James D. Lechleiter

Chemical Calcium Indicators

Our understanding of the underlying mechanisms of Ca2+ signaling as well as our appreciation for its ubiquitous role in cellular processes has been rapidly advanced, in large part, due to the development of fluorescent Ca2+ indicators. In this chapter, we discuss some of the most common chemical Ca2+ indicators that are widely used for the investigation of intracellular Ca2+ signaling. Advantages, limitations and relevant procedures will be presented for each dye including their spectral qualities, dissociation constants, chemical forms, loading methods and equipment for optimal imaging. Chemical indicators now available allow for intracellular Ca2+ detection over a very large range (<50 nM to >50 microM). High affinity indicators can be used to quantify Ca2+ levels in the cytosol while lower affinity indicators can be optimized for measuring Ca2+ in subcellular compartments with higher concentrations. Indicators can be classified into either single wavelength or ratiometric dyes. Both classes require specific lasers, filters, and/or detection methods that are dependent upon their spectral properties and both classes have advantages and limitations. Single wavelength indicators are generally very bright and optimal for Ca2+ detection when more than one fluorophore is being imaged. Ratiometric indicators can be calibrated very precisely and they minimize the most common problems associated with chemical Ca2+ indicators including uneven dye loading, leakage, photobleaching, and changes in cell volume. Recent technical advances that permit in vivo Ca2+ measurements will also be discussed.

Germline mutations in TMEM127 confer susceptibility to pheochromocytoma

Pheochromocytomas, which are catecholamine-secreting tumors of neural crest origin, are frequently hereditary. However, the molecular basis of the majority of these tumors is unknown. We identified the transmembrane-encoding gene TMEM127 on chromosome 2q11 as a new pheochromocytoma susceptibility gene. In a cohort of 103 samples, we detected truncating germline TMEM127 mutations in approximately 30% of familial tumors and about 3% of sporadic-appearing pheochromocytomas without a known genetic cause. The wild-type allele was consistently deleted in tumor DNA, suggesting a classic mechanism of tumor suppressor gene inactivation. Pheochromocytomas with mutations in TMEM127 are transcriptionally related to tumors bearing NF1 mutations and, similarly, show hyperphosphorylation of mammalian target of rapamycin (mTOR) effector proteins. Accordingly, in vitro gain-of-function and loss-of-function analyses indicate that TMEM127 is a negative regulator of mTOR. TMEM127 dynamically associates with the endomembrane system and colocalizes with perinuclear (activated) mTOR, suggesting a subcompartmental-specific effect. Our studies identify TMEM127 as a tumor suppressor gene and validate the power of hereditary tumors to elucidate cancer pathogenesis.

PI 3-kinase regulation of dopamine uptake

The magnitude and duration of dopamine (DA) signaling is defined by the amount of vesicular release, DA receptor sensitivity, and the efficiency of DA clearance, which is largely determined by the DA transporter (DAT). DAT uptake capacity is determined by the number of functional transporters on the cell surface as well as by their turnover rate. Here we show that inhibition of phosphatidylinositol (PI) 3‐kinase with LY294002 induces internalization of the human DAT (hDAT), thereby reducing transport capacity. Acute treatment with LY294002 reduced the maximal rate of [3H]DA uptake in rat striatal synaptosomes and in human embryonic kidney (HEK) 293 cells stably expressing the hDAT (hDAT cells). In addition, LY294002 caused a significant redistribution of the hDAT from the plasma membrane to the cytosol. Conversely, insulin, which activates PI 3‐kinase, increased [3H]DA uptake and blocked the amphetamine‐induced hDAT intracellular accumulation, as did transient expression of constitutively active PI 3‐kinase. The LY294002‐induced reduction in [3H]DA uptake and hDAT cell surface expression was inhibited by expression of a dominant negative mutant of dynamin I, indicating that dynamin‐dependent trafficking can modulate transport capacity. These data implicate DAT trafficking in the hormonal regulation of dopaminergic signaling, and suggest that a state of chronic hypoinsulinemia, such as in diabetes, may alter synaptic DA signaling by reducing the available cell surface DATs.

Amphetamine-induced Dopamine Efflux A VOLTAGE-SENSITIVE AND INTRACELLULAR Na+-DEPENDENT MECHANISM

Amphetamine (AMPH) elicits its behavioral effects by acting on the dopamine (DA) transporter (DAT) to induce DA overflow into the synaptic cleft. Facilitated exchange diffusion is the classical model used to describe AMPH-induced DA efflux. This model hypothesizes that AMPH-induced DA efflux is mediated by DAT and results from the transport of AMPH into the cell followed by a counter movement of DA out to the extracellular compartment. To further characterize the action of AMPH, we used the patch clamp technique in the whole-cell configuration combined with amperometry on human embryonic kidney HEK-293 cells stably transfected with the human DAT (DAT cells). In DAT cells, AMPH-induced DAT-mediated currents were blocked by cocaine. We demonstrate that DA efflux mediated by DAT is voltage-dependent, electrogenic, and dependent on intracellular Na+ concentration in the recording electrode. Intracellular Na+ fluorescence, as measured by confocal microscopy using a Na+-sensitive dye, was enhanced by AMPH application. Furthermore, the ability of AMPH to induce DA efflux was regulated by intracellular Na+ concentration and correlated with the size of the DAT-mediated, AMPH-induced ion flux across the plasma membrane. In the absence of intracellular Na+ but the presence of high intracellular Cl−, AMPH-induced inward currents elicited DA efflux proportionally to their dimension and duration. Thus, we propose that AMPH-induced DA efflux depends on two correlated transporter processes. First, AMPH binds to the DAT and is transported, thereby causing an inward current. Second, because of this AMPH-induced inward current, Na+ becomes more available intracellularly to the DAT, thereby enhancing DAT-mediated reverse transport of DA.

A heteroplasmic, not homoplasmic, mitochondrial DNA mutation promotes tumorigenesis via alteration in reactive oxygen species generation and apoptosis.

Mitochondrial alteration has been long proposed to play a major role in tumorigenesis. Recently, mitochondrial DNA (mtDNA) mutations have been found in a variety of cancer cells. In this study, we examined the contribution of mtDNA mutation and mitochondrial dysfunction in tumorigenesis first using human cell lines carrying a frame-shift at NADH dehydrogenase (respiratory complex I) subunit 5 gene (ND5); the same homoplasmic mutation was also identified in a human colorectal cancer cell line earlier. With increasing mutant ND5 mtDNA content, respiratory function including oxygen consumption and ATP generation through oxidative phosphorylation declined progressively, while lactate production and dependence on glucose increased. Interestingly, the reactive oxygen species (ROS) levels and apoptosis exhibited antagonistic pleiotropy associated with mitochondrial defects. Furthermore, the anchorage-dependence phenotype and tumor-forming capacity of cells carrying wild-type and mutant mtDNA were tested by growth assay in soft agar and subcutaneous implantation of the cells in nude mice. Surprisingly, the cell line carrying the heteroplasmic ND5 mtDNA mutation showed significantly enhanced tumor growth, while cells with homoplasmic form of the same mutation inhibited tumor formation. Similar results were obtained from the analysis of a series of mouse cell lines carrying a nonsense mutation at ND5 gene. Our results indicate that the mtDNA mutations might play an important role in the early stage of cancer development, possibly through alteration of ROS generation and apoptosis.

Adiponectin activates AMP-activated protein kinase in muscle cells via APPL1/LKB1-dependent and phospholipase C/Ca2+/Ca2+/calmodulin-dependent protein kinase kinase-dependent pathways

The binding of the adaptor protein APPL1 to adiponectin receptors is necessary for adiponectin-induced AMP-activated protein kinase (AMPK) activation in muscle, yet the underlying molecular mechanism remains unknown. Here we show that in muscle cells adiponectin and metformin induce AMPK activation by promoting APPL1-dependent LKB1 cytosolic translocation. APPL1 mediates adiponectin signaling by directly interacting with adiponectin receptors and enhances LKB1 cytosolic localization by anchoring this kinase in the cytosol. Adiponectin also activates another AMPK upstream kinase Ca2+/calmodulin-dependent protein kinase kinase by activating phospholipase C and subsequently inducing Ca2+ release from the endoplasmic reticulum, which plays a minor role in AMPK activation. Our results show that in muscle cells adiponectin is able to activate AMPK via two distinct mechanisms as follows: a major pathway (the APPL1/LKB1-dependent pathway) that promotes the cytosolic localization of LKB1 and a minor pathway (the phospholipase C/Ca2+/Ca2+/calmodulin-dependent protein kinase kinase-dependent pathway) that stimulates Ca2+ release from intracellular stores.

Multi-photon laser scanning microscopy using an acoustic optical deflector.

Multi-photon laser scanning microscopes have many advantages over single-photon systems. However, the speed and flexibility of currently available multi-photon microscopes are limited by the use of mechanical mirrors to steer pulsed radiation for fluorophore excitation. Here, we describe the multi-photon adaptation of a confocal microscope that uses an acoustic optical deflector (AOD) for beam steering. AODs are capable of very rapid scanning and, in addition, offer the flexibility of zooming, panning, and being adjustable for slow image acquisition. Because of the highly dispersive nature of AODs, pulsed radiation must be temporally compressed by introducing negative dispersion into the beam path. More critically, pulsed radiation must also be spatially compressed by introducing lateral dispersion into the beam path. This was accomplished by using two prisms in the external beam path and by introducing a third prism element subsequent to the AOD. The end result is an AOD-based multi-photon microscope that is capable of rapid imaging of physiological events as well as slow detection of weakly fluorescent biological samples.

Chronic Rapamycin Restores Brain Vascular Integrity and Function Through NO Synthase Activation and Improves Memory in Symptomatic Mice Modeling Alzheimer’s Disease

Vascular pathology is a major feature of Alzheimer’s disease (AD) and other dementias. We recently showed that chronic administration of the target-of-rapamycin (TOR) inhibitor rapamycin, which extends lifespan and delays aging, halts the progression of AD-like disease in transgenic human (h)APP mice modeling AD when administered before disease onset. Here we demonstrate that chronic reduction of TOR activity by rapamycin treatment started after disease onset restored cerebral blood flow (CBF) and brain vascular density, reduced cerebral amyloid angiopathy and microhemorrhages, decreased amyloid burden, and improved cognitive function in symptomatic hAPP (AD) mice. Like acetylcholine (ACh), a potent vasodilator, acute rapamycin treatment induced the phosphorylation of endothelial nitric oxide (NO) synthase (eNOS) and NO release in brain endothelium. Administration of the NOS inhibitor L-NG-Nitroarginine methyl ester reversed vasodilation as well as the protective effects of rapamycin on CBF and vasculature integrity, indicating that rapamycin preserves vascular density and CBF in AD mouse brains through NOS activation. Taken together, our data suggest that chronic reduction of TOR activity by rapamycin blocked the progression of AD-like cognitive and histopathological deficits by preserving brain vascular integrity and function. Drugs that inhibit the TOR pathway may have promise as a therapy for AD and possibly for vascular dementias.

Impact of Mitochondrial Ca2+ Cycling on Pattern Formation and Stability

Energization of mitochondria significantly alters the pattern of Ca2+ wave activity mediated by activation of the inositol (1,4,5) trisphosphate (IP3) receptor (IP3R) in Xenopus oocytes. The number of pulsatile foci is reduced and spiral Ca2+ waves are no longer observed. Rather, target patterns of Ca2+ release predominate, and when fragmented, fail to form spirals. Ca2+ wave velocity, amplitude, decay time, and periodicity are also increased. We have simulated these experimental findings by supplementing an existing mathematical model with a differential equation for mitochondrial Ca2+ uptake and release. Our calculations show that mitochondrial Ca2+ efflux plays a critical role in pattern formation by prolonging the recovery time of IP3Rs from a refractory state. We also show that under conditions of high energization of mitochondria, the Ca2+ dynamics can become bistable with a second stable stationary state of high resting Ca2+ concentration.

Calcineurin interacts with PERK and dephosphorylates calnexin to relieve ER stress in mammals and frogs

Background The accumulation of misfolded proteins within the endoplasmic reticulum (ER) triggers a cellular process known as the Unfolded Protein Response (UPR). One of the earliest responses is the attenuation of protein translation. Little is known about the role that Ca2+ mobilization plays in the early UPR. Work from our group has shown that cytosolic phosphorylation of calnexin (CLNX) controls Ca2+ uptake into the ER via the sarco-endoplasmic reticulum Ca2+-ATPase (SERCA) 2b. Methodology/Principal Findings Here, we demonstrate that calcineurin (CN), a Ca2+ dependent phosphatase, associates with the (PKR)-like ER kinase (PERK), and promotes PERK auto-phosphorylation. This association, in turn, increases the phosphorylation level of eukaryotic initiation factor-2 α (eIF2-α) and attenuates protein translation. Data supporting these conclusions were obtained from co-immunoprecipitations, pull-down assays, in-vitro kinase assays, siRNA treatments and [35S]-methionine incorporation measurements. The interaction of CN with PERK was facilitated at elevated cytosolic Ca2+ concentrations and involved the cytosolic domain of PERK. CN levels were rapidly increased by ER stressors, which could be blocked by siRNA treatments for CN-Aα in cultured astrocytes. Downregulation of CN blocked subsequent ER-stress-induced increases in phosphorylated elF2-α. CN knockdown in Xenopus oocytes predisposed them to induction of apoptosis. We also found that CLNX was dephosphorylated by CN when Ca2+ increased. These data were obtained from [γ32P]-CLNX immunoprecipitations and Ca2+ imaging measurements. CLNX was dephosphorylated when Xenopus oocytes were treated with ER stressors. Dephosphorylation was pharmacologically blocked by treatment with CN inhibitors. Finally, evidence is presented that PERK phosphorylates CN-A at low resting levels of Ca2+. We further show that phosphorylated CN-A exhibits decreased phosphatase activity, consistent with this regulatory mechanism being shut down as ER homeostasis is re-established. Conclusions/Significance Our data suggest two new complementary roles for CN in the regulation of the early UPR. First, CN binding to PERK enhances inhibition of protein translation to allow the cell time to recover. The induction of the early UPR, as indicated by increased P-elF2α, is critically dependent on a translational increase in CN-Aα. Second, CN dephosphorylates CLNX and likely removes inhibition of SERCA2b activity, which would aid the rapid restoration of ER Ca2+ homeostasis.