Robert A. Colvin

Cytosolic zinc buffering and muffling: Their role in intracellular zinc homeostasis

Our knowledge of the molecular mechanisms of intracellular homeostatic control of zinc ions is now firmly grounded on experimental findings gleaned from the study of zinc proteomes and metallomes, zinc transporters, and insights from the use of computational approaches. A cells repertoire of zinc homeostatic molecules includes cytosolic zinc-binding proteins, transporters localized to cytoplasmic and organellar membranes, and sensors of cytoplasmic free zinc ions. Under steady state conditions, a primary function of cytosolic zinc-binding proteins is to buffer the relatively large zinc content found in most cells to a cytosolic zinc(ii) ion concentration in the picomolar range. Under non-steady state conditions, zinc-binding proteins and transporters act in concert to modulate transient changes in cytosolic zinc ion concentration in a process that is called zinc muffling. For example, if a cell is challenged by an influx of zinc ions, muffling reactions will dampen the resulting rise in cytosolic zinc ion concentration and eventually restore the cytosolic zinc ion concentration to its original value by shuttling zinc ions into subcellular stores or by removing zinc ions from the cell. In addition, muffling reactions provide a potential means to control changes in cytosolic zinc ion concentrations for purposes of cell signalling in what would otherwise be considered a buffered environment not conducive for signalling. Such intracellular zinc ion signals are known to derive from redox modifications of zinc-thiolate coordination environments, release from subcellular zinc stores, and zinc ion influx via channels. Recently, it has been discovered that metallothionein binds its seven zinc ions with different affinities. This property makes metallothionein particularly well positioned to participate in zinc buffering and muffling reactions. In addition, it is well established that metallothionein is a source of zinc ions under conditions of redox signalling. We suggest that the biological functions of transient changes in cytosolic zinc ion concentrations (presumptive zinc signals) complement those of calcium ions in both spatial and temporal dimensions.

Zinc Transport in the Brain: Routes of Zinc Influx and Efflux in Neurons

Studies of the routes of entry and exit for zinc in different tissues and cell types have shown that zinc can use several pathways of exit or entry. In neurons, known pathways include (1) presynaptic release along with glutamate when synaptic vesicles empty their contents into the synaptic cleft, (2) voltage-gated L-type Ca(2+) channels and glutamate-gated channels that provide an entry route when cells are depolarized and that mediate extracellular zinc toxicity and (3) a plasma membrane transporter potentially present in all neurons important for cellular zinc homeostasis. The least understood of these pathways, in terms of mechanism, is the transporter pathway. The kinetics of zinc uptake in cultured neurons under resting conditions are consistent with and suggest the existence of a saturable transporter in the plasma membrane. The proteins responsible for plasma membrane zinc transport have not yet been definitely identified. Likely candidates include two proteins identified by molecular cloning termed zinc transporter 1 and divalent cation transporter DCT1. Both proteins have been shown to be expressed in the brain, but only DCT1 is clearly demonstrated to be a transport protein, whereas zinc transporter 1 may only modulate zinc transport in association with as-yet-unidentified proteins. Understanding the mechanism and neuromodulation of plasma membrane zinc transport will be an important first step toward a complete understanding of neuronal zinc homeostasis.

Zinquin identifies subcellular compartmentalization of zinc in cortical neurons. Relation to the trafficking of zinc and the mitochondrial compartment

Zinquin (Zn(2+) selective fluorophore), when used to visualize intracellular Zn(2+), typically shows brightly fluorescent perinuclear endosome-like structures, presumably identifying Zn(2+) containing organelles. In this study, zinquin identified numerous and widespread sites of Zn(2+) compartmentalization in primary cultures of embryonic rat cortical neurons. Nuclear fluorescence, however, was absent. We labeled neuronal mitochondria with MitoTracker Green in the presence of zinquin and show that the fluorescent patterns of MitoTracker Green and zinquin were distinct and clearly different in both the perinuclear region and in processes. The mitochondrial compartment was much larger than the sum of the areas of zinquin fluorescence, as indicated by the small amount (<10% MitoTracker Green over zinquin) of overlap of MitoTracker Green on zinquin. Zinquin fluorescence was unaffected by carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP) treatment. The zinquin fluorescent objects were generally spherical in shape with a average diameter of about 0.6 mum. Most fluorescent objects, nearly two thirds on average, appeared to be docked, but both anterograde and retrograde movements were observed by time lapse image analysis. Although some fluorescent objects moved as much as 1 mum in 5 min, typical movements were smaller, usually 0.5 mum or less. Colchicine treatment caused striking aggregation of MitoTracker Green most noticeable in the perinuclear region. Zinquin fluorescence similarly showed reduced distribution throughout the cytoplasm, suggesting that zinquin fluorescent structures were associated with microtubules. Treatment with cytochalasin D had little noticeable effect on either the pattern of zinquin and MitoTracker Green fluorescence or their coincidence. Thus, numerous Zn(2+) sequestering organelles/structures are present in perinuclear regions and processes of cultured neurons and are sometimes found coincident with mitochondria. We demonstrated real time trafficking of sequestered Zn(2+), using zinquin fluorescence, apparently associated with an endosome-like compartment or protein complexes in the cytosol.

Characterization of a plasma membrane zinc transporter in rat brain

Many studies now show that zinc plays a critical and unique role in central nervous system development and function. The cellular mechanisms of zinc efflux and influx are largely unknown and few models exist that describe cellular zinc transport in the brain. This report provides convincing evidence of a zinc transporter in plasma membrane vesicles isolated from rat brain. Zinc uptake was saturable (Km = 15 microM; Vmax = 10 nmol/mg per 30 s), was seen in the absence of ATP, and was unaffected by gradients for other ions such as Na+ or K+. Increasing the ionic strength of the extravesicular media with Na+, K+, or choline+ reduced zinc uptake approximately 50%. Whereas, increasing extravesicular H+ concentration (pH = 5) resulted in near complete inhibition of zinc uptake. Intravesicular zinc was rapidly released upon lowering extravesicular concentrations of zinc with the heavy metal chelator O-phenanthroline (1 mM). The results are consistent with a freely-reversible transport of zinc across the plasma membrane of neurons.

Na+/Ca2+ exchange activity is increased in Alzheimer's disease brain tissues

These studies were performed to determine the changes that occur in Na+/Ca2+ exchange activity in Alzheimers disease (AD) brain tissues. Cerebral plasma membrane vesicles were purified by sucrose density gradient centrifugation from frozen postmortem hippocampal/temporal cortex tissue slices derived from age matched brains of normal, AD and non-Alzheimer dementia (NAD) origin (autopsy confirmed). Membrane marker assays (Na/K ATPase, muscarinic receptor, cytochrome c oxidase) revealed no change in membrane purity across different preparations. Thin-section electron microscopy revealed predominantly intact unilamellar vesicles. Vesicles were preincubated for 15 min (37 degrees C) in buffer containing 132 mM NaCl, 5 mM KCl, 1.3 mM MgCl2, 10 mM glucose and 10 mM HEPES (pH 7.4). Ca2+ uptake was initiated by diluting vesicles 20-fold with buffer containing either 132 mM NaCl or 132 mM choline chloride and 45CaCl2 then terminated by addition of 200 microM LaCl3 and rapid filtration. Ca2+ content increased rapidly at first and then maintained a steady plateau for up to 5 min. When the Ca2+ ionophore A23187 (10 microM) with 100 microM EGTA was added after 4 min, Ca2+ content was reduced to 10% of its original value. Ruthenium red (10 microM) had no effect on Ca2+ content. Na(+)-dependent Ca2+ uptake (Ca2+ content measured in choline chloride minus that measured in NaCl) was increased in AD brains as evidenced by both an increase in the initial rise in Ca2+ content and in elevated values of peak plateau Ca2+ content.(ABSTRACT TRUNCATED AT 250 WORDS)

Evidence for a zinc/proton antiporter in rat brain

The data presented in this paper are consistent with the existence of a plasma membrane zinc/proton antiport activity in rat brain. Experiments were performed using purified plasma membrane vesicles isolated from whole rat brain. Incubating vesicles in the presence of various concentrations of 65Zn2+ resulted in a rapid accumulation of 65Zn2+. Hill plot analysis demonstrated a lack of cooperativity in zinc activation of 65Zn2+ uptake. Zinc uptake was inhibited in the presence of 1 mM Ni2+, Cd2+, or CO2+. Calcium (1 mM) was less effective at inhibiting 65Zn2+ uptake and Mg2+ and Mn2+ had no effect. The initial rate of vesicular 65Zn2+ uptake was inhibited by increasing extravesicular H+ concentration. Vesicles preloaded with 65Zn2+ could be induced to release 65Zn2+ by increasing extravesicular H+ or addition of 1 mM nonradioactive Zn2+. Hill plot analysis showed a lack of cooperativity in H+ activation of 65Zn2+ release. Based on the Hill analyses, the stoichiometry of transport may include Zn2+/Zn2+ exchange and Zn2+/H+ antiport, the latter being potentially electrogenic. Zinc/proton antiport may be an important mode of zinc uptake into neurons and contribute to the reuptake of zinc to replenish presynaptic vesicle stores after stimulation.

Silencing of ZnT1 reduces Zn2+ efflux in cultured cortical neurons.

Zinc dyshomeostasis in brain might be involved in the pathogenesis of a series of brain diseases such as Alzheimers disease and stroke. It is essential that the level of intracellular free Zn2+ in neurons is tightly controlled to maintain a narrow window of optimal concentration. The plasma membrane bound transporter ZnT1 is suggested to lower intracellular Zn2+ concentration. In this study, the function of ZnT1 in cultured cortical neurons was studied. Using vector-based shRNA interference, the expression of this protein was reduced approximately 40% in cultured rat cortical neurons when measured by immunofluorescence using a ZnT1 antibody. Changes in intracellular Zn2+ levels were tracked in individual neurons by microfluorometry using the Zn2+ selective fluorophore, FluoZin3. Unopposed Zn2+ efflux was measured by first loading cultured cortical neurons with Zn2+ then reducing extracellular Zn2+ to near zero by addition of EDTA. Reducing ZnT1 expression caused Zn2+ efflux to decrease compared with the Zn2+ efflux measured in nonsense transfected neurons, suggesting that ZnT1 plays a direct role in Zn2+ efflux. ZnT1 dependent Zn2+ efflux rate was higher in the first 10 min than at later time periods suggesting that ZnT1-mediated efflux was heavily dependent on the intracellular free Zn2+ concentration and/or required an outwardly directed Zn2+ gradient.

Mechanisms of Zn2+ efflux in cultured cortical neurons

Zinc dyshomeostasis in brain might be involved in the pathogenesis of brain diseases such as Alzheimer’s disease and stroke. Resting neurons tightly regulate and maintain low to subnanomolar levels of intracellular free Zn2+, but mechanisms of normal Zn2+ homeostasis are poorly understood. In this study, the mechanisms of transporter‐mediated Zn2+ extrusion across the plasma membrane of cultured cortical neurons were studied. Changes in intracellular free Zn2+ levels were tracked in individual neurons by microfluorometry using a Zn2+ selective fluorophore, FluoZin3. Unopposed Zn2+ efflux was measured by first loading cultured cortical neurons with Zn2+ then reducing extracellular Zn2+ to near zero by addition of EDTA. Studies revealed that the primary means of Zn2+ efflux in cortical neurons required both extracellular Na+ and Ca2+. The actions of either Na+ or Ca2+ on Zn2+ efflux were blunted in the absence of the other cation. Reversed Na+ gradients could induce Zn2+ uptake. The Na+ dependence of Zn2+ efflux was not affected by a small pHo shift (7.6–8); whereas an effect of Ca2+ was not observed at pHo 8. In summary, a Na+, Ca2+/Zn2+ exchanger mechanism is proposed to be the primary transport mechanism that extrudes Zn2+ when neuronal intracellular free Zn2+ levels rise.

Lymphatic drainage system of the brain: A novel target for intervention of neurological diseases

The belief that the vertebrate brain functions normally without classical lymphatic drainage vessels has been held for many decades. On the contrary, new findings show that functional lymphatic drainage does exist in the brain. The brain lymphatic drainage system is composed of basement membrane-based perivascular pathway, a brain-wide glymphatic pathway, and cerebrospinal fluid (CSF) drainage routes including sinus-associated meningeal lymphatic vessels and olfactory/cervical lymphatic routes. The brain lymphatic systems function physiological as a route of drainage for interstitial fluid (ISF) from brain parenchyma to nearby lymph nodes. Brain lymphatic drainage helps maintain water and ion balance of the ISF, waste clearance, and reabsorption of macromolecular solutes. A second physiological function includes communication with the immune system modulating immune surveillance and responses of the brain. These physiological functions are influenced by aging, genetic phenotypes, sleep-wake cycle, and body posture. The impairment and dysfunction of the brain lymphatic system has crucial roles in age-related changes of brain function and the pathogenesis of neurovascular, neurodegenerative, and neuroinflammatory diseases, as well as brain injury and tumors. In this review, we summarize the key component elements (regions, cells, and water transporters) of the brain lymphatic system and their regulators as potential therapeutic targets in the treatment of neurologic diseases and their resulting complications. Finally, we highlight the clinical importance of ependymal route-based targeted gene therapy and intranasal drug administration in the brain by taking advantage of the unique role played by brain lymphatic pathways in the regulation of CSF flow and ISF/CSF exchange.

Visualizing Metal Content and Intracellular Distribution in Primary Hippocampal Neurons with Synchrotron X-Ray Fluorescence.

Increasing evidence suggests that metal dyshomeostasis plays an important role in human neurodegenerative diseases. Although distinctive metal distributions are described for mature hippocampus and cortex, much less is known about metal levels and intracellular distribution in individual hippocampal neuronal somata. To solve this problem, we conducted quantitative metal analyses utilizing synchrotron radiation X-Ray fluorescence on frozen hydrated primary cultured neurons derived from rat embryonic cortex (CTX) and two regions of the hippocampus: dentate gyrus (DG) and CA1. Comparing average metal contents showed that the most abundant metals were calcium, iron, and zinc, whereas metals such as copper and manganese were less than 10% of zinc. Average metal contents were generally similar when compared across neurons cultured from CTX, DG, and CA1, except for manganese that was larger in CA1. However, each metal showed a characteristic spatial distribution in individual neuronal somata. Zinc was uniformly distributed throughout the cytosol, with no evidence for the existence of previously identified zinc-enriched organelles, zincosomes. Calcium showed a peri-nuclear distribution consistent with accumulation in endoplasmic reticulum and/or mitochondria. Iron showed 2–3 distinct highly concentrated puncta only in peri-nuclear locations. Notwithstanding the small sample size, these analyses demonstrate that primary cultured neurons show characteristic metal signatures. The iron puncta probably represent iron-accumulating organelles, siderosomes. Thus, the metal distributions observed in mature brain structures are likely the result of both intrinsic neuronal factors that control cellular metal content and extrinsic factors related to the synaptic organization, function, and contacts formed and maintained in each region.