Neuromodulation and Mitochondrial Transport: Live Imaging in Hippocampal Neurons over Long Durations

To understand the relationship between mitochondrial transport and neuronal function, it is critical to observe mitochondrial behavior in live cultured neurons for extended durations^1-3. This is now possible through the use of vital dyes and fluorescent proteins with which cytoskeletal components, organelles, and other structures in living cells can be labeled and then visualized via dynamic fluorescence microscopy. For example, in embryonic chicken sympathetic neurons, mitochondrial movement was characterized using the vital dye rhodamine 123⁴. In another study, mitochondria were visualized in rat forebrain neurons by transfection of mitochondrially targeted eYFP⁵. However, imaging of primary neurons over minutes, hours, or even days presents a number of issues. Foremost among these are: 1) maintenance of culture conditions such as temperature, humidity, and pH during long imaging sessions; 2) a strong, stable fluorescent signal to assure both the quality of acquired images and accurate measurement of signal intensity during image analysis; and 3) limiting exposure times during image acquisition to minimize photobleaching and avoid phototoxicity.Here, we describe a protocol that permits the observation, visualization, and analysis of mitochondrial movement in cultured hippocampal neurons with high temporal resolution and under optimal life support conditions. We have constructed an affordable stage-top incubator that provides good temperature regulation and atmospheric gas flow, and also limits the degree of media evaporation, assuring stable pH and osmolarity. This incubator is connected, via inlet and outlet hoses, to a standard tissue culture incubator, which provides constant humidity levels and an atmosphere of 5-10% CO₂/air. This design offers a cost-effective alternative to significantly more expensive microscope incubators that don''t necessarily assure the viability of cells over many hours or even days. To visualize mitochondria, we infect cells with a lentivirus encoding a red fluorescent protein that is targeted to the mitochondrion. This assures a strong and persistent signal, which, in conjunction with the use of a stable xenon light source, allows us to limit exposure times during image acquisition and all but precludes photobleaching and phototoxicity. Two injection ports on the top of the stage-top incubator allow the acute administration of neurotransmitters and other reagents intended to modulate mitochondrial movement. In sum, lentivirus-mediated expression of an organelle-targeted red fluorescent protein and the combination of our stage-top incubator, a conventional inverted fluorescence microscope, CCD camera, and xenon light source allow us to acquire time-lapse images of mitochondrial transport in living neurons over longer durations than those possible in studies deploying conventional vital dyes and off-the-shelf life support systems.     

MicroRNA-101 Regulates Amyloid Precursor Protein Expression in Hippocampal Neurons

The amyloid precursor protein (APP) and its proteolytic product amyloid beta (Aβ) are associated with both familial and sporadic forms of Alzheimer disease (AD). Aberrant expression and function of microRNAs has been observed in AD. Here, we show that in rat hippocampal neurons cultured in vitro, the down-regulation of Argonaute-2, a key component of the RNA-induced silencing complex, produced an increase in APP levels. Using site-directed mutagenesis, a microRNA responsive element (RE) for miR-101 was identified in the 3′-untranslated region (UTR) of APP. The inhibition of endogenous miR-101 increased APP levels, whereas lentiviral-mediated miR-101 overexpression significantly reduced APP and Aβ load in hippocampal neurons. In addition, miR-101 contributed to the regulation of APP in response to the proinflammatory cytokine interleukin-1β (IL-lβ). Thus, miR-101 is a negative regulator of APP expression and affects the accumulation of Aβ, suggesting a possible role for miR-101 in neuropathological conditions.     

Scutellarin Blocks Sodium Current in Freshly Isolated Mouse Hippocampal CA1 Neurons

Scutellarin (Scu), the main bioactive component of Erilgeron breviscapus, protects the brain against ischemic damages. To explore the therapeutic mechanism of Scu, we investigated the impact of Scu on sodium current (I _Na) of freshly isolated mouse hippocampal CA1 neurons using the whole-cell patch clamp technique. Results showed that Scu inhibited I _Na in concentration- and holding potential-dependent manners. At 50 μM, Scu markedly shifted the steady state inactivation curve of I _Na towards a more negative potential, slowed down the recovery of I _Na from inactivation state, and elicited a frequency-dependent block of I _Na. The shape of the current–voltage (I–V) curve and the steady state activation curve of I _Na were unaffected by Scu treatment. These findings suggest that Scu is capable of inhibiting I _Na in neurons through predominantly affecting the inactivated state of I _Na. Inhibition of Na⁺ channels provides a novel pharmacological basis for the anti-ischemic application of Scu.     

Wnt-5a Ligand Modulates Mitochondrial Fission-Fusion in Rat Hippocampal Neurons

The Wnt signaling pathway plays an important role in developmental processes, including embryonic patterning, cell specification, and cell polarity. Wnt components participate in the development of the central nervous system, and growing evidence indicates that this pathway also regulates the function of the adult nervous system. In this study, we report that Wnt-5a, a noncanonical Wnt ligand, is a potent activator of mitochondrial dynamics and induces acute fission and fusion events in the mitochondria of rat hippocampal neurons. The effect of Wnt-5a was inhibited in the presence of sFRP, a Wnt scavenger. Similarly, the canonical Wnt-3a ligand had no effect on mitochondrial fission-fusion events, suggesting that this effect is specific for Wnt-5a alone. We also show that the Wnt-5a effects on mitochondrial dynamics occur with an increase in both intracellular and mitochondrial calcium (Ca²⁺), which was correlated with an increased phosphorylation of Drp1(Ser-616) and a decrease of Ser-637 phosphorylation, both indicators of mitochondrial dynamics. Electron microscope analysis of hippocampal tissues in the CA1 region showed an increase in the number of mitochondria present in the postsynaptic region, and this finding correlated with a change in mitochondrial morphology. We conclude that Wnt-5a/Ca²⁺ signaling regulates the mitochondrial fission-fusion process in hippocampal neurons, a feature that might help to further understand the role of Wnt-related pathologies, including neurodegenerative diseases associated with mitochondrial dysfunction, and represents a potentially important link between impaired metabolic function and degenerative disorders.     

A Comparative Quantitative Assessment of Axonal and Dendritic mRNA Transport in Maturing Hippocampal Neurons

Translation of mRNA in axons and dendrites enables a rapid supply of proteins to specific sites of localization within the neuron. Distinct mRNA-containing cargoes, including granules and mitochondrial mRNA, are transported within neuronal projections. The distributions of these cargoes appear to change during neuronal development, but details on the dynamics of mRNA transport during these transitions remain to be elucidated. For this study, we have developed imaging and image processing methods to quantify several transport parameters that can define the dynamics of RNA transport and localization. Using these methods, we characterized the transport of mitochondrial and non-mitochondrial mRNA in differentiated axons and dendrites of cultured hippocampal neurons varying in developmental maturity. Our results suggest differences in the transport profiles of mitochondrial and non-mitochondrial mRNA, and differences in transport parameters at different time points, and between axons and dendrites. Furthermore, within the non-mitochondrial mRNA pool, we observed two distinct populations that differed in their fluorescence intensity and velocity. The net axonal velocity of the brighter pool was highest at day 7 (0.002±0.001 µm/s, mean ± SEM), raising the possibility of a presynaptic requirement for mRNA during early stages of synapse formation. In contrast, the net dendritic velocity of the brighter pool increased steadily as neurons matured, with a significant difference between day 12 (0.0013±0.0006 µm/s ) and day 4 (−0.003±0.001 µm/s) suggesting a postsynaptic role for mRNAs in more mature neurons. The dim population showed similar trends, though velocities were two orders of magnitude higher than of the bright particles. This study provides a baseline for further studies on mRNA transport, and has important implications for the regulation of neuronal plasticity during neuronal development and in response to neuronal injury.     

Mitochondrial Trafficking in Neurons

Neurons, perhaps more than any other cell type, depend on mitochondrial trafficking for their survival. Recent studies have elucidated a motor/adaptor complex on the mitochondrial surface that is shared between neurons and other animal cells. In addition to kinesin and dynein, this complex contains the proteins Miro (also called RhoT1/2) and milton (also called TRAK1/2) and is responsible for much, although not necessarily all, mitochondrial movement. Elucidation of the complex has permitted inroads for understanding how this movement is regulated by a variety of intracellular signals, although many mysteries remain. Regulating mitochondrial movement can match energy demand to energy supply throughout the extraordinary architecture of these cells and can control the clearance and replenishing of mitochondria in the periphery. Because the extended axons of neurons contain uniformly polarized microtubules, they have been useful for studying mitochondrial motility in conjunction with biochemical assays in many cell types.     

The Ferroxidase Hephaestin But Not Amyloid Precursor Protein is Required for Ferroportin-Supported Iron Efflux in Primary Hippocampal Neurons

Iron efflux in mammalian cells is mediated by the ferrous iron exporter ferroportin (Fpn); Fpn plasma membrane localization and function are supported by a multicopper ferroxidase and/or the soluble amyloid precursor protein (sAPP). Fpn and APP are ubiquitously expressed in all cell types in the central nervous system including neurons. In contrast, neuronal ferroxidase(s) expression has not been well characterized. Using primary cultures of hippocampal neurons, we examined the molecular mechanism of neuronal Fe efflux in detail. Developmental increases of Fpn, APP, and the ferroxidase hephaestin (Hp) were observed in hippocampal neurons. Iron efflux in these neurons depended on the level of Fpn localized at the cell surface; as noted, Fpn stability is supported by ferroxidase activity, an enzymatic activity that is required for Fe efflux. Iron accumulation increases and iron efflux decreases in Hp knockout neurons. In contrast, suppression of endogenous APP by RNAi knockdown does not affect surface Fpn stability or Fe efflux. These data support the model that the neuronal ferroxidase Hp plays a unique role in support of Fpn-mediated Fe efflux in primary hippocampal neurons. Our data also demonstrate that Hp ferroxidase activity relies on copper bioavailability, which suggests neuronal iron homeostasis will be modulated by cellular copper status.     

Imaging of Mitochondrial and Non-Mitochondrial Responses in Cultured Rat Hippocampal Neurons Exposed to Micromolar Concentrations of TMRM

Tetramethylrhodamine methyl ester (TMRM) is a fluorescent dye used to study mitochondrial function in living cells. Previously, we reported that TMRM effectively labeled mitochondria of neurons deep within mouse brain slices. Use of micromolar concentration of dye, which was required to get sufficient staining for two-photon imaging, resulted in typical fluctuations of TMRM. With prolonged exposure, we recorded additional responses in some neurons that included slow oscillations and propagating waves of fluorescence. (Note: We use the terms “fluctuation” to refer to a change in the fluorescent state of an individual mitochondrion, “oscillation” to refer to a localized change in fluorescence in the cytosol, and “wave” to refer to a change in cytosolic fluorescence that propagated within a cell. Use of these terms does not imply any underlying periodicity.) In this report we describe similar results using cultured rat hippocampal neurons. Prolonged exposure of cultures to 2.5 µM TMRM produced a spontaneous increase in fluorescence in some neurons, but not glial cells, after 45–60 minutes that was followed by slow oscillations, waves, and eventually apoptosis. Spontaneous increases in fluorescence were insensitive to high concentrations of FCCP (100 µM) and thapsigargin (10 µM) indicating that they originated, at least in part, from regions outside of mitochondria. The oscillations did not correlate with changes in intracellular Ca²⁺, but did correlate with differences in fluorescence lifetime of the dye. Fluorescence lifetime and one-photon ratiometric imaging of TMRM suggested that the spontaneous increase and subsequent oscillations were due to movement of dye between quenched (hydrophobic) and unquenched (hydrophilic) compartments. We propose that these movements may be correlates of intracellular events involved in early stages of apoptosis.     

Perforin Promotes Amyloid Beta Internalisation in Neurons

Studies on the mechanisms of neuronal amyloid-β (Aβ) internalisation are crucial for understanding the neuropathological progression of Alzheimer’s disease (AD). We here investigated how extracellular Aβ peptides are internalised and focused on three different pathways: (i) via endocytic mechanisms, (ii) via the receptor for advanced glycation end products (RAGE) and (iii) via the pore-forming protein perforin. Both Aβ₄₀ and Aβ₄₂ were internalised in retinoic acid differentiated neuroblastoma (RA-SH-SY5Y) cells. A higher concentration was required for Aβ₄₀ (250 nM) compared with Aβ₄₂ (100 nM). The internalised Aβ₄₀ showed a dot-like pattern of distribution whereas Aβ₄₂ accumulated in larger and distinct formations. By confocal microscopy, we showed that Aβ₄₀ and Aβ₄₂ co-localised with mitochondria, endoplasmic reticulum (ER) and lysosomes. Aβ treatment of human primary cortical neurons (hPCN) confirmed our findings in RA-SH-SY5Y cells, but hPCN were less sensitive to Aβ; therefore, a 20 (Aβ₄₀) and 50 (Aβ₄₂) times higher concentration was needed for inducing uptake. The blocking of endocytosis completely inhibited the internalisation of Aβ peptides in RA-SH-SY5Y cells and hPCN, indicating that this is a major pathway by which Aβ enters the cells. In addition, the internalisation of Aβ₄₂, but not Aβ₄₀, was reduced by 55 % by blocking RAGE. Finally, for the first time we showed that pore formation in cell membranes by perforin led to Aβ internalisation in hPCN. Understanding how Aβ is internalised sheds light on the pathological role of Aβ and provides further ideas of inhibitory strategies for preventing Aβ internalisation and the spreading of neurodegeneration in AD.     

人源性神经营养素-6对体外培养海马神经元存活的促进作用

分离出一周SD乳鼠海马组织,进行离体海马细胞培养;在培养基中加入神经营养素-6成熟肽片段,通过神经元特异性烯醇化酶免疫组化染色,计数存活海马神经元数量,与对照组比较,观察神经营养素-6对神经元存活的促进作用。结果显示,神经营养素-6实验组海马神经元存活数目显著高于对照组。表明人源性神经营养素-6可以促进体外培养神经元的存活。     

海马神经元的瞬间外向钾电流

瞬间外向钾电流(IA)具有快速激活和失活等特征,是动作电位复极化早期外向钾离子电流的主要成分,广泛分布在海马神经元,树突处尤为突出.该电流通过减慢去极化速度和延缓动作电位的产生等作用,调节突触的输入和动作电位的反向传播,从而在信号整合及突触可塑性等过程中扮演重要角色.很多人类疾病,如癫痫性疾病等,和海马神经元的IA电流有关.     

Estrogens Attenuate and Corticosterone Exacerbates Excitotoxicity, Oxidative Injury, and Amyloid β-Peptide Toxicity in Hippocampal Neurons

Abstract: Steroid hormones, particularly estrogens and glucocorticoids, may play roles in the pathogenesis of neurodegenerative disorders, but their mechanisms of action are not known. We report that estrogens protect cultured hippocampal neurons against glutamate toxicity, glucose deprivation, FeSO₄ toxicity, and amyloid β-peptide (Aβ) toxicity. The toxicity of each insult was significantly attenuated in cultures pretreated for 2 h with 100 n M -10 µ M 17β-estradiol, estriol, or progesterone. In contrast, corticosterone exacerbated neuronal injury induced by glutamate, FeSO₄, and Aβ. Several other steroids, including testosterone, aldosterone, and vitamin D, had no effect on neuronal vulnerability to the different insults. The protective actions of estrogens and progesterone were not blocked by actinomycin D or cycloheximide. Lipid peroxidation induced by FeSO₄ and Aβ was significantly attenuated in neurons and isolated membranes pretreated with estrogens and progesterone, suggesting that these steroids possess antioxidant activities. Estrogens and progesterone also attenuated Aβ- and glutamate-induced elevation of intracellular free Ca²⁺ concentrations. We conclude that estrogens, progesterone, and corticosterone can directly affect neuronal vulnerability to excitotoxic, metabolic, and oxidative insults, suggesting roles for these steroids in several different neurodegenerative disorders.     

Microtubule-dependent Recruitment of Staufen-Green Fluorescent Protein into Large RNA-containing Granules and Subsequent Dendritic Transport in Living Hippocampal Neurons

Dendritic mRNA transport and local translation at individual potentiated synapses may represent an elegant way to form synaptic memory. Recently, we characterized Staufen, a double-stranded RNA-binding protein, in rat hippocampal neurons and showed its presence in large RNA-containing granules, which colocalize with microtubules in dendrites. In this paper, we transiently transfect hippocampal neurons with human Staufen-green fluorescent protein (GFP) and find fluorescent granules in the somatodendritic domain of these cells. Human Stau-GFP granules show the same cellular distribution and size and also contain RNA, as already shown for the endogenous Stau particles. In time-lapse videomicroscopy, we show the bidirectional movement of these Staufen-GFP–labeled granules from the cell body into dendrites and vice versa. The average speed of these particles was 6.4 μm/min with a maximum velocity of 24.3 μm/min. Moreover, we demonstrate that the observed assembly into granules and their subsequent dendritic movement is microtubule dependent. Taken together, we have characterized a novel, nonvesicular, microtubule-dependent transport pathway involving RNA-containing granules with Staufen as a core component. This is the first demonstration in living neurons of movement of an essential protein constituent of the mRNA transport machinery.     

Calcium Phosphate Transfection of Primary Hippocampal Neurons

Calcium phosphate precipitation is a convenient and economical method for transfection of cultured cells. With optimization, it is possible to use this method on hard-to-transfect cells like primary neurons. Here we describe our detailed protocol for calcium phosphate transfection of hippocampal neurons cocultured with astroglial cells.     

Toxicity of Dopamine to Striatal Neurons In Vitro and Potentiation of Cell Death by a Mitochondrial Inhibitor

Abstract: Intrastriatal injections of the mitochondrial toxins malonate and 3-nitropropionic acid produce selective cell death similar to that seen in transient ischemia and Huntington's disease. The extent of cell death can be attenuated by pharmacological or surgical blockade of cortical glutamatergic input. It is not known, however, if dopamine contributes to toxicity caused by inhibition of mitochondrial function. Exposure of primary striatal cultures to dopamine resulted in dose-dependent death of neurons. Addition of medium supplement containing free radical scavengers and antioxidants decreased neuronal loss. At high concentrations of the amine, cell death was predominantly apoptotic. Methyl malonate was used to inhibit activity of the mitochondrial respiratory chain. Neither methyl malonate (50 µ M ) nor dopamine (2.5 µ M ) caused significant toxicity when added individually to cultures, whereas simultaneous addition of both compounds killed 60% of neurons. Addition of antioxidants and free radical scavengers to the incubation medium prevented this cell death. Dopamine (up to 250 µ M ) did not alter the ATP/ADP ratio after a 6-h incubation. Methyl malonate, at 500 µ M , reduced the ATP/ADP ratio by ∼30% after 6 h; this decrease was not augmented by coincubation with 25 µ M dopamine. Our results suggest that dopamine causes primarily apoptotic death of striatal neurons in culture without damaging cells by an early adverse action on oxidative phosphorylation. However, when combined with minimal inhibition of mitochondrial function, dopamine neurotoxicity is markedly enhanced.