Manganese Neurotoxicity: Lessons Learned from Longitudinal Studies in Nonhuman Primates

1Manganese is an essential micronutrient that has a broad role in macromolecular metabolism. Mn plays a role in immune response, blood sugar homeostasis, adenosine triphosphate (ATP) regulation, reproduction, digestion, and bone growth. It is a necessary component of metalloenzymes such as Mn superoxide dismutase, arginase, phosphoenol-pyruvate decarboxylase, and glutamine synthetase (GS). GS, an enzyme that converts glutamate into glutamine, is thought to be associated with up to 80% of brain Mn. Mn has a heterogeneous distribution throughout the brain. In the normal human brain, Mn is most concentrated in the globus pallidus, caudate, and putamen and is less concentrated in cortical areas. Intraneuronal axonal transport and potassium-evoked 54Mn release in rat amygdala demonstrate that Mn may participate in neuronal function and neuro-transmission. Mn can also enter neuronal terminals through calcium channels. The consumption of a Mn-deficient diet produced seizures in rats, demonstrating the importance of Mn in neuronal function. Mn deficiency, although rare, can cause developmental defects including malformation of bones, altered macro-molecular metabolism, and reduced fertility.

2Mn is ubiquitous in the environment. It is present at 0.1% in the earth’s crust, and it is also a constituent of soil, ranging in concentrations from 40 to 900 mg/kg. It is released into the environment as a product of industrial activities, the use of the Mn-containing pesticide maneb, and through the use of methyl-cyclopentadienyl manganese tricarbonyl (MMT) as a gasoline antiknock agent. Mn is present in a variety of foods, with the highest concentrations found in nuts, legumes, and blueberries. An estimated safe level of daily intake is 2–5 mg Mn (for review, see ).

3Increased Mn concentrations in the brain can occur from a variety of conditions, with contributions to human morbidity stemming from occupational, iatrogenic, medical, and environmental exposures. Occupational Mn exposure is well documented; for example, excessive Mn exposure occurs in occupations such as ferro alloy smelting, welding, mining, battery assembly, and the manufacture of glass and ceramics.

4Iatrogenic Mn exposure occurs in individuals receiving total parenteral nutrition resulting in increased concentrations of Mn in the brain. Further, exposure to at-risk populations with compromised or immature blood–brain barriers or underdeveloped excretory pathways, such as children, can also result in increased brain Mn levels after total parenteral nutrition. In support of this concept, Mn has been shown to accumulate in the brain of postnatal day (PN) 21 rat pups to a greater extent than PN70 young adult rats after 21 days of oral exposure to Mn.

5Mn is excreted primarily through the bile. Individuals with a decreased capacity for biliary Mn excretion also experience Mn neurotoxicity. Increased Mn has been measured in the blood and brains of individuals with cirrhosis. Further, increases in brain Mn measured by magnetic resonance imaging (MRI) has been documented in patients with liver dysfunction and patients with liver failure.

6Recently, Mn exposure has also been associated with use of methcathinone (ephedrone) contaminated with the oxidant potassium permanganate added to the preparation. Ephedrone is used intravenously as a drug of abuse, and case reports suggest that the rapid onset of hypokinesia, dysarthria, dystonia, and postural dysfunction in these recreational drug users is a product of Mn neurotoxicity.

7The use of MMT as a gasoline antiknock agent has raised concerns over potential increased environmental exposures of the general population to Mn. Elevated average indoor and outdoor air concentrations of Mn have been reported near expressways in Montreal, Quebec, Canada, which uses MMT in gasoline, compared with rural communities in which MMT use is infrequent. The contribution from the use of MMT to environmental levels of Mn to humans is an important issue, as an increased environmental Mn burden as a consequence of Mn generated from MMT-containing gasoline and industrial activities may be associated with an increased prevalence of movement abnormalities and parkinsonism.

8At high levels of exposure, Mn can produce multiple symptoms of neurotoxicity. first described progressive motor abnormalities in workers occupationally exposed to Mn oxide. Mn poisoning has since been associated with an extra-pyramidal syndrome including kinetic tremor, bradykinesia, rigidity, dystonia, and specific gait disturbances. In the absence of continued exposure, motor deficits can persist for many years. In addition to these motor abnormalities, neuro psychologic symptoms including deficits in working memory, concentration, and spatial orientation also occur early in Mn neurotoxicity. A study conducted in Bangladesh showed a significant association between exposure to high levels of Mn in well water and deficits in cognitive function assessed by verbal scores and measures of intellectual ability of children. Importantly, a diagnosis of Parkinson disease (PD) at an earlier average age was correlated with elevated ambient Mn concentrations related to industrial emissions in a Canadian study. In addition, increased air concentrations of Mn derived from mining and industrial activities in Mexico were statistically significantly associated with deficits in movement coordination and hand position changes. Further, an increased prevalence of PD in a population in Brescia, Italy, was associated with increased Mn concentrations in settled dust resulting from nearby ferroalloy plants.

9Mn is known to have 11 oxidation states, although only two oxidation states—Mn2+ and Mn3+—are common in biological systems. Mn2+ is the most stable, although Mn3+ most efficiently oxidizes intra-cellular substrates such as dopamine. Mn3+ binds to and is transported by transferrin, whereas it is postulated that Mn2+ must use other transporter systems to enter cells. Gastrointestinal absorption and inhalation are the two main routes by which Mn is absorbed into the human body.

10Approximately 1–5% of gastrointestinal Mn is absorbed. Sixty to 70% of inhaled Mn is expelled from the lung by mucocilliary movement and is swallowed, although Mn absorption also occurs directly in the lung. Importantly, Mn can be transported directly into the brain during inhalation via the olfactory tract. Mn is mainly excreted through the bile. Under normal conditions, Mn concentrations in the blood are between 0.8 and 2.1 μg/L. After excessive Mn exposures, average blood Mn levels have been documented at 10–40 μg/L, but maximum reported values from one study measured blood Mn in newborns at approximately 90 μg/L and in the birthing mother approximately 150 μg/L. In blood, Mn is present as a free ion, and it is also bound to serum proteins and metal binding proteins such as transferrin.

11Mn enters the brain at the capillary endothelium across the choroid plexus. Calcium has been shown to play an important role in the regulation of Mn transport at the blood–brain barrier. Additionally, divalent metal ion transporter 1 (DMT1)-mediated transport, the N-methyl-d-aspartate (NMDA) receptor channel, transferrin, diffusion, and active transport also contribute to Mn transport across the blood–brain barrier (for review, see ). The importance of the transferrin pathway in brain uptake of Mn is underscored by observations of increased Mn levels in the brains of rats following iron deficiency, which up-regulates transferrin-mediated transport.

12Once Mn has crossed the blood–brain barrier, the average half-life of Mn in the rat brain is 51–74 days. In nonhuman primates, available data suggest that the rate of elimination in the brain has region specificity; however, estimates of half-life range from = 33 days to 53 days after inhalational and subcutaneous Mn exposure, respectively. The normal concentration of Mn in the brain is, on average, 0.26 μg/g wet weight, although Mn distribution throughout the brain is known to be heterogeneous. In nonhuman primates chronically exposed to Mn by intravenous injection and inhalation, Mn has been observed to accumulate throughout the brain in vivo using T1-weighted MRI. The region of the brain in which Mn accumulates to the greatest extent is the globus pallidus. Using inhalation as a route of exposure, demonstrated that the globus pallidus accumulates Mn concentrations in a dose-dependent fashion and ranges from 1.6- to 6.0-fold relative to control animals after occupationally relevant exposures. An intravenous injection paradigm of Mn exposure produced an approximately 5-fold increase in pallidal Mn concentrations.

13Mn can be taken up into astrocytes and neurons. showed that astrocytic uptake of Mn takes place at least in part via DMT1. Neuronal uptake of Mn involves transferrin as well as utilization of specific transporter systems such as the dopamine transporter (DAT). Intracellularly, Mn accumulates in the mitochondria, and Mn accumulation in the nucleus has been demonstrated. Utilization of divalent cation transporters such as the calcium uniporter and the positive valence of Mn ion may explain the localization of Mn to electron-rich mitochondria. Axonal transport of Mn occurs, which contributes to the observations of elevated concentrations of Mn efferent to iron-rich regions of the brain that have high concentrations of the transferrin receptor.

14recently compared the pharmacokinetics of Mn entry into and distribution throughout the brain in rodents and nonhuman primates as measured by T1-weighted MRI. That study demonstrated that for a given dose, Mn transport into nonhuman primate brain is greater than the absorption into the brain of rodents. Additionally, these authors demonstrated that the distribution of Mn throughout the brain differs between nonhuman primates and rodents. explained these differences between species as a product of Mn transport from the cerebrospinal fluid at the choroid plexus into the brain. The differences in brain anatomy between rodents and non human primates and differential positioning of brain regions such as the striatum relative to brain ventricles may drive the unique patterns of regional accumulation of Mn described in rodents and nonhuman primates. The similarities in brain structure between nonhuman primates and humans offer the opportunity to study Mn neurotoxicity in an animal model that recapitulates the pattern of human brain Mn accumulation.

15The disruption of mitochondrial activity by Mn is well documented. Mn2+ uses the calcium uniporter to gain entrance into mitochondria. In the mitochondria, the bulk of Mn is bound to the inner mitochondrial membrane or matrix proteins. This allows Mn to directly interact with proteins involved in oxidative phosphorylation. Mn has been shown in vitro to interfere with ATP synthesis, particularly by inhibiting the function of the F1ATPase and at higher concentrations by inhibiting complex I. Importantly, the speciation of Mn is a critical determinant of its interactions. Trivalent Mn inhibits complex I more potently than does divalent Mn, and Mn3+ more efficiently oxidizes intra cellular substrates such as dopamine. However, divalent Mn is the predominant species found in tissue. Mn has been associated with suppression of ATP-dependent calcium waves in astrocytes, suggesting that Mn promotes the potentially disruptive mitochondrial sequestration of calcium. Nuclear magnetic resonance spectroscopy has been used in culture to show that Mn decreases ATP/adenosine diphosphate ratios and impairs glucose metabolism and metabolic activity in the globus pallidus.

16The generation of reactive oxygen species is intimately linked to mitochondrial activities, and there is much evidence to support the role of reactive oxygen species in Mn neurotoxicity. Rats treated with Mn for 7 days by oral gavage showed increased striatal concentrations of ascorbic acid and gluta thione (GSH), antioxidants that when increased signal the presence of an elevated burden from reactive oxygen species. A later study conducted in rats exposed to Mn by inhalation confirmed the presence of markers of oxidative stress, with decreased GSH and increased metallothionine in the Mn-exposed animals. Nonhuman primate studies further substantiated the association between Mn exposure and increases in metallothionine and decreases in GSH. Addition of Mn to cultured astrocytes increases 2′,7′-dichlorofluoroscein fluorescence indicative of increased reactive oxygen species, and coincubation of N-acetylcysteine blocks Mn toxicity.

17Not only has Mn exposure been associated with markers of oxidative stress, but activation of signaling pathways involved in the response to oxidative stress has been documented as well. In PC12 cells, Mn induced the expression of nuclear factor kappa B (NFκB) and AP-1, further providing support for an oxidative basis in Mn neurotoxicity. Pretreatment with either vitamin E or a NFκB inhibitor has been shown to protect against Mn toxicity in mesencephalic cells. The intracellular consequences of Mn-induced increases in oxidative stress are becoming clearer as we learn more about the mechanisms of Mn neurotoxicity. Recent studies have shown that in dopaminergic cells, Mn can promote apoptosis by a caspase 3–dependent activation of protein kinase C delta. Mn neurotoxicity may involve oxidative stress and apoptosis, interconnected pathways that have been linked to the pathophysiology of neurodegenerative disease. Microarray data from the frontal cortex of Mn-exposed nonhuman primates shows that the expression of genes involved in apoptosis and inflammation is increased relative to control animals.

18Much research has endeavored to describe the mechanisms by which Mn produces adverse motor function outcomes reminiscent of idiopathic PD. Because Mn-exposed individuals often exhibit motor symptoms such as kinetic tremor and dystonia not typically associated with PD and because Mn-exposed individuals generally do not respond to levodopa therapy, the gold standard for PD treatment, it is believed that Mn neurotoxicity involves pathologic mechanisms distinct from those of PD. Available human autopsy data suggest that the nigrostriatal system is not degenerated after Mn exposure. Mn may cause dysfunction of the dopamine neuronal terminal that may set the stage for susceptibility to a later insult. However, the evidence for Mn-induced dopaminergic neuronal dysfunction is incomplete. It is known that in vitro Mn can promote autooxidation of dopamine, which leads to the creation of reactive dopa mine quinones. However, early rodent data offer conflicting evidence on the influence of Mn exposure on catecholamine concentrations. In nonhuman primates, high cumulative exposures to Mn > 300 mg/kg show reduced striatal concentrations of dopamine; however, studies using lower doses have demonstrated no effect of Mn on dopamine levels. Additional evidence from nonhuman primate data suggests a Mn-induced post synaptic decrease of D2-like dopamine receptor levels. However, the limited number of studies that have examined the effect of Mn in the context of chronic exposure leave the relationship between Mn, the dopaminergic system, and the development of motor dysfunction an open question.

19There is a persistent notion in the literature that a component of Mn neurotoxicity may be modified by dys regulation of excitatory glutamatergic neurotransmission. Increased glutamate levels in the brain have been documented in rodents exposed to Mn. showed that Mn increases glutamate in some areas such as the frontal cortex but decreases it in the globus pallidus, but additional research suggests that glutamate levels are not affected by Mn exposure in rodents. These inconsistencies are most likely a consequence of varied Mn doses, routes of administration, dosing intervals and durations, species-based differences in pharmacokinetics, and differences in the brain regions analyzed. In vitro studies have implicated Mn in the reduction of glutamate transporter expression and astrocytic glutamate uptake. These alterations could negatively impact synaptic glutamate metabolism and distribution. Additional limited data are available on the effect of chronic Mn exposure in nonhuman primates and its effects on the glutamatergic systems. However, available evidence suggests that nonhuman primates exposed to Mn via inhalation for 13 weeks did not have altered glutamate levels in the globus pallidus, caudate, and putamen but did express increased mRNA expression and decreased protein levels of GLAST and GLT, the two main astrocytic glutamate transporters, and GS, the enzyme that converts glutamate into glutamine, in multiple brain regions.

20Evidence for the involvement of GABAergic systems in Mn neurotoxicity stems from early observations that Mn exposure in rodents increases striatal GABA (γ-aminobutyric acid) concentrations. Later studies showed that chronic Mn administration to rodents produced an increase in striatal GABA concentrations and abolished age-dependent declines in glutamic acid decarboxylase, the enzyme that converts glutamate to GABA. Additional rodent studies further supported an association between Mn exposure and increased brain GABA concentrations. However, other rodent studies complicated this picture by showing that Mn decreases striatal and frontal cortex GABA levels. Moreover, other rodent studies show no association between GABA levels and Mn exposure. Little information is available regarding the GABAergic system in the context of Mn exposure in nonhuman primates. One study available in rhesus monkeys chronically exposed to Mn by inhalation indicates that in multiple brain regions Mn does not alter GABA levels. Another study that used monthly injections of Mn oxide in nonhuman primates for 2 years showed no effect of Mn on GABAa receptor levels.

21Accumulating evidence indicates that Mn may indirectly affect neuronal function by damaging glial cells. Autopsy data from humans exposed to Mn indicate the presence of astrocyte activation in the brain (for review, see ). These associations have been recapitulated in animal models. Astrogliosis and Alzheimer type II astrocytes have been demonstrated in nonhuman primates treated acutely and chronically with Mn. In cultured astrocytes treated with Mn, cellular swelling was observed within 24 hr of Mn exposure. Further, Mn disrupts intracellular calcium homeostasis and intercellular calcium waves in primary astrocytes. Increased inducible nitric oxide synthase (iNOS) mRNA and protein and increased release of nitric oxide have also been observed after Mn exposure in primary astrocytes, microglial cells, mesencephalic cells, and Mn-exposed mice. The nitric oxide pathway is intimately linked with NMDA receptor-mediated excitotoxic processes and microglial activation. One group has demonstrated in cultured astrocytes, in rodents, and in nonhuman primates that Mn exposure causes a decrease in astrocytic glutamate transporters and the astrocytic enzyme GS.

22Microglia are also affected by Mn exposure. Microglial cells treated with Mn readily release hydrogen peroxide. In rats treated acutely with Mn, observed an increase in the trans locator protein (18 kilodaltons) (TSPO-18kDa) (previously known as the peripheral benzodiazepine receptor). In the brain, TSPO-18kDa is a microglial and astrocytic protein that has been used as a marker of gliosis and is useful in identifying areas of brain injury and inflammation. An increase in proinflammatory genes such as tumor necrosis factor-α, iNOS, and activated inflammatory proteins such as P-p38, P-ERK, and P-JNK have been measured in primary rat glial cells after Mn exposure. Mn also potentiates lipopolysaccharide-induced increases in proinflammatory cytokines in microglial cultures and increases in nitric oxide production. This association between Mn exposure and inflammatory gene expression is supported by microarray data from the frontal cortex of Mn-exposed nonhuman primates that show that chronic Mn exposure increases of the expression of inflammatory genes such as osteopontin and the interferon-γ receptor.

23Much of the available data that inform our understanding of the mechanisms of Mn neurotoxicity is derived from rodent studies. Little work has been comprehensively conducted analyzing the effects of chronic Mn exposure in nonhuman primates. We have analyzed one of the largest cohorts of nonhuman primates ever used to study chronic Mn neuro toxicity. Experiments are still under way; however, the available data thus far support the involvement of dopaminergic nigrostriatal dysfunction, selective astrocytic modulation of glutamate processing in the globus pallidus, and multiple indices of neurotoxicity in brain regions not generally associated with Mn exposure. Here, we summarize the findings from behavioral, neuroimaging, and neuro pathologic studies that provide important insights on mechanisms suggested by previous studies and more pathways not previously thought to be operational in Mn neuro toxicity. The average blood Mn concentrations of the animals we used were in the 65–85 μg/L range. This concentration is within the upper range of blood Mn concentrations documented in human populations such as those living near Mn mining areas, in mothers and newborns and in children in communities in which MMT is used in gasoline, and as a result of parenteral nutrition.

24Because humans exposed to high levels of Mn experience behavioral abnormalities and cognitive deficits, we sought to characterize the involvement of behavioral and cognitive disturbances in nonhuman primates exposed to 3.3–5.0 mg Mn/kg/week for approximately 10 months. Animals (cynomolgus macaques) were trained on a series of tasks that measure behavior and cognitive performance. After approximately 20 weeks of exposure and continuing throughout the duration of the study, the Mn-exposed animals developed a small but statistically significant increase in the rate of PD-like behaviors on a scale developed to assess parkinsonian behaviors in non human primates. Fine motor skills were assessed by an easy task and by a difficult task. Although Mn exposure had no effect on the easy task, by the end of the observation period the animals performed poorly on the difficult task. These observations are consistent with documentation of humans exposed to Mn occupationally or as a consequence of treatment with total parenteral nutrition who suffer cognitive deficits and also perform poorly on tests of fine motor ability. The Mn-exposed animals also expressed stereotypic and compulsive-like behaviors that may be explained by involvement of dysfunctional frontostriatal circuitry.

25Few data are available that comprehensively characterize Mn neurotoxicity throughout the brain, especially in the context of chronic exposure in nonhuman primates. Therefore, we used 1H-magnetic resonance spectroscopy (1H-MRS) to longitudinally assess the potential toxic effects of chronic Mn exposure on levels of brain metabolites that are thought to reflect various aspects of neuronal and glial functioning in the brain. The concentrations of the brain metabolites creatine, N-acetylaspartate (NAA), choline, and myoinositol were characterized by 1H-MRS in the parietal cortex, striatum, thalamus, and frontal white matter. The globus pallidus could not be assessed because the iron content interferes with acquisition of the 1H-MRS signal. Data were normalized to creatine content in a particular brain region. NAA is a neuron-specific metabolite, myoinositol is used as a glial cell marker, and choline is a substituent of cell membranes. Reductions in NAA/creatine are reflective of neuronal loss and/or dysfunction, alterations in myoinositol/crea tine are suggestive of glial activation or damage, and modulation of choline/creatine can provide important information on membrane phospholipids in the context of myelin breakdown. Whereas the ratio of choline/crea tine and myoinositol/creatine were unchanged by chronic Mn exposure, the ratio of NAA/creatine was significantly reduced in the parietal cortex of Mn-exposed animals relative to baseline. A near-significant (p = 0.055) decrease was observed in frontal white matter. Reduced NAA levels also occur in pathologic conditions such as stroke and Alzheimer’s disease, and the reduction of NAA is reflective of neuronal dysfunction or degeneration. The in vivo analy sis of Mn-exposed animals by MRS further underscores observations that although Mn accumulates to the greatest extent in the globus pallidus, other regions of the brain that may accumulate Mn to a lesser extent can still be vulnerable to Mn neurotoxicity.

26Given the similarities in some of the symptoms of Mn intoxication and the clinical symptoms of PD, as well as available supporting animal research implicating the dopaminergic system in Mn neurotoxicity, we have characterized multiple aspects of the dopaminergic synapse in nonhuman primates exposed to Mn both acutely and chronically using positron emission tomography (PET) and ex vivo methods.

27In an early study, we examined the effect of acute Mn exposure on DAT levels in baboons as assessed by PET and post mortem analysis. Acute Mn exposure caused a transient increase in DAT levels within 1 week of exposure that normalized after approximately 1 month. Further, it was shown that Mn inhibits [3H]-dopamine uptake in rat striatal synaptosomes. Although Mn has no effect on the affinity (Kd) of the cocaine analog [3H]-WIN 35,428 binding to DAT, we observed a statistically significant 30% reduction of the Bmax, indicating that although Mn has no effect of the affinity of the DAT ligand, it reduces the available number of binding sites. This suggested that Mn may affect the presynaptic dopaminergic neuronal terminal through modulation of DAT function and levels.

28We have also used neuroimaging and neuropathologic techniques to comprehensively probe the effect of chronic Mn exposure in cynomolgus macaques on multiple parameters of the dopaminergic synapse. The most dramatic change observed with chronic Mn exposure was a reduction in the capacity of striatal dopamine release, measured by PET with amphetamine (AMPH) challenge. Importantly, an animal receiving the highest dose of Mn (8.3–10.0 mg/kg/week) that expressed dystonia and dyskinesias also exhibited the largest decline in AMPH-induced dopa mine release from a baseline of 44% to 8.0% release after only 7 weeks of Mn exposure. These findings indicate that Mn-induced motor function deficits are caused by mechanisms that inhibit dopamine release in the presence of normal levels of dopa mine and an intact nigrostriatal dopa mine system. This view is in contrast to theories suggesting that Mn neurotoxicity is mediated by an effect on post-synaptic dopamine receptors.

29Importantly, the use of control animals that received no Mn but did receive repeated AMPH injections as part of the PET imaging protocol provided insight on the activity of Mn at the dopaminergic synapse. Although AMPH administration was necessary to characterize the in vivo capacity for dopamine release by PET, AMPH itself causes alterations in the integrity of dopaminergic neuronal terminals. However, this image-control group showed greater deficits in DAT, vesicular monoamine transporter type 2, and dopamine and its metabolites relative to Mn-exposed animals that received equivalent amounts of AMPH normalized to body weight. This suggests that Mn may directly interact with DAT and interfere with AMPH uptake into dopaminergic neurons. This is consistent with previously published reports that Mn is a substrate for DAT and that Mn inhibits binding of a cocaine analog to DAT and inhibits dopamine uptake in striatal synaptosomes. These findings strongly suggest that the documented motor function abnormalities in Mn-exposed nonhuman primates at Mn doses given in this study are mediated by inhibition of dopa mine release in the striatum and shed new light on the molecular basis of Mn-induced parkinsonism.

30Evidence of the involvement of the glutamate and GABA neurotransmitter systems can be traced back three decades and involves studies conducted in primary cell culture models, rodents, and nonhuman primates. However, the culture and rodent data has been contradictory and inconclusive (see “Glutamatergic synapses” and “GABAergic synapses” above). Until recently, little information has been available in nonhuman primates on glutamate and GABA neuro trans mission throughout the brain. Therefore, we also recently studied the effect of Mn on glutamatergic and GABAergic systems throughout the nonhuman primate brain. Chronic Mn exposure did not alter glutamate, GABA, or the glycine (a coagonist for NMDA glutamate receptors) levels in frontal white matter, globus pallidus, caudate, putamen, hippocampus, thalamus, cortex (frontal, motor, somatosensory, parietal, visual), cerebellar lobules, and deep cerebellar nucleus (Burton NC, unpublished data). These findings are consistent with a recent report that suggested that chronic Mn exposure in nonhuman primates does not alter the concentration of glutamate or GABA in the globus pallidus, caudate, or putamen. In our recent study (Burton NC, unpublished data), Mn did not consistently promote a change in levels of NMDA receptors, GABAa receptors, or glutamate transporters. However, Mn did cause a reduction in GS in the globus pallidus, a glial-specific enzyme that converts glutamate into glutamine as part of the glutamate recycling process. The decrease in GS levels in Mn-exposed nonhuman primates is consistent with observations made in another cohort of nonhuman primates exposed to Mn via inhalation. Reductions in GS levels can cause dysregulation of the glutamate recycling pathway, and by mass action, a reduction in the conversion of glutamate into glutamine could lead to an increase in intracellular glutamate concentrations in astrocytes and a potential spillover into the synapse with the potential to cause excitotoxicity in the globus pallidus.

31The neurologic sequelae of exposure to high levels of Mn occur in multiple stages. The early phase of Mn intoxication involves a psychiatric component, characterized in part by irritability, apathy, and psychosis, and is sometimes called Mn mania. Deficits in measures of executive function, such as short-term memory and computational ability, are also early manifestations of Mn neurotoxicity. Studies show that Mn accumulates in the frontal cortex, and it has been suggested that Mn may alter cognitive domains mediated by the frontal cortex and subcortical structures. Toward this end, we performed microarray analysis of frontal cortex tissue from naïve control animals and animals exposed to 3.3–5.0 mg Mn/week. The results indicated that Mn promoted modulation of genes related to inflammation, apoptosis, cell cycle, protein turnover and folding, synaptic function, and cholesterol homeostasis. These observations suggested that Mn may promote inflammation and cell death in the frontal cortex. Further, the activation of cell cycle machinery in post-mitotic cells such as neurons disrupts normal cellular processes and has been implicated in dysfunction and cell death in Alzheimer’s disease. An imbalance in cholesterol homeostasis is associated with the development of pathologic processes present in Alzheimer’s disease. In addition, the most highly up-regulated gene was amyloid beta (A4) precursor-like protein 1 (APLP1), which is a member of the amyloid precursor protein involved in copper homeostasis and the regulation of the toxic peptide beta amyloid (Aβ)1–42. The increase in APLP1 gene expression was confirmed by immunohistochemistry and led to the further analysis of A β levels in the frontal cortex. Unexpectedly, in the juvenile animals used in this study, extracellular diffuse aggregation of A β was observed in the frontal cortex of Mn-exposed animals, a condition normally observed in the aged brain. This suggested that Mn accelerates the aging process and promotes Alzheimer’s-like neuropathology in the frontal cortex. The presence of neurodegenerative changes in the frontal cortex of Mn-exposed animals was confirmed by the presence of neuronal agyrophilic inclusions using silver staining and by morphologic changes consistent with apoptotic stigmata. Neurons of the frontal cortex of Mn-exposed animals expressed hypertrophic nuclei and vacuolar cell bodies suggestive of ongoing pathology. Hydroxynonenol staining, a marker of lipid peroxidation, was associated with apoptotic cells. Astrogliosis was also observed in the Mn-exposed animals. APLP1 is regulated by the tumor suppressor p53, as are many of the genes identified in the microarray analysis. Increased p53 and APLP1 staining colocalized with cells showing morphologically apoptotic features, suggesting the possibility that Mn may function through p53 to affect changes in APLP1 that promote extracellular A β accumulation. In addition, Mn exposure also caused an increase of approximately 30% in copper concentrations in the frontal cortex. Accumulation of brain copper has been associated with A β plaques in the AD brain. Dysregulation of copper homeostasis may be one additional mechanism by which Mn promotes neurodegenerative processes. Further studies are warranted in an appropriate cell culture system to determine the mechanistic relationship among Mn exposure, p53 levels and activity, APLP1, A β, and copper homeostasis. The gene expression changes and markers of neurodegeneration in the frontal cortex may help to explain not only the subtle cognitive deficits previously characterized in these Mn-exposed animals, but also some of the early manifestations of Mn neurotoxicity in humans related to working memory and neuropsychiatric behaviors.

32Much of the research that has informed the understanding of the mechanisms of Mn neurotoxicity is based on data generated from cell culture and research conducted in rodents. Possible mechanisms suggested by rodent data must be confirmed in higher species, especially in light of recent pharmacokinetic studies suggesting that brain Mn uptake and distribution may be dramatically different between primates and rodents. Unfortunately, studies conducted in nonhuman primates are few. The multidisciplinary study described within is the largest cohort of nonhuman primates that has been used to characterize the effects of chronic Mn exposure throughout the brain on neuroimaging, pathology, multiple neurotransmitter systems, behavior, and cognition. These data provide corroborative support that chronic Mn exposure does not alter total levels of dopamine, glutamate, or GABA. However, Mn may reduce GS levels and produce a glial-specific effect on the glutamatergic system in the globus pallidus by interfering with the recycling of glutamate, which may ultimately promote a redistribution of glutamate at the synapse. Additionally, PET studies demonstrated that Mn produces its most substantial effect on the nigrostriatal dopamine system not by producing its degeneration, but by disrupting the capacity to release dopamine. Last, MRS documented neuronal loss and/or dysfunction in the parietal cortex, and postmortem analysis showed neurodegeneration in the frontal cortex. These cortical regions were previously unsuspected to be involved in Mn neurotoxicity because they accumulate relatively low levels of Mn compared with the globus pallidus. These findings indicate that Mn neurotoxicity is not simply defined by the amount of Mn that a brain region accumulates but by the intrinsic vulnerability to injury by Mn.

33Therefore, the continuum of dysfunction caused by Mn from psychiatric to cognitive to motor abnormalities cannot be explained by dysregulation of basal ganglia structures alone. These findings suggest that historical views of Mn neurotoxicity should be revisited to account for the spectrum of Mn-induced neurologic dysfunction.