Queuosine-tRNA promotes sex-dependent learning and memory formation by maintaining codon-biased translation elongation speed.

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.

ALS-linked KIF5A ΔExon27 mutant causes neuronal toxicity through gain-of-function.

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.

Treatment with THI, an inhibitor of sphingosine-1-phosphate lyase, modulates glycosphingolipid metabolism and results therapeutically effective in experimental models of Huntington's disease.

Huntington's disease (HD) is a fatal neurodegenerative disorder with no effective cure currently available. Over the past few years our research has shown that alterations in sphingolipid metabolism represent a critical determinant in HD pathogenesis. In particular, aberrant metabolism of sphingosine-1-phosphate (S1P) has been reported in multiple disease settings, including human postmortem brains from HD patients. In this study, we investigate the potential therapeutic effect of the inhibition of S1P degradative enzyme SGPL1, by the chronic administration of the 2-acetyl-5-tetrahydroxybutyl imidazole (THI) inhibitor. We show that THI mitigated motor dysfunctions in both mouse and fly models of HD. The compound evoked the activation of pro-survival pathways, normalized levels of brain-derived neurotrophic factor, preserved white matter integrity, and stimulated synaptic functions in HD mice. Metabolically, THI restored normal levels of hexosylceramides and stimulated the autophagic and lysosomal machinery, facilitating the reduction of nuclear inclusions of both wild-type and mutant huntingtin proteins.

C9orf72-associated neurodegeneration in ALS-FTD: breaking new ground in ribosomal RNA and nucleolar dysfunction.

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.

Essential role of sympathetic endothelin A receptors for adverse cardiac remodeling.

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 [(124)I]-metaiodobenzylguanidine-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, antiadrenergic therapies may blunt potentially beneficial effects of ETAi. Taken together, this may indicate that patients with ß blocker intolerance or disturbed sympathetic nerve function could be evaluated for a potential benefit from ETAi.

Phasic dopaminergic activity exerts fast control of cholinergic interneuron firing via sequential NMDA, D2, and D1 receptor activation.

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.

How Parkinson's disease meets nucleolar stress.

Parkinson's disease (PD) is the second most common neurodegenerative disorder. Although the causes of PD are still not understood, aging is a predisposing factor and metabolic stress seems to be a common trigger. Interestingly, the response to stress conditions and quality control mechanisms is impaired in PD, as well as in other neurodegenerative disorders. Downregulation of rRNA transcription is one major strategy to maintain cellular homeostasis under stress conditions, as it limits energy consumption in disadvantageous circumstances. Altered rRNA transcription and disruption of nucleolar integrity are associated with neurodegenerative disorders, and with aging. Nucleolar stress can be triggered by genetic and epigenetic factors, and by specific signaling mechanisms, that are altered in neurodegenerative disorders. The consequences of neuronal nucleolar stress seem to depend on p53 function, the mammalian target of rapamycin (mTOR) activity and deregulation of protein translation. In this review, we will summarize findings identifying an emerging role of nucleolar stress for the onset and progression of in particular PD. Emphasis is given to similarities in molecular causes and consequences of nucleolar stress in other neurodegenerative disorders. The mechanisms by which nucleolar stress participates in PD could help identify novel risk factors, and develop new therapeutic strategies to slow down the progressive loss of neurons in neurodegenerative diseases. This article is part of a Special Issue entitled: Role of the Nucleolus in Human Disease.

A TBK1 variant causes autophagolysosomal and motoneuron pathology without neuroinflammation in mice.

Heterozygous mutations in the TBK1 gene can cause amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). The majority of TBK1-ALS/FTD patients carry deleterious loss-of-expression mutations, and it is still unclear which TBK1 function leads to neurodegeneration. We investigated the impact of the pathogenic TBK1 missense variant p.E696K, which does not abolish protein expression, but leads to a selective loss of TBK1 binding to the autophagy adaptor protein and TBK1 substrate optineurin. Using organelle-specific proteomics, we found that in a knock-in mouse model and human iPSC-derived motor neurons, the p.E696K mutation causes presymptomatic onset of autophagolysosomal dysfunction in neurons precipitating the accumulation of damaged lysosomes. This is followed by a progressive, age-dependent motor neuron disease. Contrary to the phenotype of mice with full Tbk1 knock-out, RIPK/TNF-α-dependent hepatic, neuronal necroptosis, and overt autoinflammation were not detected. Our in vivo results indicate autophagolysosomal dysfunction as a trigger for neurodegeneration and a promising therapeutic target in TBK1-ALS/FTD.

Bidirectional regulation of intravenous general anesthetic actions by α3-containing γ-aminobutyric acid A receptors.

BACKGROUND: γ-aminobutyric acid A (GABAA) receptors mediate the actions of several intravenous general anesthetics. However, the contribution of α3-containing GABAA receptors to the action of these drugs is unknown. METHODS: The authors compared anesthetic endpoints (hypnosis, immobility, hypothermia) in response to various intravenous anesthetics in mice lacking the α3 subunit of the GABAA receptor (α3 knockout) and in wild-type mice. Furthermore, the authors generated and analyzed conditional mutant mice expressing the GABAA receptor α3 subunit exclusively in noradrenergic neurons. RESULTS: α3 knockout mice displayed decreased hypnotic and hypothermic responses to etomidate and midazolam, but an increased response to pentobarbital. The hypnotic response to ketamine was unaltered, whereas the hypothermic response was increased. In contrast, the hypnotic but not the hypothermic response to medetomidine was increased. The combination of ketamine/xylazine displayed increased hypnotic, immobilizing, and hypothermic effects in α3 knockout mice. Mice expressing the α3 subunit exclusively in noradrenergic neurons were generated to assess whether the lack of α3 subunits on noradrenergic neurons may be responsible for this effect. In these mice, the increases of the hypnotic and immobilizing actions induced by ketamine/xylazine were largely absent, whereas the increase in the hypothermic action was still present. CONCLUSION: α3-containing GABAA receptors bidirectionally regulate essential anesthetic actions: they mediate anesthetic actions of etomidate and midazolam, known to selectively act at GABAA receptors, and they negatively constrain anesthetic actions of compounds with targets partly or exclusively distinct from GABAA receptors such as medetomidine, ketamine, and pentobarbital. Furthermore, our results indicate that α3-containing GABAA receptors on noradrenergic neurons may contribute to this constraint.

Deep learning-based image analysis identifies a DAT-negative subpopulation of dopaminergic neurons in the lateral Substantia nigra.

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.

Nucleolar disruption in dopaminergic neurons leads to oxidative damage and parkinsonism through repression of mammalian target of rapamycin signaling.

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 pharmacological PD model 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.

Glutamate input to noradrenergic neurons plays an essential role in the development of morphine dependence and psychomotor sensitization.

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 ß-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.

Pten ablation in adult dopaminergic neurons is neuroprotective in Parkinson's disease models.

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.

Oxidative Stress in Neurodegenerative Diseases.

Oxidative stress is typically reported in neurodegenerative diseases [...].

Genetic lesions of the noradrenergic system trigger induction of oxidative stress and inflammation in the ventral midbrain.

Parkinson's disease (PD) is a neurodegenerative disorder characterized by motor deficits caused by the loss of dopaminergic neurons in the substantia nigra (SN) and ventral tegmental area (VTA). However, clinical data revealed that not only the dopaminergic system is affected in PD. Postmortem studies showed degeneration of noradrenergic cells in the locus coeruleus (LC) to an even greater extent than that observed in the SN/VTA. Pharmacological models support the concept that modification of noradrenergic transmission can influence the PD-like phenotype induced by neurotoxins. Nevertheless, there are no existing data on animal models regarding the distant impact of noradrenergic degeneration on intact SN/VTA neurons. The aim of this study was to create a transgenic mouse model with endogenously evoked progressive degeneration restricted to noradrenergic neurons and investigate its long-term impact on the dopaminergic system. To this end, we selectively ablated the transcription initiation factor-IA (TIF-IA) in neurons expressing dopamine ß-hydroxylase (DBH) by the Cre-loxP system. This mutation mimics a condition of nucleolar stress affecting neuronal survival. TIF-IADbhCre mice were characterized by selective, progressive degeneration of noradrenergic neurons, followed by phenotypic alterations associated with sympathetic system impairment. Our studies did not show any loss of tyrosine hydroxylase (TH)-positive cells in the SN/VTA of mutant mice; however, we observed increased indices of oxidative stress, enhanced markers of glial cell activation, inflammatory processes and isolated degenerating cells positive for FluoroJade C. These results were supported by gene expression profiling of VTA and SN from TIF-IADbhCre mice, revealing that 34 out of 246 significantly regulated genes in the SN/VTA were related to PD. Overall, our results shed new light on the possible negative influence of noradrenergic degeneration on dopaminergic neurons, reinforcing the neuroprotective role of noradrenaline.

The Gata3 transcription factor is required for the survival of embryonic and adult sympathetic neurons.

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 NFkappaB) 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.

Targeted Ablation of Primary Cilia in Differentiated Dopaminergic Neurons Reduces Striatal Dopamine and Responsiveness to Metabolic Stress.

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.

Nucleolar stress controls mutant Huntington toxicity and monitors Huntington's disease progression.

Transcriptional and cellular-stress surveillance deficits are hallmarks of Huntington's disease (HD), a fatal autosomal-dominant neurodegenerative disorder caused by a pathological expansion of CAG repeats in the Huntingtin (HTT) gene. The nucleolus, a dynamic nuclear biomolecular condensate and the site of ribosomal RNA (rRNA) transcription, is implicated in the cellular stress response and in protein quality control. While the exact pathomechanisms of HD are still unclear, the impact of nucleolar dysfunction on HD pathophysiology in vivo remains elusive. Here we identified aberrant maturation of rRNA and decreased translational rate in association with human mutant Huntingtin (mHTT) expression. The protein nucleophosmin 1 (NPM1), important for nucleolar integrity and rRNA maturation, loses its prominent nucleolar localization. Genetic disruption of nucleolar integrity in vulnerable striatal neurons of the R6/2 HD mouse model decreases the distribution of mHTT in a disperse state in the nucleus, exacerbating motor deficits. We confirmed NPM1 delocalization in the gradually progressing zQ175 knock-in HD mouse model: in the striatum at a presymptomatic stage and in the skeletal muscle at an early symptomatic stage. In Huntington's patient skeletal muscle biopsies, we found a selective redistribution of NPM1, similar to that in the zQ175 model. Taken together, our study demonstrates that nucleolar integrity regulates the formation of mHTT inclusions in vivo, and identifies NPM1 as a novel, readily detectable peripheral histopathological marker of HD progression.

The CREB/CREM transcription factors negatively regulate early synaptogenesis and spontaneous network activity.

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 CREB1(Nescre)CREM(-/-) (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.