradrenergic (NA) and adrenergic neurons and compared them with the phenotype of the germline CREB mutant. Whereas the germline CREB mutant revealed increased apoptosis of NA neurons and misplacement of sympathetic precursors, the NA neuron-specific mutation unexpectedly led to reduced levels of caspase-3-dependent apoptosis in sympathetic ganglia during the period of naturally occurring neuronal death. A reduced level of p75 neurotrophin receptor expression in the absence of CREB was shown to be responsible. Thus, our analysis indicates that the activity of cell-autonomous pro-survival signalling is operative in developing sympathetic neurons in the absence of CREB. [ABSTRACT FROM AUTHOR]

Sympathetic neurons are generated through a succession of differentiation steps that initially lead to noradrenergic neurons innervating different peripheral target tissues. Specific targets, like sweat glands in rodent footpads, induce a change from noradrenergic to cholinergic transmitter phenotype. Here, we show that cytokines acting through the gp130 receptor are present in sweat glands. Selective elimination of the gp130 receptor in sympathetic neurons prevents the acquisition of cholinergic and peptidergic features (VAChT, ChT1, VIP) without affecting other properties of sweat gland innervation. The vast majority of cholinergic neurons in the stellate ganglion, generated postnatally, are absent in gp130-deficient mice. These results demonstrate an essential role of gp130-signaling in the target-dependent specification of the cholinergic neurotransmitter phenotype. [ABSTRACT FROM AUTHOR]