Neuronal Deletion Research Articles

Extracellular signal-regulated kinase 2 has duality in function between neuronal and astrocyte expression following neonatal hypoxic-ischaemic cerebral injury.

Key points This study identifies phosphorylated extracellular signal‐regulated kinase (ERK) to be immediately diminished followed by a rapid if transient increase for up to 4 h following hypoxic–ischaemic insult (HI) in the neonatal mouse.Phosphorylated ERK up‐regulation was prevented with systemic injection of the mitogen‐activated protein kinase kinase (MEK) inhibitor SL327. Treatment with SL327 both pre‐ and post‐HI gave a strong reduction in the number of dying cells and microgliosis.By utilising transgenic mouse mutations, we observe that neuronal ERK2 significantly contributes to tissue damage, while ERK1 and astrocytic ERK2 are neuroprotective.Compared to global inactivation, selective cell‐specific interference with ERK activity could result in stronger neuroprotection. Hypoxia–ischaemia (HI) is a major cause of neonatal brain injury resulting in cerebral palsy, epilepsy, cognitive impairment and other neurological disabilities. The role of extracellular signal‐regulated kinase (ERK) isoforms and their mitogen‐activated protein kinase kinase (MEK)‐dependent phosphorylation in HI has previously been explored but remains unresolved at cellular level. This is pertinent given the growing awareness of the role of non‐neuronal cells in neuroprotection. Using a modified Rice–Vannucci model of HI in the neonatal mouse we observed time‐ and cell‐dependent ERK phosphorylation (pERK), with strongly up‐regulated pERK immunoreactivity first in periventricular white matter axons within 15–45 min of HI, followed by forebrain astrocytes and neurons (1–4 h post‐HI), and return to baseline by 16 h. We explored the effects of pharmacological ERK blockade through the MEK inhibitor SL327 on neonatal HI‐brain damage following HI alone (30 or 60 min) or lipopolysaccharide (LPS)‐sensitised HI insult (30 min). Global inhibition of ERK phosphorylation with systemically applied SL327 abolished forebrain pERK immunoreactivity, and significantly reduced cell death and associated microglial activation at 48 h post‐HI. We then explored the effects of cell‐specific ERK2 deletion alone or in combination with global ERK1 knockout under the same conditions of HI insult. Neuronal ERK2 deletion strongly decreased infarct size, neuronal cell death and microglial activation in grey matter following both HI alone or LPS‐sensitised HI. ERK1 deletion attenuated the protective effect of neuronal ERK2 deletion. Removal of astroglial ERK2 produced a reverse response, with a 3‐ to 4‐fold increase in microglial activation and cell death. Our data suggest a cell‐specific and time‐dependent role of ERK in neonatal HI, with a predominant, neurotoxic effect of neuronal ERK2, which is counteracted by neuroprotection by ERK1 and astrocytic ERK2. Overall, global pharmacological inhibition of ERK phosphorylation is strongly neuroprotective.

Food anticipatory activity on a calorie-restricted diet is independent of Sirt1.

A number of studies have demonstrated that the Sirtuin family member, Sirt1, is a key integrator of growth, metabolism, and lifespan. Sirt1 directly interacts with and deacetylates key regulators of the circadian clock, positioning it to be an important link between feeding and circadian rhythms. In fact, one study suggests that Sirt1 is necessary for behavioral anticipation of limited daily food availability, a circadian process termed food anticipatory activity (FAA). In their study, mice overexpressing Sirt1 had enhanced FAA, while mice lacking Sirt1 had little to no FAA. Based on the supposition that Sirt1 was indeed required for FAA, we sought to use Sirt1 deletion to map the neural circuitry responsible for FAA. We began by inactivating Sirt1 using the cell-type specific Cre-driver lines proopiomelanocortin, but after observing no effect on body weight loss or FAA we then moved on to more broadly neuronal Cre drivers Ca2+/calmodulin-dependent protein kinase II and nestin. As neither of these neuronal deletions of Sirt1 had impaired FAA, we then tested 1) a broad postnatal tamoxifen-inducible deletion, 2) a complete, developmental knockout of Sirt1, and 3) a gene replacement, catalytically inactive, form of Sirt1; but all of these mice had FAA similar to controls. Therefore, our findings suggest that FAA is completely independent of Sirt1.

LXRs Protect DRG Neurons from High-Fat Diet–Induced ER Stress and Allodynia

Painful diabetic neuropathy (DN) affecting small sensory neurons is one of the most common complications of obesity and type 2 diabetes. While emerging clinical evidence demonstrates that DN is associated with dyslipidemia, the complex molecular and cellular neurobiology triggering the disease is unknown. Emerging evidence demonstrates the important contribution of complex pathways including endoplasmic reticulum stress and inflammation. Recent OMICs data analyzing peripheral nervous tissue (Dorsal root ganglia (DRG) and associated nerves) of obese individuals have also highlighted alterations in lipid nuclear receptors, which provide novel insight into the onset of DN. Liver X Receptors (LXR) α and β are nuclear transcription factors that respond to cholesterol or fatty acid metabolites. Nuclear LXRs control gene programs in liver cells, adipocytes, and macrophages to regulate lipid metabolism, inflammation and ER stress though the specific physiological role of LXRs in neurons is difficult to interpret given its broad tissue expression. Our tissue specific deletion model unmasks an important role of LXR in small sensory neurons in type 2 DN pathogenesis. Our results show that LXR deletion in sensory neurons worsens the hypersensitivity induced by a High fat Diet (HFD) associated with changes in DRG gene. In addition, treatment with the LXR agonist GW4965 improves the HFD-induced mechanical allodynia in vivo and protects mice DRG sensory neurons from lipid-induced ER stress in vitro and in vivo (ribo-tag model), further confirming a protective role for LXR in DN. Our data suggest that the lipid sensor LXRs in small sensory neurons could respond to dietary lipid to potentially maintain normal PNS function in the face of high-fat nutrition. Disclosure V. Aubert: None.

Neuronal Deletion of the Mammalian Indy Homolog (Slc13a5) Increases Energy Expenditure in Mice

INDY (I’m Not Dead Yet) is a transporter of TCA cycle intermediates, mediating cellular citrate uptake and is highly expressed in liver and brain. Reduced expression of Indy in lower organisms extended lifespan, reduced whole body fat content and increased mitochondrial biogenesis. In mammals, whole body deletion of the mammalian Indy homolog (mIndy, Slc13a5) increased energy expenditure and protected mice from diet and aging induced obesity and insulin resistance. Here, we address the role of neuronal mIndy in energy homeostasis. We generated a neuronal mIndy knockout (NINKO) mouse model by crossing mIndy-floxed to NestinCre mice. Deletion of mIndy in neurons led to an increase in energy expenditure compared to NestinCre mice after 8 weeks of high fat diet (HFD) feeding (average 24h EE: NestinCre: 11.5±0.5 kcal/h/kg lean mass (lm); NINKO: 13.6±0.6 kcal/h/kg lm; p<0.001). The respiratory exchange ratio decreased from 0.78±0.01 in NestinCre mice to 0.77±0.01 in NINKO mice during the light phase (p<0.001), indicating augmented lipid oxidation in NINKO mice. Body temperature increased in NINKO compared to NestinCre mice (NestinCre: 37.0±0.1°C, NINKO: 37.3±0.1°C at 6pm; p<0.05). Fat mass was reduced and lean mass increased significantly in NINKO mice (NestinCre: 60±1% lm, 31±1% fat mass; NINKO: 63±1% lm, 27±1% fat mass after 8 weeks of HFD feeding; p<0.05). Hyperinsulinemic-euglycemic clamp studies showed improved insulin sensitivity in NINKO mice (steady state GINF NestinCre: 26±2 mg/kg/min; NINKO: 34.6±2 mg/kg/min; p<0.05). Together, these data suggest that neuronal mIndy is a critical regulator of energy and glucose homeostasis in mammals. Further studies will address the mechanisms involved in the effect. Disclosure A. Kurzbach: None. D.M. Willmes: None. T. Schumann: None. C. Henke: None. N. El-Agroudy: None. A. Kleinridders: None. A.L. Birkenfeld: None.

Shank2 Deletion in Parvalbumin Neurons Leads to Moderate Hyperactivity, Enhanced Self-Grooming and Suppressed Seizure Susceptibility in Mice.

Shank2 is an abundant postsynaptic scaffolding protein implicated in neurodevelopmental and psychiatric disorders, including autism spectrum disorders (ASD). Deletion of Shank2 in mice has been shown to induce social deficits, repetitive behaviors, and hyperactivity, but the identity of the cell types that contribute to these phenotypes has remained unclear. Here, we report a conditional mouse line with a Shank2 deletion restricted to parvalbumin (PV)-positive neurons (Pv-Cre;Shank2fl/fl mice). These mice display moderate hyperactivity in both novel and familiar environments and enhanced self-grooming in novel, but not familiar, environments. In contrast, they showed normal levels of social interaction, anxiety-like behavior, and learning and memory. Basal brain rhythms in Pv-Cre;Shank2fl/fl mice, measured by electroencephalography, were normal, but susceptibility to pentylenetetrazole (PTZ)-induced seizures was decreased. These results suggest that Shank2 deletion in PV-positive neurons leads to hyperactivity, enhanced self-grooming and suppressed brain excitation.

Targeting of microRNA-21-5p protects against seizure damage in a kainic acid-induced status epilepticus model via PTEN-mTOR

ObjectiveStudies have shown that microRNAs play a role in the development of epilepsy by regulating downstream target messenger (m)RNA. The present study aims to determine the changes associated with microRNA-21-5p (miR-21-5p) during epileptogenesis in a kainic acid rat model, and to assess whether the PTEN-mTOR pathway is a target of miR-21-5p. MethodReverse transcription polymerase chain reaction (RT-PCR) was used to examine the quantitative expressions of miR-21-5p and PTEN, and Western blotting was used to test the activity of mTOR in the acute, latent, and chronic stages of epileptogenesis. The antagomir of miR-21-5p was injected into the intracerebroventricular space using a microsyringe. Neuronal death and epilepsy discharge were assessed by Nissl staining and electroencephalography (EEG), respectively. The Morris water maze (MWM) was used to assess the cognitive impairment in rats after status epilepticus (SE). ResultsBoth miR-21-5p and mTOR were upregulated and PTEN was downregulated in rats during acute, latent, and chronic stages of epileptogenesis when compared with those of the control. After using antagomir miR-21-5p in vivo, miR-21-5p and mTOR decreased and the expression of PTEN increased compared with that in the SE model. The silencing of miR-21-5p diminished the number of abnormal spikes on EEG and decreased the number of neuron deletions on Nissl staining. The cognitive and memory impairment caused by epilepsy could also be improved after miR-21-5p knockdown in vivo. ConclusionThe results of the present study demonstrate that PTEN-mTOR is the target of miR-21-5p in a kainic acid model of epilepsy. The knockout of miR-21-5p decreases the neuronal damage in stages of epileptogenesis. The miR-21-5p/PTEN/mTOR axis may be a potential target for preventing and treating seizures and epileptic damage.

TCPTP Regulates Insulin Signaling in AgRP Neurons to Coordinate Glucose Metabolism With Feeding.

Insulin regulates glucose metabolism by eliciting effects on peripheral tissues as well as the brain. Insulin receptor (IR) signaling inhibits AgRP-expressing neurons in the hypothalamus to contribute to the suppression of hepatic glucose production (HGP) by insulin, whereas AgRP neuronal activation attenuates brown adipose tissue (BAT) glucose uptake. The tyrosine phosphatase TCPTP suppresses IR signaling in AgRP neurons. Hypothalamic TCPTP is induced by fasting and degraded after feeding. Here we assessed the influence of TCPTP in AgRP neurons in the control of glucose metabolism. TCPTP deletion in AgRP neurons (Agrp-Cre;Ptpn2fl/fl ) enhanced insulin sensitivity, as assessed by the increased glucose infusion rates, and reduced HGP during hyperinsulinemic-euglycemic clamps, accompanied by increased [14C]-2-deoxy-d-glucose uptake in BAT and browned white adipose tissue. TCPTP deficiency in AgRP neurons promoted the intracerebroventricular insulin-induced repression of hepatic gluconeogenesis in otherwise unresponsive food-restricted mice, yet had no effect in fed/satiated mice where hypothalamic TCPTP levels are reduced. The improvement in glucose homeostasis in Agrp-Cre;Ptpn2fl/fl mice was corrected by IR heterozygosity (Agrp-Cre;Ptpn2fl/fl ;Insrfl/+ ), causally linking the effects on glucose metabolism with the IR signaling in AgRP neurons. Our findings demonstrate that TCPTP controls IR signaling in AgRP neurons to coordinate HGP and brown/beige adipocyte glucose uptake in response to feeding/fasting.

Remote Memory and Cortical Synaptic Plasticity Require Neuronal CCCTC-Binding Factor (CTCF).

The molecular mechanism of long-term memory has been extensively studied in the context of the hippocampus-dependent recent memory examined within several days. However, months-old remote memory maintained in the cortex for long-term has not been investigated much at the molecular level yet. Various epigenetic mechanisms are known to be important for long-term memory, but how the 3D chromatin architecture and its regulator molecules contribute to neuronal plasticity and systems consolidation is still largely unknown. CCCTC-binding factor (CTCF) is an 11-zinc finger protein well known for its role as a genome architecture molecule. Male conditional knock-out mice in which CTCF is lost in excitatory neurons during adulthood showed normal recent memory in the contextual fear conditioning and spatial water maze tasks. However, they showed remarkable impairments in remote memory in both tasks. Underlying the remote memory-specific phenotypes, we observed that female CTCF conditional knock-out mice exhibit disrupted cortical LTP, but not hippocampal LTP. Similarly, we observed that CTCF deletion in inhibitory neurons caused partial impairment of remote memory. Through RNA sequencing, we observed that CTCF knockdown in cortical neuron culture caused altered expression of genes that are highly involved in cell adhesion, synaptic plasticity, and memory. These results suggest that remote memory storage in the cortex requires CTCF-mediated gene regulation in neurons, whereas recent memory formation in the hippocampus does not.SIGNIFICANCE STATEMENT CCCTC-binding factor (CTCF) is a well-known 3D genome architectural protein that regulates gene expression. Here, we use two different CTCF conditional knock-out mouse lines and reveal, for the first time, that CTCF is critically involved in the regulation of remote memory. We also show that CTCF is necessary for appropriate expression of genes, many of which we found to be involved in the learning- and memory-related processes. Our study provides behavioral and physiological evidence for the involvement of CTCF-mediated gene regulation in the remote long-term memory and elucidates our understanding of systems consolidation mechanisms.

Brain region-specific disruption of Shank3 in mice reveals a dissociation for cortical and striatal circuits in autism-related behaviors

We previously reported a new line of Shank3 mutant mice which led to a complete loss of Shank3 by deleting exons 4−22 (Δe4−22) globally. Δe4−22 mice display robust ASD-like behaviors including impaired social interaction and communication, increased stereotypical behavior and excessive grooming, and a profound deficit in instrumental learning. However, the anatomical and neural circuitry underlying these behaviors are unknown. We generated mice with Shank3 selectively deleted in forebrain, striatum, and striatal D1 and D2 cells. These mice were used to interrogate the circuit/brain-region and cell-type specific role of Shank3 in the expression of autism-related behaviors. Whole-cell patch recording and biochemical analyses were used to study the synaptic function and molecular changes in specific brain regions. We found perseverative exploratory behaviors in mice with deletion of Shank3 in striatal inhibitory neurons. Conversely, self-grooming induced lesions were observed in mice with deletion of Shank3 in excitatory neurons of forebrain. However, social, communicative, and instrumental learning behaviors were largely unaffected in these mice, unlike what is seen in global Δe4−22 mice. We discovered unique patterns of change for the biochemical and electrophysiological findings in respective brain regions that reflect the complex nature of transcriptional regulation of Shank3. Reductions in Homer1b/c and membrane hyper-excitability were observed in striatal loss of Shank3. By comparison, Shank3 deletion in hippocampal neurons resulted in increased NMDAR-currents and GluN2B-containing NMDARs. These results together suggest that Shank3 may differentially regulate neural circuits that control behavior. Our study supports a dissociation of Shank3 functions in cortical and striatal neurons in ASD-related behaviors, and it illustrates the complexity of neural circuit mechanisms underlying these behaviors.

Huntington's disease pattern of transcriptional dysregulation in the absence of mutant huntingtin is produced by knockout of neuronal GLT-1

GLT-1 is the major glutamate transporter in the brain, and is expressed in astrocytes and in axon terminals in the hippocampus, cortex, and striatum. Neuronal GLT-1 accounts for only 5–10% of total brain GLT-1 protein, and its function is uncertain. In HD, synaptic dysfunction of the corticostriate synapse is well-established. Transcriptional dysregulation is a key feature of HD. We hypothesized that deletion of neuronal GLT-1, because it is expressed in axon terminals in the striatum, might produce a synaptopathy similar to that present in HD. If true, then some of the gene expression changes observed in HD might also be observed in the neuronal GLT-1 knockout. In situ hybridization using 33P labeled oligonucleotide probes was carried out to assess localization and expression of a panel of genes known to be altered in expression in HD. We found changes in the expression of cannabinoid receptors 1 and 2, preproenkaphalin, and PDE10A in the striatum of mice in which the GLT-1 gene was inactivated in neurons by expression of synapsin-Cre, compared to wild-type littermates. These changes in expression were observed at 12 weeks of age but not at 6 weeks of age. No changes in DARPP-32, PDE1B, NGFIA, or β-actin expression were observed. In addition, we found widespread alteration in expression of the dynamin 1 gene. The changes in expression in the neuronal GLT-1 knockout of genes thought to exemplify HD transcriptional dysregulation suggest an overlap in the synaptopathy caused by neuronal GLT-1 deletion and HD. These data further suggest that specific changes in expression of cannabinoid receptors, preproenkephalin, and PDE10A, considered to be the hallmark of HD transcriptional dysregulation, may be produced by an abnormality of glutamate homeostasis under the regulation of neuronal GLT-1, or a synaptic disturbance caused by that abnormality, independently of mutation in huntingtin.

From Brain to Pancreas: Beneficial Effects of the Neuronal (Pro)renin Receptor Deletion in High‐fat Diet Induced Type II Diabetes

We recently reported that the (pro)renin receptor (PRR) is a key component of the brain renin‐angiotensin system, mediating the majority of Ang II formation, and plays a pivotal role in the development of hypertension. Its importance in obesity‐related metabolic syndrome is, however, unknown. We hypothesize that brain PRR plays a regulatory role in high‐fat diet (HFD) induced metabolic syndrome. To test our hypothesis, neuron‐specific PRR knockout (PRRKO) mice and wildtype (WT) littermates were fed with either HFD (60% calories from fat) or normal fat chow (NFD, 10% calories from fat) with matching calories for 16 weeks. Weekly body weight (BW) and monthly fasting blood glucose (FBG) measurements were recorded and end point glucose tolerance (GTT) and insulin sensitivity tests (IST) were performed. Liver samples were taken and paraffin‐embedded for histology. Plasma insulin and leptin were measured by ELISA using the UC Davis Mouse Metabolic Phenotyping Core service. Neuronal PRR deletion attenuated the elevation of FBG (127.12±10.46 vs. 167.77±16.57 mg/dl, p=0.039) induced by HFD. Glucose tolerance was significantly improved in PRRKO compared with WT following 16 weeks of HFD (AUC: 20557±894 vs. 29994±2976, p=0.006), while there was no significant difference in the IST between the groups. We evaluated the pancreatic islet number and size using H&E staining and found that pancreatic islet size was significantly increased following 6 weeks of HFD (33908±5890 mm, p=0.0005) compared with the NFD (11468±1246 mm) in the WT mice, and was reduced to normal size after 16 weeks of HFD (10991±1389 mm). The islet size was significantly smaller in PRRKO (17059±2968 mm, p=0.034) compared with WT mice following 6 weeks of HFD. More importantly, islets in PRRKO mice remained a similar size following 16 weeks of HFD when compared with WT mice in NFD, indicating that PRRKO may protect against islet hypertrophy in HFD. However, no change in islet number was observed among all groups. Sixteen weeks of HFD slightly increased the fasting plasma insulin level (740±100 pg/ml) compared with the NFD (437±59 pg/ml) in WT mice (p=0.05). Interestingly, the insulin level was significantly higher in the PRRKO (1292±234 pg/ml, p=0.05) compared to the WT mice following 16 weeks of HFD, suggesting a potential beneficial effect of neuronal PRRKO in promoting insulin secretion. Plasma leptin levels were significantly elevated in both WT (25983±3389 pg/ml) and PRRKO (32208±8516 pg/ml) mice following 16 weeks of HFD compared to NFD controls (5014±1022 pg/ml); however, there was no difference between these two groups. Our data indicates that PRR deletion in the neurons attenuates the development of HFD‐induced diabetes, protects against glucose intolerance, pancreatic islet hypertrophy and function during HFD possibly by promoting insulin secretion. We conclude that neuronal PRR plays a regulatory role in the development HFD‐induced type II diabetes.Support or Funding InformationNIH/NHLBI (R01HL122770) and Innovative Research Grant (17IRG33370128) to Y. FengThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Potassium Chloride Cotransporter‐3 (KCC3) in Motor Neurons

The K‐Cl cotransporter KCC3 is an electroneutral membrane transporter of the SLC12A gene family. It uses the K+ gradient established by the Na+/K+ ATPase to efflux K+, Cl−, and obligatory water from cells. KCC3 loss‐of‐function (LOF) mutations have been found to cause the disease Agenesis of the Corpus Callosum with Peripheral Neuropathy (ACCPN). ACCPN patients suffer from intellectual disability, dysmorphic features, hallucinations, degrading ambulation, and a severe, progressive sensorimotor neuropathy. Previous literature has confirmed that both global KCC3 knock‐out mice and neuron‐specific knock‐out (KO) mice recapitulate the severe sensorimotor deficits seen in patients. Furthermore, mice with KCC3 deleted in a subset of sensory neurons (parvalbumin‐positive proprioceptive neurons) also show a locomotor deficit. Thus, KCC3 dysfunction in sensory neurons may be a key to ACCPN pathogenesis; however, it is unknown if KCC3 dysfunction in sensory neurons could account for the entire range of patient symptoms, that is, both sensory and motor neuropathy. From the spinal cord, motor neurons extend their axons to the periphery where they synapse onto skeletal muscles. Reciprocally, sensory neurons, specifically proprioceptive neurons, bring information from muscles back to the spinal cord, where they synapse onto motor neurons and interneurons. Therefore, KCC3 LOF directly in motor neurons, in sensory neurons, or in both types of neurons could be responsible for the ACCPN phenotype.To investigate the participation of motor neurons, we used a mouse line that expresses the CRE recombinase under the control of the HB9 promoter. HB9 is a transcription factor expressed during peak motor neuron development at mouse embryonic days E9.5–10.5. We crossed HB9‐CRE mice with a KCC3‐floxed line to generate mice with KCC3 deleted in motor neurons (HB9‐CRE x KCC3‐flox). We then subjected cohorts of HB9‐positive KCC3‐flox mice versus HB9‐negative KCC3‐flox mice to locomotor tests. The HB9‐positive KCC3‐flox mice did not show any deficits in the accelerated rotarod test, which assesses balance and overall locomotion performance, nor in the elevated balance beam task, which assesses finer movement coordination. Absence of a phenotype indicates that targeting KCC3 deletion in motor neurons does not contribute to the development of neuropathy. These data suggest that the motor phenotype observed in ACCPN patients might be secondary to the sensory deficit. Such an indirect effect is possible as motor neurons need proper sensory feedback from muscles to appropriately coordinate posture and movement.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

P.1.018 - The impact of selective CREB deletion in serotonergic neurons on the effects of fluoxetine administration and antidepressive behaviour

The majority of current antidepressant therapies is based on the enhancement of monoaminergic transmission observed directly after drug administration, yet alleviation of depressive symptoms occurs weeks later. Therefore, the mechanism of molecular changes arising during chronic antidepressant treatment is intensively researched. Many studies have pointed to an important role of cAMP response element binding (CREB) protein in the mechanism of antidepressant drug action, however the data are inconclusive. Generally, an increase of CREB level or activity after chronic antidepressant treatment was postulated, but in other studies opposite effects were observed [1]. Moreover, CREB-deficient animals show antidepressant-like phenotype. However, the feedback from knock-out studies could have been disturbed by cAMP response element modulator (CREM) upregulation occurring upon CREB loss [2], which was not taken into consideration. In our studies we investigated the role of CREB in the mechanism of antidepressant treatment using novel, inducible transgenic mouse model lacking CREB selectively in serotonergic neurons [3], maintained in CREM-deficient background (Creb1TPH2CreERT2Crem-/- mice). The animals have been phenotypically characterized showing no impairments in their basal behavior. However, single Creb1TPH2CreERT2 mutants of both sexes presented drug-resistant phenotype in tail suspension test after fluoxetine treatment, whereas male but not female double mutants (Creb1TPH2CreERT2Crem-/-) reacted to the drug similar to control animals [4]. The aim of current studies was to assess the molecular changes induced by chronic fluoxetine (10 mg/kg i.p., 21 days, 1x daily) treatment in hippocampus and prefrontal cortex (PFC). Animals were sacrificed 24h after last injection and the brain structures were collected. Assessment of mRNA expression for Creb1 and Bdnf genes was measured by RT-PCR using TaqMan probes. The levels of phosphorylated CREB, total CREB and BDNF proteins were studied using Western Blot. The results were analyzed using two-way ANOVA, followed by Fisher’s LSD test. We observed no effect in mRNA expression of Creb1 and Bdnf genes in control animals after fluoxetine treatment, nor in any of studied mutants. Moreover, no changes in phosphorylation of CREB or total CREB protein were visible in any of studied group. On the other hand, BDNF protein was strongly increased in response to drug in hippocampus of control males and females (by 212% and 120%, respectively), and in PFC of control females only (by 47%). Both treated and non-treated Creb1TPH2CreERT2 single mutant mice did not show any changes in BDNF in both studied brain structures; the result was significantly different from the effect observed in control animals after fluoxetine administration. Creb1TPH2CreERT2Crem-/- double mutants presented only non-significant tendency of increased BDNF protein level in hippocampus (both males and females) and PFC (females only). Despite the absence of any effect in CREB mRNA or protein levels observed in both mutant lines, the BDNF upregulation in fluoxetine-treated control mice suggests increased activity of CREB in hippocampus after antidepressant treatment as observed by other investigators [5]. Lack of molecular or behavioral changes after fluoxetine treatment in Creb1TPH2CreERT2 single mutant animals suggests that compensatory role of CREM upregulation in the inducible line may have different effects from that observed previously in constitutive CREB knockouts.

(110) - Optogenetic silencing of CGRPα-expressing sensory neurons alleviates neuropathic pain

Calcitonin gene-related peptide (CGRP) α-expressing sensory neurons are essential for thermal sensation in both the naïve and injured state, but for mechanical sensation only after injury. Previously, it was demonstrated that chemical ablation of these neurons decreased the heat hypersensitivity that develops following both neuropathic and inflammatory injuries. To determine if the silencing of intact neurons results in similar or different behavioral outcomes, and to further investigate the involvement of this cell population in spared nerve injury (SNI), postoperative pain and Complete Freund's Adjuvant (CFA) inflammation, we used mice that selectively express Archaerhodopsin (Arch) in CGRPα-positive neurons. We report that the presence of intact CGRPα neurons after injury results in different behavioral phenotypes than when these neurons are ablated before injury. Further, our results reveal that intact peripheral CGRPα neurons are required for SNI-induced mechanical and thermal hyperalgesia, but not inflammatory-induced mechanical and heat hyperalgesia. In addition, we find that CGRPα peptide signaling does not play a role in SNI-mediated mechanical or thermal hyperalgesias, but instead is responsible for inflammation-induced mechanical and heat hyperalgesias. Furthermore, our data show that isolated sensory neurons are sensitized to mechanical stimuli after SNI and that this sensitization can be reversed by Arch activation. These results reveal previously unknown functions for CGRPα-expressing neurons in the establishment of hyperalgesia following neuropathic injury. Furthermore, transient peripheral silencing of these neurons evades the compensatory mechanisms or circuitry changes that might occur from permanent deletion of CGRPα-expressing neurons, thus revealing critical targets for the treatment of neuropathic pain.

AgRP Neurons Require Carnitine Acetyltransferase to Regulate Metabolic Flexibility and Peripheral Nutrient Partitioning

AgRP neurons control peripheral substrate utilizationand nutrient partitioning during conditions of energy deficit and nutrient replenishment, although the molecular mechanism is unknown. We examined whether carnitine acetyltransferase (Crat) in AgRP neurons affects peripheral nutrient partitioning. Crat deletion in AgRP neurons reduced food intake and feeding behavior and increased glycerol supply to the liver during fasting, as a gluconeogenic substrate, which was mediated by changes to sympathetic output and peripheral fatty acid metabolism in the liver. Crat deletion in AgRP neurons increased peripheral fatty acid substrate utilization and attenuated the switch to glucose utilization after refeeding, indicating altered nutrient partitioning. Proteomic analysis in AgRP neurons shows that Crat regulatesprotein acetylation and metabolic processing. Collectively, our studies highlight that AgRP neurons require Crat to provide the metabolic flexibility to optimize nutrient partitioning and regulate peripheral substrate utilization, particularly during fasting and refeeding.

Neuronal PTEN deletion in adult cortical neurons triggers progressive growth of cell bodies, dendrites, and axons

Deletion of the phosphatase and tensin (PTEN) gene in neonatal mice leads to enlargement of the cell bodies of cortical motoneurons (CMNs) in adulthood (Gutilla et al., 2016). Here, we assessed whether PTEN deletion in adult mice would trigger growth of mature neurons. PTEN was deleted by injecting AAV-Cre into the sensorimotor cortex of adult transgenic mice with a lox-P flanked exon 5 of the PTEN gene and Cre-dependent reporter gene tdTomato. PTEN-deleted CMN's identified by tdT expression and retrograde labeling with fluorogold (FG) were significantly enlarged four months following PTEN deletion, and continued to increase in size through the latest time intervals examined (12–15 months post-deletion). Sholl analyses of tdT-positive pyramidal neurons revealed increases in dendritic branches at 6 months following adult PTEN deletion, and greater increases at 12 months. 12 months after adult PTEN deletion, axons in the medullary pyramids were significantly larger and G-ratios were higher. Mice with PTEN deletion exhibited no overt neurological symptoms and no seizures. Assessment of motor function on the rotarod and cylinder test revealed slight impairment of coordination with unilateral deletion; however, mice with bilateral PTEN deletion in the motor cortex performed better than controls on the rotarod at 8 and 10 months post-deletion. Our findings demonstrate that robust neuronal growth can be induced in fully mature cortical neurons long after the developmental period has ended and that this continuous growth occurs without obvious functional impairments.

SGK1/FOXO3 Signaling in Hypothalamic POMC Neurons Mediates Glucocorticoid-Increased Adiposity.

Although the central nervous system has been implicated in glucocorticoid-induced gain of fat mass, the underlying mechanisms are poorly understood. The aim of this study was to investigate the possible involvement of hypothalamic serum- and glucocorticoid-regulated kinase 1 (SGK1) in glucocorticoid-increased adiposity. It is well known that SGK1 expression is induced by acute glucocorticoid treatment, but it is interesting that we found its expression to be decreased in the arcuate nucleus of the hypothalamus, including proopiomelanocortin (POMC) neurons, following chronic dexamethasone (Dex) treatment. To study the role of SGK1 in POMC neurons, we produced mice that developed or experienced adult-onset SGK1 deletion in POMC neurons (PSKO). As observed in Dex-treated mice, PSKO mice exhibited increased adiposity and decreased energy expenditure. Mice overexpressing constitutively active SGK1 in POMC neurons consistently had the opposite phenotype and did not experience Dex-increased adiposity. Finally, Dex decreased hypothalamic α-melanocyte-stimulating hormone (α-MSH) content and its precursor Pomc expression via SGK1/FOXO3 signaling, and intracerebroventricular injection of α-MSH or adenovirus-mediated FOXO3 knockdown in the arcuate nucleus largely reversed the metabolic alterations in PSKO mice. These results demonstrate that POMC SGK1/FOXO3 signaling mediates glucocorticoid-increased adiposity, providing new insights into the mechanistic link between glucocorticoids and fat accumulation and important hints for possible treatment targets for obesity.

Differential contribution of POMC and AgRP neurons to the regulation of regional autonomic nerve activity by leptin

ObjectivesThe autonomic nervous system is critically involved in mediating the control by leptin of many physiological processes. Here, we examined the role of the leptin receptor (LepR) in proopiomelanocortin (POMC) and agouti-related peptide (AgRP) neurons in mediating the effects of leptin on regional sympathetic and parasympathetic nerve activity. MethodsWe analyzed how deletion of the LepR in POMC neurons (POMCCre/LepRfl/fl mice) or AgRP neurons (AgRPCre/LepRfl/fl mice) affects the ability of leptin to increase sympathetic and parasympathetic nerve activity. We also studied mice lacking the catalytic p110α or p110β subunits of phosphatidylinositol-3 kinase (PI3K) in POMC neurons. ResultsLeptin-evoked increase in sympathetic nerve activity subserving thermogenic brown adipose tissue was partially blunted in mice lacking the LepR in either POMC or AgRP neurons. On the other hand, loss of the LepR in AgRP, but not POMC, neurons interfered with leptin-induced sympathetic nerve activation to the inguinal fat depot. The increase in hepatic sympathetic traffic induced by leptin was also reduced in mice lacking the LepR in AgRP, but not POMC, neurons whereas LepR deletion in either AgRP or POMC neurons attenuated the hepatic parasympathetic nerve activation evoked by leptin. Interestingly, the renal, lumbar and splanchnic sympathetic nerve activation caused by leptin were significantly blunted in POMCCre/LepRfl/fl mice, but not in AgRPCre/LepRfl/fl mice. However, loss of the LepR in POMC or AgRP neurons did not interfere with the ability of leptin to increase sympathetic traffic to the adrenal gland. Furthermore, ablation of the p110α, but not the p110β, isoform of PI3K from POMC neurons eliminated the leptin-elicited renal sympathetic nerve activation.Finally, we show trans-synaptic retrograde tracing of both POMC and AgRP neurons from the kidneys. ConclusionsPOMC and AgRP neurons are differentially involved in mediating the effects of leptin on autonomic nerve activity subserving various tissues and organs.

Ultradeep mapping of neuronal mitochondrial deletions in Parkinson's disease

Mitochondrial DNA (mtDNA) deletions accumulate with age in postmitotic cells and are associated with aging and neurodegenerative disorders such as Parkinson's disease. Although the exact mechanisms by which deletions form remain elusive, the dominant theory is that they arise spontaneously at microhomologous sites and undergo clonal expansion. We characterize mtDNA deletions at unprecedented resolution in individual substantia nigra neurons from individuals with Parkinson's disease, using ultradeep sequencing. We show that the number of deleted mtDNA species per neuron is substantially higher than previously reported. Moreover, each deleted mtDNA species shows significant differences in sequence composition compared with the remaining mtDNA population, which is highly consistent with independent segregation and clonal expansion. Deletion breakpoints occur consistently in regions of sequence homology, which may be direct or interrupted stretches of tandem repeats. While our results support a crucial role for misannealing in deletion generation, we find no overrepresentation of the 3′-repeat sequence, an observation that is difficult to reconcile with the current view of replication errors as the source of mtDNA deletions.

Alpha2delta-1 in SF1+ Neurons of the Ventromedial Hypothalamus Is an Essential Regulator of Glucose and Lipid Homeostasis

The central mechanisms controlling glucose and lipid homeostasis are inadequately understood. We show that α2δ-1 is an essential regulator of glucose and lipid balance, acting in steroidogenic factor-1 (SF1) neurons of the ventromedial hypothalamus (VMH). These effects are body weight independent and involve regulation of SF1+ neuronal activity and sympathetic output to metabolic tissues. Accordingly, mice with α2δ-1 deletion in SF1 neurons exhibit glucose intolerance, altered lipolysis, and decreased cholesterol content in adipose tissue despite normal energy balance regulation. Profound reductions in the firing rate of SF1 neurons, decreased sympathetic output, and elevated circulating levels of serotonin are associated with these alterations. Normal calcium currents but reduced excitatory postsynaptic currents in mutant SF1 neurons implicate α2δ-1 in the promotion of excitatory synaptogenesis separate from its canonical role as a calcium channel subunit. Collectively, these findings identify an essential mechanism that regulates VMH neuronal activity and glycemic and lipid control and may be a target for tackling metabolic disease.