Heat shock protein responses to aging and proteotoxicity in the olfactory bulb

The olfactory bulb is one of the most vulnerable brain regions in age‐related proteinopathies. Proteinopathic stress is mitigated by the heat shock protein (Hsp) family of chaperones. Here, we describe age‐related decreases in Hsc70 in the olfactory bulb of the female rat and higher levels of Hsp70 and Hsp25 in middle and old age than at 2–4 months. To model proteotoxic and oxidative stress in the olfactory bulb, primary olfactory bulb cultures were treated with the proteasome inhibitors lactacystin and MG132 or the pro‐oxidant paraquat. Toxin‐induced increases were observed in Hsp70, Hsp25, and Hsp32. To determine the functional consequences of the increase in Hsp70, we attenuated Hsp70 activity with two mechanistically distinct inhibitors. The Hsp70 inhibitors greatly potentiated the toxicity of sublethal lactacystin or MG132 but not of paraquat. Although ubiquitinated protein levels were unchanged with aging in vivo or with sublethal MG132 in vitro, there was a large, synergistic increase in ubiquitinated proteins when proteasome and Hsp70 functions were simultaneously inhibited. Our study suggests that olfactory bulb cells rely heavily on Hsp70 chaperones to maintain homeostasis during mild proteotoxic, but not oxidative insults, and that Hsp70 prevents the accrual of ubiquitinated proteins in these cells.

     

Expression of astrocytic genes coding for proteins implicated in neural excitation and brain edema is altered after acute liver failure

In vitro and in vivo studies have suggested that reduced astrocytic uptake of neuronally released glutamate, alterations in expression of glial fibrillary acidic protein (GFAP) and aquaporin‐4 (AQP‐4) contribute to brain edema in acute liver failure (ALF). However, there is no evidence to date to suggest that these alterations occur in patients with ALF. We analyzed the mRNA expression of excitatory amino acid transporters (EAAT‐1, EAAT‐2), GFAP, and AQP‐4 in the cerebral cortex obtained at autopsy from eight patients with ALF and from seven patients with no evidence of hepatic or neurological disorders by real‐time PCR, and protein expression was assessed using immunoblotting and immunohistochemistry. We demonstrated a significant decrease in GFAP mRNA and protein levels in ALF patients compared to controls. While the loss of EAAT‐2 protein in ALF samples was post‐translational in nature, EAAT‐1 protein remained within normal limits. Immunohistochemistry confirmed that, in all cases, the losses of EAAT‐2 and GFAP were uniquely astrocytic in their localization. AQP‐4 mRNA expression was significantly increased and its immunohistochemistry demonstrated increased AQP‐4 immunoreactivity in the glial end‐feet process surrounding the microvessels. These findings provide evidence of selective alterations in the expression of genes coding for key astrocytic proteins implicated in central nervous system (CNS) excitability and brain edema in human ALF.

     

Inhibition of microglial activation contributes to propofol‐induced protection against post‐cardiac arrest brain injury in rats

It has been suggested that propofol can modulate microglial activity and hence may have potential roles against neuroinflammation following brain ischemic insult. However, whether and how propofol can inhibit post‐cardiac arrest brain injury via inhibition of microglia activation remains unclear. A rat model of asphyxia cardiac arrest (CA) was created followed by cardiopulmonary resuscitation. CA induced marked microglial activation in the hippocampal CA1 region, revealed by increased OX42 and P2 class of purinoceptor 7 (P2X7R) expression, as well as p38 MAPK phosphorylation. Morris water maze showed that learning and memory deficits following CA could be inhibited or alleviated by pre‐treatment with the microglial inhibitor minocycline or propofol. Microglial activation was significantly suppressed likely via the P2X7R/p‐p38 pathway by propofol. Moreover, hippocampal neuronal injuries after CA were remarkably attenuated by propofol. In vitro experiment showed that propofol pre‐treatment inhibited ATP‐induced microglial activation and release of tumor necrosis factor‐α and interleukin‐1β. In addition, propofol protected neurons from injury when co‐culturing with ATP‐treated microglia. Our data suggest that propofol pre‐treatment inhibits CA‐induced microglial activation and neuronal injury in the hippocampus and ultimately improves cognitive function.

     

Ionizing radiation causes increased tau phosphorylation in primary neurons

Radiotherapy is the major treatment modality for primary and metastatic brain tumors which involves the exposure of brain to ionizing radiation. Ionizing radiation can induce various detrimental pathophysiological effects in the adult brain, and Alzheimer's disease and related neurodegenerative disorders are considered to be late effects of radiation. In this study, we investigated whether ionizing radiation causes changes in tau phosphorylation in cultured primary neurons similar to that in Alzheimer's disease. We demonstrated that exposure to 0.5 or 2 Gy γ rays causes increased phosphorylation of tau protein at several phosphorylation sites in a time‐ and dose‐dependent manner. Consistently, we also found ionizing radiation causes increased activation of GSK3β, c‐Jun N‐terminal kinase and extracellular signal‐regulated kinase before radiation‐induced increase in tau phosphorylation. Specific inhibitors of these kinases almost fully blocked radiation‐induced tau phosphorylation. Our studies further revealed that oxidative stress plays an important role in ionizing radiation‐induced tau phosphorylation, likely through the activation of c‐Jun N‐terminal kinase and extracellular signal‐regulated kinase, but not GSK3β. Overall, our studies suggest that ionizing radiation may cause increased risk for development of Alzheimer's disease by promoting abnormal tau phosphorylation.

     

Enteric GFAP expression and phosphorylation in Parkinson's disease

Enteric glial cells (EGCs) are in many respects similar to astrocytes of the central nervous system and express similar proteins including glial fibrillary acidic protein (GFAP). Changes in GFAP expression and/or phosphorylation have been reported during brain damage or central nervous system degeneration. As in Parkinson's disease (PD) the enteric neurons accumulate α‐synuclein, and thus are showing PD‐specific pathological features, we undertook the present survey to study whether the enteric glia in PD become reactive by assessing the expression and phosphorylation levels of GFAP in colonic biopsies. Twenty‐four PD, six progressive supranuclear palsy (PSP), six multiple system atrophy (MSA) patients, and 21 age‐matched healthy controls were included. The expression levels and the phosphorylation state of GFAP were analyzed in colonic biopsies by western blot. Additional experiments were performed using real‐time PCR for a more precise analysis of the GFAP isoforms expressed by EGCs. We showed that GFAPκ was the main isoform expressed in EGCs. As compared to control subjects, patients with PD, but not PSP and MSA, had significant higher GFAP expression levels in their colonic biopsies. The phosphorylation level of GFAP at serine 13 was significantly lower in PD patients compared to control subjects. By contrast, no change in GFAP phosphorylation was observed between PSP, MSA and controls. Our findings provide evidence that enteric glial reaction occurs in PD and further reinforce the role of the enteric nervous system in the initiation and/or the progression of the disease.

     

Neuroprotective effect of exercise in rat hippocampal slices submitted to in vitro ischemia is promoted by decrease of glutamate release and pro‐apoptotic markers

The role of physical exercise as a neuroprotective agent against ischemic injury has been extensively discussed. Nevertheless, the mechanisms underlying the effects of physical exercise on cerebral ischemia remain poorly understood. Here, we investigate the hypothesis that physical exercise increases ischemic tolerance by decreasing the induction of cellular apoptosis and glutamate release. Rats (n = 50) were submitted to a swimming exercise protocol for 8 weeks. Hippocampal slices were then submitted to oxygen and glucose deprivation. Cellular viability, pro‐apoptotic markers (Caspase 8, Caspase 9, Caspase 3, and apoptosis‐inducing factor), and glutamate release were analyzed. The percentage of cell death, the amount of glutamate release, and the expression of the apoptotic markers were all decreased in the exercise group when compared to the sedentary group after oxygen and glucose deprivation. Our results suggest that physical exercise protects hippocampal slices from the effects of oxygen and glucose deprivation, probably by a mechanism involving both the decrease of glutamatergic excitotoxicity and apoptosis induction.

     

A novel caffeoyl triterpene attenuates cerebral ischemic injury with potent anti‐inflammatory and hypothermic effects

Despite the intense efforts in searching for stroke therapies, an urgent need still exists to explore novel neuroprotective agents for ischemic stroke that have high efficacy and wide therapeutic time‐window. Here, we provide the first demonstration that 28‐O‐caffeoyl betulin (B‐CA), a novel derivative of naturally occurring caffeoyl triterpene, could significantly alleviate brain infarction and neurological deficit when given as late as 6 h after transient middle cerebral artery occlusion in the rat. Moreover, post‐ischemia B‐CA administration exhibited long‐term (14 days post stroke) protective effects on both brain infarction and functional (i.e., motor and sensory) deficits. Protective B‐CA effects correlated with decreased inflammatory responses as indicated by inhibition of microglia and astrocyte activation [stained with ionized calcium‐binding adapter molecule 1 (Iba‐1) and glial fibrillary acidic protein (GFAP) antibody, respectively], as well as suppression of tumor necrosis factor‐α, interleukin‐1β, and cyclooxygenase‐2 overproduction in the ipsilateral cortex of ischemic rat. B‐CA administration caused significant hypothermia in the focal cerebral ischemic rat, which may contribute to its ameliorative effects on brain damage and inflammation. In view of its potency in wide therapeutic time‐window, robust anti‐inflammatory and hypothermic effects, this novel caffeoyl triterpene derivative may lead toward the development of effective therapeutic strategies for the treatment of ischemic stroke.

     

HIF prolyl hydroxylase inhibition prior to transient focal cerebral ischaemia is neuroprotective in mice

This study investigated the effects of 2‐(1‐chloro‐4‐hydroxyisoquinoline‐3‐carboxamido) acetic acid (IOX3), a selective small molecule inhibitor of hypoxia‐inducible factor (HIF) prolyl hydroxylases, on mouse brains subject to transient focal cerebral ischaemia. Male, 8‐ to 12‐week‐old C57/B6 mice were subjected to 45 min of middle cerebral artery occlusion (MCAO) either immediately or 24 h after receiving IOX3. Mice receiving IOX3 at 20 mg/kg 24 h prior to the MCAO had better neuroscores and smaller blood–brain barrier (BBB) disruption and infarct volumes than mice receiving the vehicle, whereas those having IOX3 at 60 mg/kg showed no significant changes. IOX3 treatment immediately before MCAO was not neuroprotective. IOX3 up‐regulated HIF‐1α, and increased EPO expression in mouse brains. In an in vitro BBB model (RBE4 cell line), IOX3 up‐regulated HIF‐1α and delocalized ZO‐1. Pre‐treating IOX3 on RBE4 cells 24 h before oxygen–glucose deprivation had a protective effect on endothelial barrier preservation with ZO‐1 being better localized, while immediate IOX3 treatment did not. Our study suggests that HIF stabilization with IOX3 before cerebral ischaemia is neuroprotective partially because of BBB protection, while immediate application could be detrimental. These results provide information for studies aimed at the therapeutic activation of HIF pathway for neurovascular protection from cerebral ischaemia.

     

A conserved sequence in calmodulin regulated spectrin‐associated protein 1 links its interaction with spectrin and calmodulin to neurite outgrowth

Calmodulin regulated spectrin‐associated protein 1 (CAMSAP1) is a vertebrate microtubule‐binding protein, and a representative of a family of cytoskeletal proteins that arose with animals. We reported previously that the central region of the protein, which contains no recognized functional domain, inhibited neurite outgrowth when over‐expressed in PC12 cells [Baines et al., Mol. Biol. Evol. 26 (2009), p. 2005]. The CKK domain (DUF1781) binds microtubules and defines the CAMSAP/ssp4 family of animal proteins (Baines et al. 2009). In the central region, three short well‐conserved regions are characteristic of CAMSAP‐family members. One of these, CAMSAP‐conserved region 1 (CC1), bound to both βIIΣ1‐spectrin and Ca²⁺/calmodulin in vitro. The binding of Ca²⁺/calmodulin inhibited spectrin binding. Transient expression of CC1 in PC12 cells inhibited neurite outgrowth. siRNA knockdown of CAMSAP1 inhibited neurite outgrowth in PC12 cells or primary cerebellar granule cells: this could be rescued in PC12 cells by wild‐type CAMSAP1‐enhanced green fluorescent protein, but not by a CC1 mutant. We conclude that CC1 represents a functional region of CAMSAP1, which links spectrin‐binding to neurite outgrowth.

     

Critical role of peripheral drug actions in experience‐dependent changes in nucleus accumbens glutamate release induced by intravenous cocaine

Recent studies reveal that cocaine experience results in persistent neuroadaptive changes within glutamate (Glu) synapses in brain areas associated with drug reward. However, it remains unclear whether cocaine affects Glu release in drug‐naive animals and how it is altered by drug experience. Using high‐speed amperometry with enzyme‐based and enzyme‐free biosensors in freely moving rats, we show that an initial intravenous cocaine injection at a low self‐administering dose (1 mg/kg) induces rapid, small and transient Glu release in the nucleus accumbens shell (NAc), which with subsequent injections rapidly becomes a much stronger, two‐component increase. Using cocaine‐methiodide, cocaine's analog that does not cross the blood–brain barrier, we confirm that the initial cocaine‐induced Glu release in the NAc has a peripheral neural origin. Unlike cocaine, Glu responses induced by cocaine‐methiodide rapidly habituate following repeated exposure. However, after cocaine experience this drug induces cocaine‐like Glu responses. Hence, the interoceptive actions of cocaine, which essentially precede its direct actions in the brain, play a critical role in experience‐dependent alterations in Glu release, cocaine‐induced neural sensitization and may contribute to cocaine addiction.

     

Evidence for an angiotensin‐(1‐7) neuropeptidase expressed in the brain medulla and CSF of sheep

Angiotensin‐(1‐7) [Ang‐(1‐7)] is an alternative product of the brain renin‐angiotensin system that exhibits central actions to lower blood pressure and improve baroreflex sensitivity. We previously identified a peptidase that metabolizes Ang‐(1‐7) to the inactive metabolite product Ang‐(1‐4) in CSF of adult sheep. This study purified the peptidase 1445‐fold from sheep brain medulla and characterized this activity. The peptidase was sensitive to the chelating agents o‐phenanthroline and EDTA, as well as the mercury compound p‐chloromercuribenzoic acid (PCMB). Selective inhibitors to angiotensin‐converting enzyme, neprilysin, neurolysin, and thimet oligopeptidase did not attenuate activity; however, the metallopeptidase agent JMV‐390 was a potent inhibitor of Ang‐(1‐7) hydrolysis (Ki = 0.8 nM). Kinetic studies using ¹²⁵I‐labeled Ang‐(1‐7), Ang II, and Ang I revealed comparable apparent K_m values (2.6, 2.8, and 4.3 μM, respectively), but a higher apparent V_max for Ang‐(1‐7) (72 vs. 30 and 6 nmol/min/mg, respectively; p < 0.01). HPLC analysis of the activity confirmed the processing of unlabeled Ang‐(1‐7) to Ang‐(1‐4) by the peptidase, but revealed < 5% hydrolysis of Ang II or Ang I, and no hydrolysis of neurotensin, bradykinin or apelin‐13. The unique characteristics of the purified neuropeptidase may portend a novel pathway to influence actions of Ang‐(1‐7) within the brain.

     

Maternal obesity impairs brain glucose metabolism and neural response to hyperglycemia in male rat offspring

Hypothalamic appetite regulators neuropeptide Y (NPY) and pro‐opiomelanocortin (POMC) are modulated by glucose. This study investigated how maternal obesity disturbs glucose regulation of NPY and POMC, and whether this deregulation is linked to abnormal hypothalamic glucose uptake‐lactate conversion. As post‐natal high‐fat diet (HFD) can exaggerate the effects of maternal obesity, its additional impact was also investigated. Female Sprague Dawley rats were fed a HFD (20 kJ/g) to model maternal obesity. At weaning, male pups were fed chow or HFD. At 9 weeks, in vivo hypothalamic NPY and POMC mRNA responses to acute hyperglycemia were measured; while hypothalami were glucose challenged in vitro to assess glucose uptake‐lactate release and related gene expression. Maternal obesity dampened in vivo hypothalamic NPY response to acute hyperglycemia, and lowered in vitro hypothalamic glucose uptake and lactate release. When challenged with 20 mM glucose, hypothalamic glucose transporter 1, monocarboxylate transporters, lactate dehydrogenase‐b, NPY and POMC mRNA expression were down‐regulated in offspring exposed to maternal obesity. Post‐natal HFD consumption reduced in vitro lactate release and monocarboxylate transporter 2 mRNA, but increased POMC mRNA levels when challenged with 20 mM glucose. Overall, maternal obesity produced stronger effects than post‐natal HFD consumption to impair hypothalamic glucose metabolism. However, they both disturbed NPY response to hyperglycemia, potentially leading to hyperphagia.

     

Expression of the synaptic exocytosis‐regulating molecule complexin 2 in taste buds and its participation in peripheral taste transduction

Taste information from type III taste cells to gustatory neurons is thought to be transmitted via synapses. However, the molecular mechanisms underlying taste transduction through this pathway have not been fully elucidated. In this study, to identify molecules that participate in synaptic taste transduction, we investigated whether complexins (Cplxs), which play roles in regulating membrane fusion in synaptic vesicle exocytosis, were expressed in taste bud cells. Among four Cplx isoforms, strong expression of Cplx2 mRNA was detected in type III taste cells. To investigate the function of CPLX2 in taste transduction, we observed taste responses in CPLX2‐knockout mice. When assessed with electrophysiological and behavioral assays, taste responses to some sour stimuli in CPLX2‐knockout mice were significantly lower than those in wild‐type mice. These results suggested that CPLX2 participated in synaptic taste transduction from type III taste cells to gustatory neurons.

     

Identification of precursor microRNAs within distal axons of sensory neuron

A set of specific precursor microRNAs (pre‐miRNAs) are reported to localize into neuronal dendrites, where they could be processed locally to control synaptic protein synthesis and plasticity. However, it is not clear whether specific pre‐miRNAs are also transported into distal axons to autonomously regulate intra‐axonal protein synthesis. Here, we show that a subset of pre‐miRNAs, whose mature miRNAs are enriched in axonal compartment of sympathetic neurons, are present in axons of neurons both in vivo and in vitro by quantitative PCR and by in situ hybridization. Some pre‐miRNAs (let 7c‐a and pre‐miRs‐16, 23a, 25, 125b‐1, 433, and 541) showed elevated axonal levels, while others (pre‐miRs‐138‐2, 185, and 221) were decreased in axonal levels following injury. Dicer and KSRP proteins are also present in distal axons, but Drosha is found restricted to the cell body. These findings suggest that specific pre‐miRNAs are selected for localization into distal axons of sensory neurons and are presumably processed to mature miRNAs in response to extracellular stimuli. This study supports the notion that local miRNA biogenesis effectively provides another level of temporal control for local protein synthesis in axons.

     

Cortical‐layer‐specific effects of PACAP and tPA on interneuron migration during post‐natal development of the cerebellum

During early post‐natal development of the cerebellum, granule neurons (GN) execute a centripetal migration toward the internal granular layer, whereas basket and stellate cells (B/SC) migrate centrifugally to reach their final position in the molecular layer (ML). We have previously shown that pituitary adenylate cyclase‐activating polypeptide (PACAP) stimulates in vitro the expression and release of the serine protease tissue‐type plasminogen activator (tPA) from GN, but the coordinated role of PACAP and tPA during interneuron migration has not yet been investigated. Here, we show that endogenous PACAP is responsible for the transient arrest phase of GN at the level of the Purkinje cell layer (PCL) but has no effect on B/SC. tPA is devoid of direct effect on GN motility in vitro, although it is widely distributed along interneuron migratory routes in the ML, PCL, and internal granular layer. Interestingly, plasminogen activator inhibitor 1 reduces the migration speed of GN in the ML and PCL, and that of B/SC in the ML. Taken together, these results reveal for the first time that tPA facilitates the migration of both GN and fast B/SC at the level of their intersection in the ML through degradation of the extracellular matrix.