Increased acetylcholine and glutamate efflux in the prefrontal cortex following intranasal orexin‐A (hypocretin‐1)

Orexin/hypocretin neurons of the lateral hypothalamus and perifornical area are integrators of physiological function. Previous work from our laboratory and others has shown the importance of orexin transmission in cognition. Age‐related reductions in markers of orexin function further suggest that this neuropeptide may be a useful target for the treatment of age‐related cognitive dysfunction. Intranasal administration of orexin‐A (OxA) has shown promise as a therapeutic option for cognitive dysfunction. However, the neurochemical mechanisms of intranasal OxA administration are not fully understood. Here, we use immunohistochemistry and in vivo microdialysis to define the effects of acute intranasal OxA administration on: (i) activation of neuronal populations in the cortex, basal forebrain, and brainstem and (ii) acetylcholine (AC h) and glutamate efflux in the prefrontal cortex (PFC ) of Fischer 344/Brown Norway F1 rats. Acute intranasal administration of OxA significantly increased c‐Fos expression, a marker for neuronal activation, in the PFC and in subpopulations of basal forebrain cholinergic neurons. Subsequently, we investigated the effects of acute intranasal OxA on neurotransmitter efflux in the PFC and found that intranasal OxA significantly increased both AC h and glutamate efflux in this region. These findings were independent from any changes in c‐Fos expression in orexin neurons, suggesting that these effects are not resultant from direct activation of orexin neurons. In total, these data indicate that intranasal OxA may enhance cognition through activation of distinct neuronal populations in the cortex and basal forebrain and through increased neurotransmission of AC h and glutamate in the PFC .

     

Clenbuterol reduces GABAergic transmission in prefrontal cortex layer 5/6 pyramidal neurons of juvenile rat via reducing action potentials firing frequency of GABAergic interneurons

Beta‐adrenoceptors (β₂‐AR s) have beneficial effects on prefrontal cortex (PFC ) working memory, however, the cellular and molecular mechanisms are unclear yet. In this study, we probed the effect of β₂‐AR ‐selective agonist clenbuterol (Clen) on synaptic transmission in layer 5/6 pyramidal neurons of PFC . Bath application of Clen reduced spontaneous IPSC (sIPSC ) frequency without effects on sEPSC s. Clen did not alter the frequency and amplitude of miniature IPSC s (mIPSC s), but exerted heterogeneous effects on evoked IPSC s (eIPSC s) recorded from PFC layer 5/6 pyramidal neurons. Clen decreased the firing rate of action potentials of fast‐spiking GABA ergic interneurons. Clen‐induced hyperpolarization of fast‐spiking GABA ergic interneurons required potentiation of an inward rectifier K⁺ channels. Clen‐induced hyperpolarization of fast‐spiking interneurons was dependent on Gs protein rather than cAMP and protein kinase A. Our findings demonstrate that Clen (10 μM) enhances inward rectifier K⁺ channels via Gs protein to cause membrane hyperpolarization of fast‐spiking GABA ergic interneurons resulting in reduction of action potentials firing rate to reduce GABA ergic transmission.

     

Vesicular GABA transporter (VGAT) transports β‐alanine

Vesicular GABA transporter (VGAT) is expressed in GABAergic and glycinergic neurons, and is responsible for vesicular storage and subsequent exocytosis of these inhibitory amino acids. In this study, we show that VGAT recognizes β‐alanine as a substrate. Proteoliposomes containing purified VGAT transport β‐alanine using Δψ but not ΔpH as a driving force. The Δψ‐driven β‐alanine uptake requires Cl^?. VGAT also facilitates Cl^? uptake in the presence of β‐alanine. A previously described VGAT mutant (Glu213Ala) that disrupts GABA and glycine transport similarly abrogates β‐alanine uptake. These findings indicated that VGAT transports β‐alanine through a mechanism similar to those for GABA and glycine, and functions as a vesicular β‐alanine transporter.

     

The effects of nesfatin‐1 in the paraventricular nucleus on gastric motility and its potential regulation by the lateral hypothalamic area in rats

The current study investigated the effects of nesfatin‐1 in the hypothalamic paraventricular nucleus (PVN) on gastric motility and the regulation of the lateral hypothalamic area (LHA). Using single unit recordings in the PVN, we show that nesfatin‐1 inhibited the majority of the gastric distention (GD)‐excitatory neurons and excited more than half of the GD‐inhibitory (GD‐I) neurons in the PVN, which were weakened by oxytocin receptor antagonist H4928. Gastric motility experiments showed that administration of nesfatin‐1 in the PVN decreased gastric motility, which was also partly prevented by H4928. The nesfatin‐1 concentration producing a half‐maximal response (EC50) in the PVN was lower than the value in the dorsomedial hypothalamic nucleus, while nesfatin‐1 in the reuniens thalamic nucleus had no effect on gastric motility. Retrograde tracing and immunofluorescent staining showed that nucleobindin‐2/nesfatin‐1 and fluorogold double‐labeled neurons were observed in the LHA. Electrical LHA stimulation changed the firing rate of GD‐responsive neurons in the PVN. Pre‐administration of an anti‐ nucleobindin‐2/nesfatin‐1 antibody in the PVN strengthened gastric motility and decreased the discharging of the GD‐I neurons induced by electrical stimulation of the LHA. These results demonstrate that nesfatin‐1 in the PVN could serve as an inhibitory factor to inhibit gastric motility, which might be regulated by the LHA.

     

Reduced blood‐brain barrier expression of fatty acid‐binding protein 5 is associated with increased vulnerability of APP/PS1 mice to cognitive deficits from low omega‐3 fatty acid diets

Lower levels of the cognitively beneficial docosahexaenoic acid (DHA) are often observed in Alzheimer's disease (AD) brains. Brain DHA levels are regulated by the blood‐brain barrier (BBB) transport of plasma‐derived DHA, a process facilitated by fatty acid‐binding protein 5 (FABP5). This study reports a 42.1 ± 12.6% decrease in the BBB transport of ¹⁴C‐DHA in 8‐month‐old AD transgenic mice (APPswe,PSEN1?E9) relative to wild‐type mice, associated with a 34.5 ± 6.7% reduction in FABP5 expression in isolated brain capillaries of AD mice. Furthermore, short‐term spatial and recognition memory deficits were observed in AD mice on a 6‐month n‐3 fatty acid‐depleted diet, but not in AD mice on control diet. This intervention led to a dramatic reduction (41.5 ± 11.9%) of brain DHA levels in AD mice. This study demonstrates FABP5 deficiency and impaired DHA transport at the BBB are associated with increased vulnerability to cognitive deficits in mice fed an n‐3 fatty acid‐depleted diet, in line with our previous studies demonstrating a crucial role of FABP5 in BBB transport of DHA and cognitive function.

     

The role of glutamate signaling in incentive salience: second‐by‐second glutamate recordings in awake Sprague‐Dawley rats

The attribution of incentive salience to reward‐predictive stimuli has been shown to be associated with substance abuse‐like behavior such as increased drug taking. Evidence suggests that glutamate neurotransmission and sequential N‐methyl‐D‐aspartate (NMDA) activation are involved in the attribution of incentive salience. Here, we further explore the role of second‐by‐second glutamate neurotransmission in the attribution of incentive salience to reward‐predictive stimuli by measuring sign‐tracking behavior during a Pavlovian conditioned approach procedure using ceramic‐based microelectrode arrays configured for sensitive measures of extracellular glutamate in awake behaving Sprague‐Dawley rats. Specifically, we show that there is an increase in extracellular glutamate levels in the prelimbic cortex (PrL) and the nucleus accumbens core (NAcC) during sign‐tracking behavior to a food‐predictive conditioned stimulus (CS+) compared to the presentation of a non‐predictive conditioned stimulus (CS?). Furthermore, the results indicate greater increases in extracellular glutamate levels in the PrL compared to NAcC in response to the CS+, including differences in glutamate release and signal decay. Taken together, the present research suggests that there is differential glutamate signaling in the NAcC and PrL during sign‐tracking behavior to a food‐predictive CS+.

     

Coordinated activation of AMP‐activated protein kinase,extracellular signal‐regulated kinase,and autophagy regulates phorbol myristate acetate‐induced differentiation of SH‐SY5Y neuroblastoma cells

We explored the interplay between the intracellular energy sensor AMP‐activated protein kinase (AMPK), extracellular signal‐regulated kinase (ERK), and autophagy in phorbol myristate acetate (PMA)‐induced neuronal differentiation of SH‐SY5Y human neuroblastoma cells. PMA‐triggered expression of neuronal markers (dopamine transporter, microtubule‐associated protein 2, β‐tubulin) was associated with an autophagic response, measured by the conversion of microtubule‐associated protein light chain 3 (LC3)‐I to autophagosome‐bound LC3‐II, increase in autophagic flux, and expression of autophagy‐related (Atg) proteins Atg7 and beclin‐1. This coincided with the transient activation of AMPK and sustained activation of ERK. Pharmacological inhibition or RNA interference‐mediated silencing of AMPK suppressed PMA‐induced expression of neuronal markers, as well as ERK activation and autophagy. A selective pharmacological blockade of ERK prevented PMA‐induced neuronal differentiation and autophagy induction without affecting AMPK phosphorylation. Conversely, the inhibition of autophagy downstream of AMPK/ERK, either by pharmacological agents or LC3 knockdown, promoted the expression of neuronal markers, thus indicating a role of autophagy in the suppression of PMA‐induced differentiation of SH‐SY5Y cells. Therefore, PMA‐induced neuronal differentiation of SH‐SY5Y cells depends on a complex interplay between AMPK, ERK, and autophagy, in which the stimulatory effects of AMPK/ERK signaling are counteracted by the coinciding autophagic response.

     

The non‐peptidic δ‐opioid receptor agonist Tan‐67 mediates neuroprotection post‐ischemically and is associated with altered amyloid precursor protein expression,maturation and processing in mice

Tan‐67 is a selective non‐peptidic δ‐opioid receptor (DOR ) agonist that confers neuroprotection against cerebral ischemia/reperfusion (I/R)‐caused neuronal injury in pre‐treated animals. In this study, we examined whether post‐ischemic administration of Tan‐67 in stroke mice is also neuroprotective and whether the treatment affects expression, maturation and processing of the amyloid precursor protein (APP ). A focal cerebral I/R model in mice was induced by middle cerebral artery occlusion for 1 h and Tan‐67 (1.5, 3 or 4.5 mg/kg) was administered via the tail vein at 1 h after reperfusion. Alternatively, naltrindole, a selective DOR antagonist (5 mg/kg), was administered 1 h before Tan‐67 treatment. Our results showed that post‐ischemic administration of Tan‐67 (3 mg/kg or 4.5 mg/kg) was neuroprotective as shown by decreased infarct volume and neuronal loss following I/R. Importantly, Tan‐67 improved animal survival and neurobehavioral outcomes. Conversely, naltrindole abolished Tan‐67 neuroprotection in infarct volume. Tan‐67 treatment also increased APP expression, maturation and processing in the ipsilateral penumbral area at 6 h but decreased APP expression and maturation in the same brain area at 24 h after I/R. Tan‐67‐induced increase in APP expression was also seen in the ischemic cortex at 24 h following I/R. Moreover, Tan‐67 attenuated BACE ‐1 expression, β‐secretase activity and the BACE cleavage of APP in the ischemic cortex at 24 h after I/R, which was abolished by naltrindole. Our data suggest that Tan‐67 is a promising DOR ‐dependent therapeutic agent for treating I/R‐caused disorder and that Tan‐67‐mediated neuroprotection may be mediated via modulating APP expression, maturation and processing, despite an uncertain causative relationship between the altered APP and the outcomes observed.

     

Astrocytes with previous chronic exposure to amyloid β‐peptide fragment 1–40 suppress excitatory synaptic transmission

Synaptic dysfunction and neuronal death are responsible for cognitive and behavioral deficits in Alzheimer's disease (AD). It is well known that such neurological abnormalities are preceded by long‐term exposure of amyloid β‐peptide (Aβ) and/or hyperphosphorylated tau prior. In addition to the neurological deficit, astrocytes as a major glial cell type in the brain, significantly participate in the neuropathogenic mechanisms underlying synaptic modulation. Although astrocytes play a significant key role in modulating synaptic transmission, little is known on whether astrocyte dysfunction caused by such long‐term Aβ exposure affects synapse formation and function. Here, we show that synapse formation and synaptic transmission are attenuated in hippocampal‐naïve neurons co‐cultured with astrocytes that have previously experienced chronic Aβ_1‐40 exposure. In this abnormal astrocytic condition, hippocampal neurons exhibit decrements of evoked excitatory post‐synaptic currents (EPSCs) and miniature EPSC frequency. Furthermore, size of readily releasable synaptic pools and number of excitatory synapses were also significantly decreased. Contrary to these negative effects, release probability at individual synapses was significantly increased in the same astrocytic condition. Taken together, our data indicate that lower synaptic transmission caused by astrocytes previously, and chronically, exposed to Aβ1–40 is attributable to a small number of synapses with higher release probability.

     

Interferon‐γ induces leucine‐rich repeat kinase LRRK2 via extracellular signal‐regulated kinase ERK5 in macrophages

The gene encoding leucine‐rich repeat kinase 2 (LRRK2) comprises a major risk factor for Parkinson's disease. Recently, it has emerged that LRRK2 plays important roles in the immune system. LRRK2 is induced by interferon‐γ (IFN‐γ) in monocytes, but the signaling pathway is not known. Here, we show that IFN‐γ‐mediated induction of LRRK2 was suppressed by pharmacological inhibition and RNA interference of the extracellular signal‐regulated kinase 5 (ERK5). This was confirmed by LRRK2 immunostaining, which also revealed that the morphological responses to IFN‐γ were suppressed by ERK5 inhibitor treatment. Both human acute monocytic leukemia THP‐1 cells and human peripheral blood monocytes stimulated the ERK5‐LRRK2 pathway after differentiation into macrophages. Thus, LRRK2 is induced via a novel, ERK5‐dependent IFN‐γ signal transduction pathway, pointing to new functions of ERK5 and LRRK2 in human macrophages.

     

S‐allyl cysteine activates the Nrf2‐dependent antioxidant response and protects neurons against ischemic injury in vitro and in vivo

Stroke is a devastating clinical condition for which an effective neuroprotective treatment is currently unavailable. S‐allyl cysteine (SAC), the most abundant organosulfur compound in aged garlic extract, has been reported to possess neuroprotective effects against stroke. However, the mechanisms underlying its beneficial effects remain poorly defined. The present study tests the hypothesis that SAC attenuates ischemic neuronal injury by activating the nuclear factor erythroid‐2‐related factor 2 (Nrf2)‐dependent antioxidant response in both in vitro and in vivo models. Our findings demonstrate that SAC treatment resulted in an increase in Nrf2 protein levels and subsequent activation of antioxidant response element pathway genes in primary cultured neurons and mice. Exposure of primary neurons to SAC provided protection against oxygen and glucose deprivation‐induced oxidative insults. In wild‐type (Nrf2^+/+) mice, systemic administration of SAC attenuated middle cerebral artery occlusion‐induced ischemic damage, a protective effect not observed in Nrf2 knockout (Nrf2^?/?) mice. Taken together, these findings provide the first evidence that activation of the Nrf2 antioxidant response by SAC is strongly associated with its neuroprotective effects against experimental stroke and suggest that targeting the Nrf2 pathway may provide therapeutic benefit for the treatment of stroke.

     

Lipid peroxidation is essential for α‐synuclein‐induced cell death

Parkinson's disease is the second most common neurodegenerative disease and its pathogenesis is closely associated with oxidative stress. Deposition of aggregated α‐synuclein (α‐Syn) occurs in familial and sporadic forms of Parkinson's disease. Here, we studied the effect of oligomeric α‐Syn on one of the major markers of oxidative stress, lipid peroxidation, in primary co‐cultures of neurons and astrocytes. We found that oligomeric but not monomeric α‐Syn significantly increases the rate of production of reactive oxygen species, subsequently inducing lipid peroxidation in both neurons and astrocytes. Pre‐incubation of cells with isotope‐reinforced polyunsaturated fatty acids (D‐PUFAs) completely prevented the effect of oligomeric α‐Syn on lipid peroxidation. Inhibition of lipid peroxidation with D‐PUFAs further protected cells from cell death induced by oligomeric α‐Syn. Thus, lipid peroxidation induced by misfolding of α‐Syn may play an important role in the cellular mechanism of neuronal cell loss in Parkinson's disease.

     

Differential toxicity of TAR DNA‐binding protein 43 isoforms depends on their submitochondrial localization in neuronal cells

TAR DNA ‐binding protein 43 (TDP ‐43) is an RNA ‐binding protein and a major component of protein aggregates found in amyotrophic lateral sclerosis and several other neurodegenerative diseases. TDP ‐43 exists as a full‐length protein and as two shorter forms of 25 and 35 kD a. Full‐length mutant TDP ‐43s found in amyotrophic lateral sclerosis patients re‐localize from the nucleus to the cytoplasm and in part to mitochondria, where they exert a toxic role associated with neurodegeneration. However, induction of mitochondrial damage by TDP ‐43 fragments is yet to be clarified. In this work, we show that the mitochondrial 35 kD a truncated form of TDP ‐43 is restricted to the intermembrane space, while the full‐length forms also localize in the mitochondrial matrix in cultured neuronal NSC ‐34 cells. Interestingly, the full‐length forms clearly affect mitochondrial metabolism and morphology, possibly via their ability to inhibit the expression of Complex I subunits encoded by the mitochondrial‐transcribed mRNA s, while the 35 kD a form does not. In the light of the known differential contribution of the full‐length and short isoforms to generate toxic aggregates, we propose that the presence of full‐length TDP ‐43s in the matrix is a primary cause of mitochondrial damage. This in turn may cause oxidative stress inducing toxic oligomers formation, in which short TDP ‐43 forms play a major role.

     

Predominant role of plasma membrane monoamine transporters in monoamine transport in 1321N1, a human astrocytoma‐derived cell line

Monoamine neurotransmitters should be immediately removed from the synaptic cleft to avoid excessive neuronal activity. Recent studies have shown that astrocytes and neurons are involved in monoamine removal. However, the mechanism of monoamine transport by astrocytes is not entirely clear. We aimed to elucidate the transporters responsible for monoamine transport in 1321N1, a human astrocytoma‐derived cell line. First, we confirmed that 1321N1 cells transported dopamine, serotonin, norepinephrine, and histamine in a time‐ and dose‐dependent manner. Kinetics analysis suggested the involvement of low‐affinity monoamine transporters, such as organic cation transporter (OCT) 2 and 3 and plasma membrane monoamine transporter (PMAT). Monoamine transport in 1321N1 cells was not Na⁺/Cl^? dependent but was inhibited by decynium‐22, an inhibitor of low‐affinity monoamine transporters, which supported the importance of low‐affinity transporters. RT‐PCR assays revealed that 1321N1 cells expressed OCT3 and PMAT but no other neurotransmitter transporters. Another human astrocytoma‐derived cell line, U251MG, and primary human astrocytes also exhibited the same gene expression pattern. Gene‐knockdown assays revealed that 1321N1 and primary human astrocytes could transport monoamines predominantly through PMAT and partly through OCT3. These results might indicate that PMAT and OCT3 in human astrocytes are involved in monoamine clearance.

     

Dense‐core vesicle biogenesis and exocytosis in neurons lacking chromogranins A and B

Chromogranin A and B (Cgs) are considered to be master regulators of cargo sorting for the regulated secretory pathway (RSP ) and dense‐core vesicle (DCV ) biogenesis. To test this, we analyzed the release of neuropeptide Y (NPY )‐pH luorin, a live RSP reporter, and the distribution, number, and appearance of DCV s, in mouse hippocampal neurons lacking expression of CHGA and CHGB genes. qRT ‐PCR analysis showed that expression of other granin family members was not significantly altered in CgA/B^?/? neurons. As synaptic maturation of developing neurons depends on secretion of trophic factors in the RSP , we first analyzed neuronal development in standardized neuronal cultures. Surprisingly, dendritic and axonal length, arborization, synapse density, and synaptic vesicle accumulation in synapses were all normal in CgA/B^?/? neurons. Moreover, the number of DCV s outside the soma, stained with endogenous marker Secretogranin II , the number of NPY ‐pH luorin puncta, and the total amount of reporter in secretory compartments, as indicated by pH ‐sensitive NPY ‐pH luorin fluorescence, were all normal in CgA/B^?/? neurons. Electron microscopy revealed that synapses contained a normal number of DCV s, with a normal diameter, in CgA/B^?/? neurons. In contrast, CgA/B^?/? chromaffin cells contained fewer and smaller secretory vesicles with a smaller core size, as previously reported. Finally, live‐cell imaging at single vesicle resolution revealed a normal number of fusion events upon bursts of action potentials in CgA/B^?/? neurons. These events had normal kinetics and onset relative to the start of stimulation. Taken together, these data indicate that the two chromogranins are dispensable for cargo sorting in the RSP and DCV biogenesis in mouse hippocampal neurons.

     

Triheptanoin protects against status epilepticus‐induced hippocampal mitochondrial dysfunctions,oxidative stress and neuronal degeneration

Triheptanoin, the triglyceride of heptanoate, is anaplerotic (refills deficient tricarboxylic acid cycle intermediates) via the propionyl‐CoA carboxylase pathway. It has been shown to be neuroprotective and anticonvulsant in several models of neurological disorders. Here, we investigated the effects of triheptanoin against changes of hippocampal mitochondrial functions, oxidative stress and cell death induced by pilocarpine‐induced status epilepticus (SE ) in mice. Ten days of triheptanoin pre‐treatment did not protect against SE , but it preserved hippocampal mitochondrial functions including state 2, state 3 ADP , state 3 uncoupled respiration, respiration linked to ATP synthesis along with the activities of pyruvate dehydrogenase complex and oxoglutarate dehydrogenase complex 24 h post‐SE . Triheptanoin prevented the SE ‐induced reductions of hippocampal mitochondrial superoxide dismutase activity and plasma antioxidant status as well as lipid peroxidation. It also reduced neuronal degeneration in hippocampal CA 1 and CA 3 regions 3 days after SE . In addition, heptanoate significantly reduced hydrogen peroxide‐induced cell death in cultured neurons. In situ hybridization localized the enzymes of the propionyl‐CoA carboxylase pathway, specifically Pcc α, Pcc β and methylmalonyl‐CoA mutase to adult mouse hippocampal pyramidal neurons and dentate granule cells, indicating that anaplerosis may occur in neurons. In conclusion, triheptanoin appears to have anaplerotic and antioxidant effects which contribute to its neuroprotective properties.