Mn is a well known neurotoxic metal as it accumulates in basal ganglia and induces manganism, a disease with common manifestations with Parkinson's disease.
In this research we examine the biochemical pathways of Mn toxicity on neurons, how the metal is absorbed, how it accumulates in basal ganglia and possible models of treatment. We also make a comparison with Parkinson's disease.
Introduction
Manganism is an altered neurological dysfunction of the basal ganglia and symptoms often overlap with Parkinson's disease, so incorrect diagnosis can occur.
In general, the syndrome associated with manganese toxicity can be divided into three stages:
1) INITIAL STAGE or ACUTE exposition (manganese madness): is marked by both emotional and cognitive disturbances characterized by excessive excitement, mood changes, irritability, compulsive behavior and intellectual deficits. Fine motor coordination also appears to be affected at the early stages of the disease. There is some evidence that removal of a subject from exposure to Mn at this early stage may lead to a reversal of symptoms, though some of the intellectual and cognitive deficits may remain.
2) SECOND STAGE: continued exposure subsequently leads to more significant neurological injury including anorexia, weakness, more severe psychotic behavior, slurred speech, mask-like face, and a general clumsiness.
3) FINAL STAGE: is characterized by more acute and incapacitating neurological impairment including limb rigidity, mild tremors, dystonic, gait disturbance, cock-like walk, excessive salivation, sweating and disturbance of balance.
When compared to Parkinson's disease, Mn toxicity is usually associated with milder tremors at rest and subjects stand in a more upright position.
Besides, many of the initial extrapyramidal symptoms associated with Mn toxicity most likely reflect differences in the primary wound site within the CNS for the two disorders.
Toxic parkinsonism differ from the idiopathic form for the early age of onset an for the reduced worsening of the clinical situation (specifically, after elimination of the exposure, symptoms tend to reach a steady-state).
Mn preferentially accumulates in the globus pallidus. There is evidence that Mn is also deposited, though to a lesser extent, in the substantia nigra.
An useful diagnostic criterion to distinguish between manganism and Parkinson's disease consist in the fact that L-DOPA treatment is basically ineffective as treatment for manganism.
Although there is a direct relationship between the extent of exposure to Mn and the severity of the symptoms, other factors such as environment, nutrition, and genetic may also influence and facilitate the critical biochemical processes provoking tissue damage and the clinical manifestations observed.
Sources of exposure:
1. food
2. water
3. air
Among these sources of exposure, inhalation of Mn is the most threatening one for two main reasons. Firstly, there is evidence of an higher rate of Mn absorption through lungs rather than other pathways. Secondly brain uptake of the metal occurs both through direct blood uptake of transferrin-bound Mn and trough anterograde axonal transport via olfactory neurons
Mechanism of Mn-Induced Toxicity
Mn-induced neurotoxicity is mediated by disruption of mitochondria initiating both apoptosis and necrotic cell death via formation of highly reactive oxygen species.
Many of the classical signaling pathways associated with programmed cell death are seen after Mn treatment including increased TUNEL staining, internucleosomal DNA cleavage, activation of the JNK and p38 (stress activated protein kinase), activation of caspase-3 like activity, and caspase-3 dependent cleavage of PARP.
Inside the cell, Mn is rapidly taken up by the mitochondria where it promotes calcium accumulation by inhibiting both the sodium-dependent and sodium- independent exporter, thus activating the permeability transition pore (PTP).
Mn also interferes with oxidative phosphorylation by inhibiting both mitochondrial F1-ATPase and complex I, resulting in depletion of ATP and production of reactive oxygen species (ROS).
Mn action on SNC :
1. Accumulation of Mn in the globus pallidus neurons mitochondria;
2. Mn inhibition of glutamate transport leading to increase synaptic levels of glutamate;
3. Increased uptake of Mn in pallidal neurons by activated glutamate channels;
4. Production of ROS and interruption of oxydative chain;
5. Dopamine autoxidation to 6-hidroxydopamine;
6. Reduction of Catalases and GSH-peroxydases;
7. Depletion of cellular GSH;
8. Augmented production of neurotransmitters;
9. Toxic products accumulation;
10. Functional exhausting of neurons and cell death.
Unlike Parkinson’s disease, manganese preferentially damages the GABAminergic neurons in the globus pallidus rather than dopaminergic neurons.
The most likely explanation is that only the globus pallidus selectively receives glutaminergic input from the subthalamic nuclei. Glutamate being an excitatory neurotoxin is potentially capable of exacerbating the actions of Mn as the toxic mechanisms of the two are similar.
This is confirmed by the studies of Brouillet et al. in 1993 and Xu et al. in 2010 demonstrating that the noncompetitive NMDA receptor antagonist, MK-801, is capable of blocking lesions produced by intrastriatal injections of Mn.
Glutamate toxicity is caused by an increase in intracellular Ca2+ initiated by binding of glutamate to its ionotropic receptor. The accumulated intracellular Ca2+ is subsequently taken up into the mitochondria via the Ca2+ uniporter where it is stored and where it causes changes in mitochondrial membrane permeability leading to formation of ROS and possibly other intracellular cytotoxic signals.
In addition, glutamate toxicity also involves activation of several kinases including ERK1/ 2, JNK, and p38. The involvement of these kinases in the toxic action of glutamate is evidenced by the fact that inhibitors of ERK phosphorylation and p38 kinase activity prevent glutamate-induced cell death. Since Mn similarly stimulates the phosphorylation of these kinases, it is conceivable that the combined activation of these kinases further contributes to the selective impairment of neurons within the globus pallidus.
Mitochondrial disfunction induced by Mn consists in:
- Inhibition of energy transduction: enzymes of the electron transport chain such as Succinate Dehydrogenase, NADH Dehydrogenase and NADH-CytC reductase (Vulnerability of mitochondrial complex I in PC12 cells exposed to manganese, 1995) are inhibited by Mn. Husain et al. shown inhibition of Citocrome C oxidase following administration of intraperitoneal MnCl2.
A reduction in enzyme content or catalytic rate could arise, both due to direct interactions between Mn and those enzyme and to mutations of the mitochondria genome (see below).
- Mutation of the mitochondrial genome: Mn is a well known mutagen (observations on S. Cerevisae and E. Coli), like other divalent cations. Prevalence of mutagenesis in mitochondrial rather than nuclear genome is a result of the higher rate of accumulation of Mn in mitochondria and of the less efficient DNA repair system in those organelles.
Most frequent mutations are deletion and point mutations.
- Generation of free radicals and DA autoxidation: inhibition of the electron transport chain increases the production of ROS. The situation is exacerbated by the Mn-induced reduction of the activity of GSH-peroxidase and SOD (Selective vulnerability of glutathione metabolism and cellular defense mechanisms in rat striatum to manganese 1988).
Mn neurotoxicity can be mediated also by nonenzymatic autoxidation of catecholamines to 6-hydroxydopamine (6-OHDA). This would explain DA depletion from specific regions of the brain following Mn exposure (Selective lesions by manganese and extensive damage by iron after injection into rat striatum or hippocampus 1994).
Mn transporters
Limiting steps for Mn toxicity are related to its rate of uptake into the body and efficacy of the various carrier-mediated transport processes to deliver the Mn to the GABAminergic neurons in the globus pallidus.
There are various processes for the transport of Mn across cell membranes:
1) Divalent metal transporter 1 (DMT1);
2) Voltage gated Ca2+ channel;
3) the Ca2+-coupled ionotropic glutamate receptor;
4) Solute-carrier-39 (SLC39) proteins;
5) ZIP8 and ZIP14.
Among these, DMT1 is generally considered to be the major transporter for Mn although both of the two ZIP proteins are reported to have a similar affinity for the divalent form of the metal.
DMT1 related transport involves either a transferrin dependent or transferrin independent pathway.
The process of maintaining iron stores in the body begins with ferrous ion being taken up across the brush border membrane within the intestinal via a non transferrin dependent process requiring DMT1.
Passage of iron out of the enterocyte requires the transport protein ferroportin, and the ferrooxidase, hephaestin, which oxidize the iron before it enters the circulation.
Ferroportin activity is regulated by the hepatic hormone, hepcidin, which is responsible for its internalization, phosphorylation, and proteasomal degradation within enterocytes. Iron is tightly bound to transferrin within the plasma.
Mn follows a similar pattern although the enzymes responsible for its transport out of the enterocyte and its oxidation before entering the blood stream have not been identified yet.
Trivalent Mn is preferentially bound to transferrin (Manganese transport via the transferrin mechanism 2013) which escapes the first pass elimination in the liver. Normally, there is little competition between Mn and iron for binding to transferrin as iron only occupies approximately 30% of the binding sites. A portion of the blood Mn may also be bound to a2-macroglobulin but this complex is considerably weaker and Mn can readily dissociate from the complex and be removed by the liver.
Uptake into cells of both iron and Mn is accomplished by transferrin complex binding to the transferrin receptor (TfR) on the cell surface and the resulting metal/Tf/TfR complex internalized within endosomal vesicles.
These vesicles undergo acidification via a hydrogen ion ATPase pump liberating the iron and Mn from the complex. In the case of iron, the released trivalent ion is subsequently reduced by the ferroreductase, STEAP 3, and transported across the endosomal membrane by a hydrogen ion symporter again dependent on DMT1. Identification of a specific reductase for Mn has yet to be determined.
Four isoforms of DMT1 have been identified in mammalian cells encoded by a single gene. All are 12 putative membrane-spanning domain proteins but differ both in their N- and C-terminal residues with two mRNA isoforms possessing a stem loop iron response element (IRE) motif downstream from the stop codon on the message. The mRNA isoforms possessing the IRE in the 30-UTR are referred to as the +IRE isoforms of DMT1 whereas the species lacking the IRE are referred to as the -IRE species.
When intracellular iron is low, the iron response proteins, IRP1 or 2, presumably can bind to the IRE stabilizing the +IRE forms of the message. IRE presence can potentially regulate expression of the +IRE species of DMT1 though its overall impact may be secondary to other regulatory mechanisms.
Several studies indicate that the four isoforms of DMT1 are differentially localized in organelles and in distinct endosomal vesicles within cells. The unique and selective distribution of the different isoforms of DMT1 implies they likely have discrete physiological functions and, most importantly, diverse regulatory processes controlling their overall expression which are likely to be dependent on their roles in maintaining nutritional requirements of iron and other essential divalent metals.
When plasma levels of iron are low Mn accumulation increases, as serum transferrin levels, which carries manganese ions, increase (Low iron stores are related to higher blood concentrations of manganese, cobalt and cadmium in non-smoking, Norwegian women in the HUNT 2 study 2010; Increased whole blood manganese concentrations observed in children with iron deficiency anaemia 2012).
The Inflammatory Response in Regulating DMT1 Expression and Iron Accumulation
There are a lot of different regulatory mechanisms of DMT1 expression that have the potential to protect cells under a variety of stress-related or inflammatory conditions, including exposure to Mn, in which there is a release of a diverse array of proinflammatory cytokines including TNFa, interleukin-1b, and IFN-c (Manganese-induced potentiation of in vitro proinflammatory cytokine production by activated microglial cells is associated with persistent activation of p38 MAPK 2008).
The common underlying element in essentially all stress-related conditions provoking an inflammatory response is the nuclear factor, NF-kB. Thus, NF-kB may be the universal regulator of DMT1 expression in stress-related injury and account for excess deposition of iron and/or manganese at lesioned sites within the CNS (Nitric oxide transcriptionally down-regulates specific isoforms of divalent metal transporter (DMT1) via NF-kappaB 2006).
Changes in DMT1 expression can result from both transcriptional and post-translational processes which are likely to have a profound effect on the accumulation of iron and other divalent metals in the CNS during periods of stress and other inflammatory processes (Inflammation alters the expression of DMT1, FPN1 and hepcidin and causes iron accumulation in central nervous system cells 2013). This correlates with the observation of abnormal iron accumulation in a variety of neurodegenerative disorders such as Friedreich ataxia, multiple sclerosis, amyotrophic lateral sclerosis, Parkinson’s, Alzheimer’s, and Huntington’s disease and even Prion’s disease (The crucial role of metal ions in neurodegeneration: the basis for a promising therapeutic strategy 2005).
Although iron is unlikely to be the primary insult eliciting these neurological conditions, it has been suggested that the accumulated iron has the potential to contribute to and possibly accelerates the progressive damage seen in the lesioned tissue. Because expression of DMT1 may be the rate limiting step controlling iron accumulation, these studies link this transporter to progressive impairment seen in these diseases.
The same rationale may occur for Mn accumulation in humans chronically exposed to high atmospheric levels of the metal. It is feasible that the initial inflammatory distress initiated by accumulated Mn within the globus pallidus may result in a positive feedback loop instigated by stimulation of the cytokine/NF-kB pathway leading to increased expression of DMT1 and the subsequent enhanced uptake of Mn and its accumulation in nervous system cells.
Relationship Between Mn Exposure and Parkinsonism: genetics
Is well established that Mn exposure relates with neurological extra-pyramidal syndromes.
Not all workers in occupations of high Mn exposure (such as welders) develop Mn toxicity symptoms. Thus some individual may have a genetic predisposition accelerating the onset of the disorder.
Several studies (Alpha-synuclein is part of a diverse and highly conserved interaction network that includes PARK9 and manganese toxicity 2009; Parkin regulates metal transport via proteasomal degradation of the 1B isoforms of divalent metal transporter 1 2012) prove a genetic link between Mn exposure and the development of parkinsonisms as both of them involve mutations in proteins associated with early onset of the disorder.
The first of these studies relates with the post-translational regulation of DMT1 by the proteasomal pathway and the observation that the ubiquitin E3 ligase, PARKIN, which associates with early onset of Parkinson’s disease, is capable of protecting against Mn toxicity.
In this case, protection against Mn toxicity is likely caused by the fact that parkin is the E3 ligase responsible for the ubiquitination of DMT1.
During inflammatory conditions there are two processes which mutually function to potentiate expression of DMT1. As indicated, any trauma to the CNS whether it is induced by Mn or a result of a neurological insult will stimulate cytokine production by microglial cells and the subsequent activation of NF-kB.
Mutations in the parkin gene would invariably result in the inability of dopaminergic cells to efficiently degrade newly synthesized DMT1 via the proteasomal pathway resulting in the net effect of increased accumulation of Mn within the neuron.
Proteasomal pathway is reduced during the normal aging process, this situation result in diminished capacity to inactivate stress-induced increases in DMT1 expression again augmenting Mn accumulation.
The second genetic disorder linking parkinsonism with Mn toxicity relate to overexpression of the protein a-synuclein that induce formation of toxic oligomeric species in dopaminergic neurons.
Normally, A-SYNUCLEIN undergoes ubiquitination by Parkin before being degraded by the proteasomal pathway. There is a strong genetic interaction between a-synuclein and PARK9, a yeast ortholog of human Parkinson’s disease-linked gene ATP13A2 (may be a Mn transporter): co-expression of PARK9 in animals overexpressing a-synuclein was shown to protect against dopaminergic neuron loss. Most importantly, yeast PARK9 protected cells from manganese toxicity suggesting a connection between Parkinson’s disease and manganism.
Other situations that can promote Mn accumulation in basal ganglia is the augmented absorption of the metal due to anemia (if related to iron deficiency, see above), low albumin/globulin ratio, vitamines deficits, ethanol ratio and conditions of liver diseases.
Neuropathology
Olanow et al. studied the effects of Mn accumulation in basal ganglia on three adult rhesus monkeys exposed to high Mn levels through intravenous injections of MnCl2. Only two monkeys developed symptoms of Mn toxicity.
On gross examination a decoloration of the GP can be noticed.
On microscopic examination gliosis can be noticed both in GP and, even if in a less degree, in SNr. The gliosis is associated with an increased number of astrocytes showing an enlarged and irregular nucleus.
Both fenomena were presented by the three monkeys, but were more pronounced in the clinically affected ones.
The study also confirmed inefficacy of L-Dopa treatment on affected animals, thus classical PD can be separated by Mn-induced parkinsonism based on clinical manifestation, response to levodopa and neuroimaging studies.
Treatment with chelating agents: EDTA and PAS
CaNa2EDTA
This synthetic polyaminocarboxylic acid binds divalent and trivalent metal ions forming a stable chelate that is excreted in unrine.
Subministration of CaNa2EDTA does not induce hypocalcemia. EDTA is considered to be a nephrotoxicant, maybe due to inappropriate dosage of the drug (nephropoxicity should however be considered).
According to a study by Herrero Hernandez, Discalzi, Valentini, Pira et al. chelating treatment with CaNa2EDTA seems to be helpful in treatment of Mn poisoning as it reduces accumulation of ion in globus pallidus and substantia nigra.
Moreover De Paris and Caroldi observed an inhibiting action of Dopamine beta-hydroxylase and Dopamine autoxidation lead by EDTA, reducing tissue toxic effects of manganese.
Para-AminoSalicylic Acid
Jiang, Mo et al. in 2006 reported a 17-year follow-up study of effective treatment of occupational Mn parkinsonism with sodium para-aminosalicylic acid (PAS).
The patient, female and aged 50 at the time of treatment, was exposed to airborne Mn for 21 years (1963-1984). The patient had palpitations, hand tremor, lower limb myalgia, hypermyotonia, and a distinct festinating gait. She received 6 g PAS per day through an intravenous drip infusion for 4 days and rested for 3 days as one therapeutic course. Fifteen such courses were carried out between March and June 1987.
At the end of PAS treatment, her symptoms were significantly alleviated and handwriting recovered to normal.
Follow-up examination at age 67 years (in 2004) showed a general normal presentation in clinical, neurologic, brain magnetic resonance imaging, and handwriting examinations with a minor yet passable gait.
This case study suggests that PAS appears to be an effective drug for treatment of severe chronic Mn poisoning with a promising prognosis.
Authors:
- Tommaso Pierani and Luca Rodano.
Other References:
