Donald A. Fox

Bcl-xL overexpression blocks bax-mediated mitochondrial contact site formation and apoptosis in rod photoreceptors of lead-exposed mice

Photoreceptor apoptosis and resultant visual deficits occur in humans and animals with inherited and disease-, injury-, and chemical-induced retinal degeneration. A clinically relevant mouse model of progressive rod photoreceptor-selective apoptosis was produced by low-level developmental lead exposure and studied in combination with transgenic mice overexpressing Bcl-xL only in the photoreceptors. A multiparametric analysis of rod apoptosis and mitochondrial structure-function was performed. Mitochondrial cristae topography and connectivity, matrix volume, and contact sites were examined by using 3D electron tomography. Lead-induced rod-selective apoptosis was accompanied by rod Ca2+ overload, rhodopsin loss, translocation of Bax from the cytosol to the mitochondria, decreased rod mitochondrial respiration and membrane potential, mitochondrial cytochrome c release, caspase-3 activation, and an increase in the number of mitochondrial contact sites. These effects occurred without mitochondrial matrix swelling, outer membrane rupture, caspase-8 activation, or Bid cleavage. Bcl-xL overexpression completely blocked all apoptotic events, except Ca2+ overload, and maintained normal rod mitochondrial function throughout adulthood. This study presents images of mitochondrial contact sites in an in vivo apoptosis model and shows that Bcl-xL overexpression blocks increased contact sites and apoptosis. These findings extend our in vitro retinal studies with Pb2+ and Ca2+ and suggest that developmental lead exposure produced rod-selective apoptosis without mitochondrial swelling by translocating cytosolic Bax to the mitochondria, which likely sensitized the Pb2+ and Ca2+ overloaded rod mitochondria to release cytochrome c. These results have relevance for therapies in a wide variety of progressive retinal and neuronal degenerations where Ca2+ overload, lead exposure, and/or mitochondrial dysfunction occur.

Calcium Overload Triggers Rod Photoreceptor Apoptotic Cell Death in Chemical‐Induced and Inherited Retinal Degenerations

Impairment and loss of vision are major human health problems. The majority of these cases have a retinal origin.1 For example, selective rod photoreceptor cell apoptosis occurs in humans and animals with different forms of inherited retinal degenerations, cancer-associated retinopathy (CAR), lead exposure during development and adulthood, mild hypoxic-ischemia injury, or light-induced damage (TABLE 1).1–9 Alterations in cGMP phosphodiesterase (PDE) metabolism underlie many types of apoptotic retinal degenerations. In some humans with autosomal recessive retinitis pigmentosa there are mutations in the genes encoding the αand βsubunits of rod cGMP PDE while in lead poisoning there is a competitive inhibition of the cGMP PDE enzyme.8,10–13 In vitro exposure of isolated retinas to 3-isobutyl1-methylxanthine (IBMX), a competitive inhibitor of cGMP PDE, results in selective rod degeneration.11,14 The loss or inhibition of rod cGMP PDE activity results in elevated [cGMP]8,10,15 that is localized almost exclusively to rods.16,17 An increase in rod [cGMP] results in more rod cGMP-gated nonselective cation channels being opened and a sustained elevation of the rod and retinal intracellular [Ca2+].18–21 Numerous studies report that a sustained elevation of intracellular [Ca2+] results in apoptotic cell death.22,23 During the effector phase of apoptosis the mitochondrial permeability transition pore (PTP) is irreversibly opened by sustained increases in matrix Ca2+ leading to mitochondrial depolarization, release of cytochrome c, activation of caspases, chromatin cleavage and apoptotic nuclear morphology.24 The open conformation of the mitochondrial PTP is blocked by cyclosporine A (CsA).24,25 Accumulating evidence suggests that sustained elevations of rod intracellular [Ca2+], following the loss or inhibition of rod cGMP PDE activity as well as in other types of retinal degenerations, may trigger apoptosis. Rod and retinal intracellular [Ca2+] are elevated in isolated retinas exposed to IBMX or Pb2+ and in leadexposed rats.19–21,26 Ca2+ overload blockers protect against rod apoptosis induced by constant light and mild hypoxia-ischemia.4,27 In CAR an increase in rod intracellular [Ca2+] appears to be the apoptotic triggering event9 as there is an increase in an autoantibody against recoverin: a cytoplasmic Ca2+-binding protein primarily localized to the photoreceptors and bipolars cells.28 Our goals were to determine the retinal [Ca2+] and examine high molecular weight (HMW) DNA fragmentation in developing retinal degeneration (rd) mice and

Oxygen consumption in the rat outer and inner retina: light- and pharmacologically-induced inhibition.

Biochemical, physiological and histological data have established that 55-65% of retinal mitochondria are located in the photoreceptor inner segments and suggested that photoreceptors have at least a two-fold greater oxygen consumption (QO2) than the remaining inner retina. QO2 in isolated whole rat retina (QWR), outer retina (QOR) and inner retina (QIR) was measured during dark and rod-saturating light adaptation. The effects of function-specific chemical agents on QWR, QOR and QIR during dark and light adaptation were determined. In addition, the oxidation-reduction (redox) potential of cytochrome a3 of whole, outer and inner retina was measured during dark and light adaptation. During dark adaptation, the mean QWR was 1.62 mumol O2 (mg dry wt)-1 hr-1 and whole retinal level of reduced cytochrome a3 was 19%. They decreased by 24% and 37% during light adaptation, respectively. To determine QOR and QIR during dark and light adaptation, the outer retina was pharmacologically-isolated from inner retina using L-2-amino-4-phosphonobutyric acid plus kynurenic acid (APB/Kyn). Experiments in the presence or absence of APB/Kyn revealed that: (i) QOR, but not QIR, of the dark-adapted retina was decreased 37% during light adaptation, (ii) the outer and inner retina consumed 65% and 35% of the QWR during dark adaptation, respectively, and 54% and 46% of the QWR during light adaptation, respectively, (iii) the level of reduced retinal cytochrome a3 in the outer, but not inner, retina was decreased 34% during light adaptation, (iv) during light adaptation, the rate of QO2 was equal in the outer and inner retina, and (v) the effects of APB/Kyn were reversible. These results establish that the mean rate of QIR and retinal cytochrome a3 are unchanged during dark or light adaptation. In addition, they suggest that QOR:QIR in the rat may be modeled using a 65%:35% model during DA and a 55%:45% model during LA. All the function-specific agents--IBMX, lead, diltiazem, ouabain, CO2+ plus Mg2+ and verapamil--significantly decreased QWR during dark and light adaptation. A more detailed analysis revealed that IBMX and lead each selectively reduced (> or = 90%) QOR during dark adaptation whereas CO2+ plus Mg2+ and verapamil each selectively reduced (> or = 93%) QIR during dark and light adaptation. These results are consistent with the known pharmacological sites and mechanisms of these agents. Additional experiments determined that the IBMX- and lead-induced inhibition of QOR during dark adaptation resulted, either wholly or partially, from the influx of extracellular Ca2+. During dark adaptation in Ca(2+)-free medium: (i) QWR and QOR increased while QIR was unchanged, (ii) QOR was not decreased in the presence of IBMX and (iii) QOR was only partially decreased in the presence of lead.(ABSTRACT TRUNCATED AT 400 WORDS)

Low-level human equivalent gestational lead exposure produces sex-specific motor and coordination abnormalities and late-onset obesity in year-old mice.

Background Low-level developmental lead exposure is linked to cognitive and neurological disorders in children. However, the long-term effects of gestational lead exposure (GLE) have received little attention. Objectives Our goals were to establish a murine model of human equivalent GLE and to determine dose–response effects on body weight, motor functions, and dopamine neurochemistry in year-old offspring. Methods We exposed female C57BL/6 mice to water containing 0, 27 (low), 55 (moderate), or 109 ppm (high) of lead from 2 weeks prior to mating, throughout gestation, and until postnatal day 10 (PN10). Maternal and litter measures, blood lead concentrations ([BPb]), and body weights were obtained throughout the experiment. Locomotor behavior in the absence and presence of amphetamine, running wheel activity, rotarod test, and dopamine utilization were examined in year-old mice. Results Peak [BPb] were < 1, ≤ 10, 24–27, and 33–42 μg/dL in control, low-, moderate- and high-dose GLE groups at PN0–10, respectively. Year-old male but not female GLE mice exhibited late-onset obesity. Similarly, we observed male-specific decreased spontaneous motor activity, increased amphetamine-induced motor activity, and decreased rotarod performance in year-old GLE mice. Levels of dopamine and its major metabolite were altered in year-old male mice, although only forebrain utilization increased. GLE-induced alterations were consistently larger in low-dose GLE mice. Conclusions Our novel results show that GLE produced permanent male-specific deficits. The nonmonotonic dose-dependent responses showed that low-level GLE produced the most adverse effects. These data reinforce the idea that lifetime measures of dose–response toxicant exposure should be a component of the neurotoxic risk assessment process.

A selective decrease of cholinergic muscarinic receptors in the visual cortex of adult rats following developmental lead exposure

The effects of low-level developmental lead (Pb) exposure (postnatal days 0-21) on the binding of [3H]quinuclidinylbenzilate (QNB) and on acetylcholinesterase (AChE) activity in the retina, superior colliculus, lateral geniculate nucleus and visual cortex (VC) were studied in the adult rat. Maximal blood and tissue Pb concentrations (50-60 micrograms %) were reached on day 21 and decreased to control levels (4-5 micrograms %), except in the retina (12 micrograms %) and VC (18 micrograms %; 0.87 microM), by day 90. A large decrease in [3H]QNB binding (-38%) and AChE activity (-29%) was found only in the VC of Pb-exposed rats. Scatchard plots of saturation binding data revealed a decrease in the density (Bmax), but not in the affinity (Kd), of the muscarinic receptors. Pb (10(-4)-10(-9)M) had no effect on [3H]QNB binding or AChE activity in VC membrane preparations from control rats. The mechanism accounting for this selective decrease of cholinergic muscarinic receptors in the VC is presently unknown. These results, in combination with those from our psychophysical and pharmacological studies demonstrating a scopolamine supersensitivity in Pb-exposed rats, suggest that the long-term effects of developmental Pb exposure are due to a direct action of Pb on visual cortex cholinergic neurons.

Rods are selectively altered by lead: II. Ultrastructure and quantitative histology

Electroretinographic and cyclic nucleotide metabolism studies have established that low-level lead exposure during early postnatal development results in long-term selective rod deficits. To determine whether there was a corresponding selective rod photoreceptor cell degeneration we examined retinas of adult rats exposed to low-level lead during development using light and electron microscopy. In all retinal regions, a rod but not cone cell degeneration was observed. Overall, 20% of the rod cells were lost. Moreover, two specific regional differences were found. Degeneration was much greater in the inferior (-25%) than superior (-15%) retina and greater in the posterior (-22%) than peripheral (-17%) retina. The latter pattern indicates a central-peripheral gradient of degeneration. Total retinal thickness decreased 15-20%, which reflects cell loss in the outer and inner nuclear layers. Ultrastructurally, the most obvious lead-induced alterations were swollen and disorganized rod outer segments and large accumulations of beta-glycogen particles in rod photoreceptor mitochondria. Glycogen accumulations were heaviest in rod inner segment mitochondria followed by rod axon and synaptic terminal mitochondria. Possible cellular mechanisms of action responsible for these lead-induced retinal alterations include an inhibition of retinal cyclic GMP phosphodiesterase and the resultant elevation of cyclic GMP, an inhibition of intermediary metabolism, and/or an alteration in calcium metabolism. In addition, the thinning of the inner nuclear layers could be due to transneuronal degeneration. As noted in our preceding paper, the first possibility has been demonstrated in rats similarly exposed to lead. These quantitative histological results, in combination with the ERG and biochemical results in the preceding paper, demonstrate that low-level lead exposure during early postnatal development produces long-term selective rod functional deficits and degeneration.

Developmental origins of adult diseases and neurotoxicity: Epidemiological and experimental studies

To date, only a small number of commercial chemicals have been tested and documented as developmental neurotoxicants. Moreover, an increasing number of epidemiological, clinical and experimental studies suggest an association between toxicant or drug exposure during the perinatal period and the development of metabolic-related diseases and neurotoxicity later in life. The four speakers at this symposium presented their research results on different neurotoxic chemicals relating to the developmental origins of health and adult disease (DOHaD). Philippe Grandjean presented epidemiological data on children exposed to inorganic mercury and methylmercury, and discussed the behavioral outcome measures as they relate to age and stage of brain development. Donald A. Fox presented data that low-dose human equivalent gestational lead exposure produces late-onset obesity only in male mice that is associated with neurodegeneration. Didima de Groot presented results on prenatal exposure of rats to methylazoxymethanol and discussed the results in light of the etiology of western Pacific amyotrophic lateral sclerosis and Parkinson-dementia complex. Merle G. Paule addressed the long-term changes in learning, motivation and short-term memory in aged Rhesus monkeys following acute 24 h exposure to ketamine during early development. Overall, these presentations addressed fundamental issues in the emerging areas of lifetime neurotoxicity testing, differential vulnerable periods of exposure, nonmonotonic dose-response effects and neurotoxic risk assessment. The results indicate that developmental neurotoxicity results in permanent changes, thus emphasizing the need to prevent such toxicity.

Lead-induced alterations in retinal cGMP phosphodiesterase trigger calcium overload, mitochondrial dysfunction and rod photoreceptor apoptosis

Lead exposure results in the selective apoptotic loss of rods and bipolar cells. During and following developmental lead exposure rod/retinal cGMP phosphodiesterase expression and activity are delayed in onset and decreased, [Ca2+] is elevated, and mitochondrial ATP synthesis is decreased. In vitro studies, using retinas incubated in Ca2+ and/or Pb2+, demonstrate that rods selectively die by apoptosis, retinal mitochondrial ATP synthesis is decreased, mitochondrial cytochrome c is released and caspase activity is increased. These results suggest that lead-induced rod and bipolar cell apoptosis is triggered by Ca2+ and Pb2+ overload due to altered cGMP phosphodiesterase activity and that mitochondrial alterations play a central role in this process.

Alterations in retinal Na+, K+ATPase in diabetes : streptozotocin-induced and Zucker diabetic fatty rats

The temporal pattern of changes in the specific activities of retinal Na+, K(+)-ATPase (Na, K-ATPase) and Mg(2+)-ATPase (Mg-ATPase) were determined at several time intervals following the onset of diabetes in streptozotocin-induced diabetic (STZ: at 1, 2, 4 and 6 months) Long-Evans hooded rats, spontaneously diabetic Zucker diabetic fatty (ZDF: at 1, 2 and 4 months) rats and their age-matched controls. These animals were utilized as models for insulin-dependent diabetes mellitus (IDDM) and non-insulin-dependent diabetes mellitus (NIDDM), respectively. Na, K-ATPase specific activity, using 10(3) M ouabain, was decreased (-6% to -14%) at all time points after the appearance of hyperglycemia in the ZDF rat, but was reduced only after 4 and 6 months in the STZ rat (-8% and -14%, respectively). In contrast, Mg-ATPase activity was significantly increased (13%) after 4 months in the ZDF rat and after 6 months in the STZ rat (8%). The concentration-dependent inhibitory effects of ouabain (10(-9) to 10(-3) M) on the activity of Na, K-ATPase in diabetic rats and age-matched controls was used to assess the time-dependent effects of diabetes on the alpha 3-high ouabain affinity or the alpha 1-low ouabain affinity retinal Na, K-ATPase isozymes. The retinal Na, K-ATPase activity for the alpha 3 isozyme was significantly lower at all times examined for the ZDF (-5% to -26%) and STZ-induced diabetic rats (-8% to -14%). This was reflected in the markedly decreased half-maximal inhibitory concentrations (IC50) of ouabain for the alpha 3 isozyme. For example, after four months of diabetes, the mean +/- SEM IC50 values were 12 +/- 3 nM in the STZ rats and 48 +/- 6 nM in the age-matched controls and 19 +/- 3 nM in the ZDF rats and 30 +/- 4 nM in the age-matched controls. In contrast, the activity of the alpha 1 isozyme was slightly, but significantly, decreased at 2 and 4 months in the ZDF rats (-4% to -7%) and after 4 and 6 months in the STZ-induced diabetic rats (-3% to -9%) while the IC50 values were unchanged. Moreover, the Hill coefficient for the alpha 3 isozyme was decreased in both diabetic groups while it was unchanged for the alpha 1 isozyme.(ABSTRACT TRUNCATED AT 400 WORDS)

Rods are selectively altered by lead. I: Electrophysiology and biochemistry

In vitro studies have demonstrated that lead selectively and reversibly depresses the rod photoreceptor component of the electroretinogram (ERG). To determine if low-level lead exposure during early postnatal development produced long-term selective rod deficits, we examined rod and cone ERG functions and cyclic GMP and cyclic AMP metabolism in adult control and lead-exposed rats. A-wave and b-wave voltage-log intensity and latency-log intensity functions, generated from single-flash ERGs in fully dark-adapted rats, revealed that low-level lead exposure during early postnatal development caused a 23- and 18% decrease in maximum amplitude, a 1.0- and 0.5 log unit decrease in absolute sensitivity and a mean latency increase of 47- and 29%, respectively. Additional ERG experiments, using scotopically balanced stimuli and scotopic and photopic flicker fusion frequency functions, also demonstrated selective rod deficits. Cone ERGs, elicited by 30-Hz white flashes in the presence of a white background adapting light, were similar in control and lead-exposed rats. Lead exposure during early postnatal development caused cGMP levels in dark-adapted and light-adapted retinas to increase 40- and 25%, respectively, above controls whereas cyclic AMP levels remained unchanged. Light-activated cyclic GMP phosphodiesterase (cGMP-PDE) was inhibited 40% while guanylate cyclase activity was unchanged. The retinal lead concentration was 10(-6) M at the end of exposure (day 21) while at the time of ERG testing and biochemical analysis it was 10(-7) M. In vitro studies with adult control retinas incubated with 10(-9)-10(-4) M lead revealed a dose-response inhibition (10-40%) of cGMP-PDE between 10(-6)- and 10(-4) M lead and stimulation of guanylate cyclase (20-158%) only above 10(-4) M lead, indicating that cGMP-PDE is more sensitive to the direct effects of lead than the synthetic cGMP enzyme. These in vitro cyclic nucleotide metabolism results are similar to those we observed in vivo and both are consistent with the observed ERG changes. The selective rod-mediated amplitude, sensitivity and temporal deficits and the lack of effect on the cone ERGs clearly demonstrate that low-level lead exposure during early postnatal development causes a long-term selective disruption of rat rod photoreceptors. The relevance and applicability of these data to subclinical pediatric lead poisoning has yet to be established.