Thyroid hormone receptors mediate two distinct mechanisms of long-wavelength vision.

Thyroid hormone (TH) signaling plays an important role in the regulation of long-wavelength vision in vertebrates. In the retina, thyroid hormone receptor ß (thrb) is required for expression of long-wavelength-sensitive opsin (lws) in red cone photoreceptors, while in retinal pigment epithelium (RPE), TH regulates expression of a cytochrome P450 enzyme, cyp27c1, that converts vitamin A1 into vitamin A2 to produce a red-shifted chromophore. To better understand how TH controls these processes, we analyzed the phenotype of zebrafish with mutations in the three known TH nuclear receptor transcription factors (thraa, thrab, and thrb). We found that no single TH nuclear receptor is required for TH-mediated induction of cyp27c1 but that deletion of all three (thraa-/-;thrab-/-;thrb-/- ) completely abrogates its induction and the resulting conversion of A1- to A2-based retinoids. In the retina, loss of thrb resulted in an absence of red cones at both larval and adult stages without disruption of the underlying cone mosaic. RNA-sequencing analysis revealed significant down-regulation of only five genes in adult thrb-/- retina, of which three (lws1, lws2, and miR-726) occur in a single syntenic cluster. In the thrb-/- retina, retinal progenitors destined to become red cones were transfated into ultraviolet (UV) cones and horizontal cells. Taken together, our findings demonstrate cooperative regulation of cyp27c1 by TH receptors and a requirement for thrb in red cone fate determination. Thus, TH signaling coordinately regulates both spectral sensitivity and sensory plasticity.

Amentoflavone Promotes Cellular Uptake and Degradation of Amyloid-Beta in Neuronal Cells.

Deposition of fibrillar forms of amyloid ß-protein (Aß) is commonly found in patients with Alzheimer's disease (AD) associated with cognitive decline. Impaired clearance of Aß species is thought to be a major cause of late-onset sporadic AD. Aß secreted into the extracellular milieu can be cleared from the brain through multiple pathways, including cellular uptake in neuronal and non-neuronal cells. Recent studies have showed that the naturally-occurring polyphenol amentoflavone (AMF) exerts anti-amyloidogenic effects. However, its effects on metabolism and cellular clearance of Aß remain to be tested. In the present study, we demonstrated that AMF significantly increased the cellular uptake of both Aß1-40 and Aß1-42, but not inverted Aß42-1 in mouse neuronal N2a cells. Though AMF promoted internalization of cytotoxic Aß1-42, it significantly reduced cell death in our assay condition. Our data further revealed that the internalized Aß is translocated to lysosomes and undergoes enzymatic degradation. The saturable kinetic of Aß uptake and our pharmacologic experiments showed the involvement of receptor-mediated endocytosis, in part, through the class A scavenger receptors as a possible mechanism of action of AMF. Taken together, our findings indicate that AMF can lower the levels of extracellular Aß by increasing their cellular uptake and clearance, suggesting the therapeutic potential of AMF for the treatment of AD.

NaV1.9 channels in muscle afferent neurons and axons.

The exercise pressor reflex (EPR) is activated by muscle contractions to increase heart rate and blood pressure during exercise. While this reflex is beneficial in healthy individuals, the reflex activity is exaggerated in patients with cardiovascular disease, which is associated with increased mortality. Group III and IV afferents mediate the EPR and have been shown to express both tetrodotoxin-sensitive (TTX-S, NaV1.6, and NaV1.7) and -resistant (TTX-R, NaV1.8, and NaV1.9) voltage-gated sodium (NaV) channels, but NaV1.9 current has not yet been demonstrated. Using a F--containing internal solution, we found a NaV current in muscle afferent neurons that activates at around -70 mV with slow activation and inactivation kinetics, as expected from NaV1.9 current. However, this current ran down with time, which resulted, at least in part, from increased steady-state inactivation since it was slowed by both holding potential hyperpolarization and a depolarized shift of the gating properties. We further show that, following NaV1.9 current rundown (internal F-), application of the NaV1.8 channel blocker A803467 inhibited significantly more TTX-R current than we had previously observed (internal Cl-), which suggests that NaV1.9 current did not rundown with that internal solution. Using immunohistochemistry, we found that the majority of group IV somata and axons were NaV1.9 positive. The majority of small diameter myelinated afferent somata (putative group III) were also NaV1.9 positive, but myelinated muscle afferent axons were rarely labeled. The presence of NaV1.9 channels in muscle afferents supports a role for these channels in activation and maintenance of the EPR. NEW & NOTEWORTHY Small diameter muscle afferents signal pain and muscle activity levels. The muscle activity signals drive the cardiovascular system to increase muscle blood flow, but these signals can become exaggerated in cardiovascular disease to exacerbate cardiac damage. The voltage-dependent sodium channel NaV1.9 plays a unique role in controlling afferent excitability. We show that NaV1.9 channels are expressed in muscle afferents, which supports these channels as a target for drug development to control hyperactivity of these neurons.

The parkinsonian mimetic, MPP+, specifically impairs mitochondrial transport in dopamine axons.

Impaired axonal transport may play a key role in Parkinson's disease. To test this notion, a microchamber system was adapted to segregate axons from cell bodies using green fluorescent protein-labeled mouse dopamine (DA) neurons. Transport was examined in axons challenged with the DA neurotoxin, 1-methyl-4-phenylpyridinium ion (MPP+). MPP+ rapidly reduced overall mitochondrial motility in DA axons; among motile mitochondria, anterograde transport was slower yet retrograde transport was increased. Transport effects were specific for DA mitochondria, which were smaller and transported more slowly than their non-DA counterparts. MPP+ did not affect synaptophysin-tagged vesicles or any other measureable moving particle. Toxin effects on DA mitochondria were not dependent upon ATP, calcium, free radical species, JNK, or caspase3/PKC pathways but were completely blocked by the thiol-anti-oxidant N-acetyl-cysteine or membrane-permeable glutathione. Since these drugs also rescued processes from degeneration, these findings emphasize the need to develop therapeutics aimed at axons as well as cell bodies to preserve "normal" circuitry and function as long as possible.

Cell stress induced by the parkinsonian mimetic, 6-hydroxydopamine, is concurrent with oxidation of the chaperone, ERp57, and aggresome formation.

Parkinson's disease (PD) involves an irreversible degeneration of the nigrostriatal pathway. As most cases of PD are sporadic, environmental risk factors may underlie neurodegeneration in dopaminergic neurons. One such factor is 6-hydroxydopamine (6-OHDA), which is widely used as a parkinsonian mimetic. Studies have shown that 6-OHDA generates reactive oxygen species and induces cell stress, the unfolded protein response, and apoptosis. Present findings show that 6-OHDA, but not hydrogen peroxide, MPP+, or rotenone, leads to the rapid formation of high-molecular-weight species of protein disulfide isomerase-associated protein 3 (ERp57) in a dose- and time-dependent fashion. Moreover, ERp57 conjugates are blocked by N-acetylcysteine and glutathione, suggesting that they represent oxidized forms of protein. Surprisingly, conjugates are complexed with DNA, because treatment with DNase reduces their appearance. Subcellular fractionation indicates that both nuclear and mitochondrial DNA are associated with the protein. Finally, toxin-treated ERp57 rapidly forms juxtanuclear aggresome-like structures in dopaminergic cells, suggesting that ERp57 plays an early adaptive response in toxin-mediated stress. Understanding the signaling mechanisms associated with parkinsonian mimetics, as well as their temporal induction, may aid in designing better interventions in models of PD.

Perihematomal mitochondrial dysfunction after intracerebral hemorrhage.

BACKGROUND AND PURPOSE: Recent measurements in intracerebral hemorrhage (ICH) patients suggest a primary reduction in brain metabolism is responsible for reduced cerebral blood flow and low oxygen extraction surrounding the hematoma. We sought to determine whether reduced mitochondrial respiratory function could account for reduced metabolic demand in ICH patients. METHODS: Brain-tissue samples from 6 patients with acute spontaneous ICH and 6 control patients undergoing brain resection for management of seizure were evaluated. Only tissue removed from the brain adjacent to the hematoma was studied. Specimens were collected in the operating room; mitochondrial studies were begun within 1-hour. Mitochondrial oxygen consumption was measured after the addition of pyruvate, malate, and ADP, followed by oligomycin and carbonylcyanide. RESULTS: The ICH patients ranged in age from 40 to 54 years; 2 were female and half black. Hemorrhages were located in the temporal lobe (3), cerebellum (2) and parietal lobe (1). The average State 3 (active) O2 consumption for mitochondria from ICH patients was approximately 40% lower than that of control patients ( CONTROLS: 129+/-39 versus ICH: 76+/-28 nmol O2/min per mg protein). With increasing time from hemorrhage to testing there was a progressive decline in State 3 respiration. Reduced State 3 respiration was evident even at 6 hours, whereas at 72 hours, there was essentially no O2 consumption. CONCLUSIONS: These data support the hypothesis that mitochondrial dysfunction and not ischemia is responsible for reduced oxygen metabolism in ICH. They point to a new direction for investigation and development of therapeutic interventions for ICH patients.

Mitochondrial uncoupling proteins in the central nervous system.

Mitochondrial uncoupling proteins (UCPs), a subfamily of the mitochondrial transporter family, are related by sequence homology to UCP1. This protein, which is located in the inner mitochondrial membrane, dissipates the proton gradient between the intermembrane space and the mitochondrial matrix to uncouple electron transport from ATP synthesis. UCP1 (thermogenin) was first discovered in brown adipose tissue and is responsible for non-shivering thermogenesis. Expression of mRNA for three other UCP isoforms, UCP2, UCP4, and BMCP1/UCP5, has been found at high levels in brain. However, the physiological function(s) of UCPs in the brain have not been determined, although it has recently been postulated that UCPs regulate free radical flux from mitochondria by physiologically modulating mitochondrial membrane potential. In the CNS, this hypothesis has been studied primarily for UCP2. UCP2 message has been shown to be up-regulated in the CNS by stress signals such as kainate administration or ischemia, and overexpression of UCP2 has been reported to be neuroprotective against oxidative stress in vivo and in vitro, although the exact mechanism has not been fully established. In this review, studies on UCPs in the nervous system will be reviewed, and the potential roles of these intriguing proteins in acute and chronic diseases of the nervous system will be discussed.

A biologically effective fullerene (C60) derivative with superoxide dismutase mimetic properties.

Superoxide, a potentially toxic by-product of cellular metabolism, may contribute to tissue injury in many types of human disease. Here we show that a tris-malonic acid derivative of the fullerene C60 molecule (C3) is capable of removing the biologically important superoxide radical with a rate constant (k(C3)) of 2 x 10(6) mol(-1) s(-1), approximately 100-fold slower than the superoxide dismutases (SOD), a family of enzymes responsible for endogenous dismutation of superoxide. This rate constant is within the range of values reported for several manganese-containing SOD mimetic compounds. The reaction between C3 and superoxide was not via stoichiometric "scavenging," as expected, but through catalytic dismutation of superoxide, indicated by lack of structural modifications to C3, regeneration of oxygen, production of hydrogen peroxide, and absence of EPR-active (paramagnetic) products, all consistent with a catalytic mechanism. A model is proposed in which electron-deficient regions on the C60 sphere work in concert with malonyl groups attached to C3 to electrostatically guide and stabilize superoxide, promoting dismutation. We also found that C3 treatment of Sod2(-/-) mice, which lack expression of mitochondrial manganese superoxide dismutase (MnSOD), increased their life span by 300%. These data, coupled with evidence that C3 localizes to mitochondria, suggest that C3 functionally replaces MnSOD, acting as a biologically effective SOD mimetic.

The Parkinsonian mimetic, 6-OHDA, impairs axonal transport in dopaminergic axons.

6-hydroxydopamine (6-OHDA) is one of the most commonly used toxins for modeling degeneration of dopaminergic (DA) neurons in Parkinson's disease. 6-OHDA also causes axonal degeneration, a process that appears to precede the death of DA neurons. To understand the processes involved in 6-OHDA-mediated axonal degeneration, a microdevice designed to isolate axons fluidically from cell bodies was used in conjunction with green fluorescent protein (GFP)-labeled DA neurons. Results showed that 6-OHDA quickly induced mitochondrial transport dysfunction in both DA and non-DA axons. This appeared to be a general effect on transport function since 6-OHDA also disrupted transport of synaptophysin-tagged vesicles. The effects of 6-OHDA on mitochondrial transport were blocked by the addition of the SOD1-mimetic, Mn(III)tetrakis(4-benzoic acid)porphyrin chloride (MnTBAP), as well as the anti-oxidant N-acetyl-cysteine (NAC) suggesting that free radical species played a role in this process. Temporally, microtubule disruption and autophagy occurred after transport dysfunction yet before DA cell death following 6-OHDA treatment. The results from the study suggest that ROS-mediated transport dysfunction occurs early and plays a significant role in inducing axonal degeneration in response to 6-OHDA treatment.

Polyphenol amentoflavone affords neuroprotection against neonatal hypoxic-ischemic brain damage via multiple mechanisms.

Flavonoids are naturally occurring polyphenolic compounds that have many biological properties, including antioxidative, anti-inflammatory and neuroprotective effects. Here, we report that amentoflavone significantly reduced cell death induced by staurosporine, etoposide and sodium nitroprusside in neuroblastoma SH-SY5Y cells. In post-natal day 7 rats, hypoxic-ischemic (H-I) brain damage induced by unilateral carotid ligation and hypoxia resulted in distinct features of neuronal cell death including apoptosis and necrosis. In this model, a systemic administration of amentoflavone (30 mg/kg) markedly reduced the H-I-induced brain tissue loss with a wide therapeutic time window up to 6 h after the onset of hypoxia. Amentoflavone blocked the activation of caspase 3, characteristic of apoptosis, and the proteolytic cleavage of its substrates following H-I injury. Amentoflavone also reduced the excitotoxic/necrotic cell death after H-I injury in vivo and after oxygen/glucose deprivation in mouse mixed cultures in vitro. Treatment of mouse microglial cells with amentoflavone resulted in a significant decrease in the lipopolysaccharide-induced production of nitric oxide and induction of inducible nitric oxide synthase and cyclo-oxygenase-2. Furthermore, amentoflavone decreased the inflammatory activation of microglia after H-I injury when assessed by the microglial-specific marker OX-42. These data demonstrate for the first time that amentoflavone strongly protects the neonatal brain from H-I injury by blocking multiple cellular events leading to brain damage.

Astrocyte mitochondria in in vitro models of ischemia.

There is growing evidence that preservation of mitochondrial respiratory function during cerebral ischemia-reperfusion predicts the ultimate extent of tissue injury. Because neurons are selectively vulnerable to ischemic injury, many studies have focused on neuronal mitochondrial dysfunction in ischemia. However, positron emission tomography (PET) studies in animals and humans suggest that non-neuronal cells such as astrocytes may also experience mitochondrial metabolic compromise that contributes to ischemic necrosis. Astrocytes carry out a number of functions that are critical to normal nervous system function, including uptake of neurotransmitters, regulation of pH and ion concentrations, and metabolic support of neurons. Mitochondria are important for many of these actions. We have used a cell culture model of stroke, oxygen-glucose deprivation (OGD), to study the response of astrocyte mitochondria to ischemia, and to evaluate how changes in astrocyte mitochondrial function might affect neuronal survival and recovery after ischemia.

Cofactors of mitochondrial enzymes attenuate copper-induced death in vitro and in vivo.

Copper toxicity contributes to neuronal death in Wilson's disease and has been speculatively linked to the pathogenesis of Alzheimer's and prion diseases. We examined copper-induced neuronal death with the goal of developing neuroprotective strategies. Copper catalyzed an increase in hydroxyl radical generation in solution, and the addition of 20 microM copper for 22 hours to murine neocortical cell cultures induced a decrease in ATP levels and neuronal death without glial death. This selective neuronal death was associated with activation of caspase-3 and was reduced by free radical scavengers and Z-Val-Ala-Asp fluoromethylketone, consistent with free radical-mediated injury leading to apoptosis. Pyruvate dehydrogenase is especially vulnerable to inhibition by oxygen free radicals, and the upstream metabolites, pyruvate, phosphoenolpyruvate, and 2-phosphoglycerate were elevated in cortical cells after toxic exposure to copper. One approach to protecting pyruvate dehydrogenase from oxidative attack might be to enhance binding to cofactors. Addition of thiamine, dihydrolipoic acid, or pyruvate reduced copper-induced neuronal death. To test efficacy in vivo, we added 1% thiamine to the drinking water of Long Evans Cinnamon rats, an animal model of Wilson's disease. This thiamine therapy markedly extended life span from 6.0 +/- 1.6 months to greater than 16 months.

The estrogen receptor is not essential for all estrogen neuroprotection: new evidence from a new analog.

We synthesized an estrogen analog, ZYC-5, lacking activity at the classical estrogen receptor and examined its neuroprotective potential against necrosis induced by N-methyl-d-aspartate (NMDA) and apoptosis/necrosis induced by the NMDA receptor antagonist (+)-3-(2-carboxypiperazine-4-yl)-propyl-1-phosphonic acid (CPP). ZYC-5 protected cortical neurons in a dose-dependent manner, and the neuroprotection was more robust than with 17beta-estradiol. The effect of ZYC-5 was not mediated by the classical estrogen receptor, because it was unaffected by the antagonists 4-hydroxytamoxifen and ICI 182,780. The ZYC-5 protection against excitotoxicity was not directly mediated through the NMDA receptor, because there was no effect of ZYC-5 on NMDA current or the intracellular calcium increase induced by NMDA. Results obtained with the free-radical-sensitive dye, dihydroethidium, suggested that the neuroprotection of ZYC-5 was partly related to its radical scavenging properties. Although some of estrogen's neuroprotective effects may depend upon the estrogen receptor, our results suggest the possibility of neuroprotection without hormonal side effects.