Paolo Zatta

Roles of the metallothionein family of proteins in the central nervous system.

Metallothioneins (MTs) constitute a family of proteins characterized by a high heavy metal [Zn(II), Cu(I)] content and also by an unusual cysteine abundance. Mammalian MTs are comprised of four major isoforms designated MT-1 trough MT-4. MT-1 and MT-2 are expressed in most tissues including the brain, whereas MT-3 (also called growth inhibitory factor) and MT-4 are expressed predominantly in the central nervous system and in keratinizing epithelia, respectively. All MT isoforms have been implicated in disparate physiological functions, such as zinc and copper metabolism, protection against reactive oxygen species, or adaptation to stress. In the case of MT-3, an additional involvement of this isoform in neuromodulatory events and in the pathogenesis of Alzheimers disease has also been suggested. It is essential to gain insight into how MTs are regulated in the brain in order to characterize MT functions, both in normal brain physiology, as well as in pathophysiological states. The focus of this review concerns the biology of the MT family in the context of their expression and functional roles in the central nervous system.

The role of metals in neurodegenerative processes: aluminum, manganese, and zinc.

Until the last decade, little attention was given by the neuroscience community to the neurometabolism of metals. However, the neurobiology of heavy metals is now receiving growing interest, since it has been linked to major neurodegenerative diseases. In the present review some metals that could possibly be involved in neurodegeneration are discussed. Two of them, manganese and zinc, are essential metals while aluminum is non-essential. Aluminum has long been known as a neurotoxic agent. It is an etiopathogenic factor in diseases related to long-term dialysis treatment, and it has been controversially invoked as an aggravating factor or cofactor in Alzheimers disease as well as in other neurodegenerative diseases. Manganese exposure can play an important role in causing Parkinsonian disturbances, possibly enhancing physiological aging of the brain in conjunction with genetic predisposition. An increased environmental burden of manganese may have deleterious effects on more sensitive subgroups of the population, with sub-threshold neurodegeneration in the basal ganglia, generating a pre-Parkinsonian condition. In the case of zinc, there has as yet been no evidence that it is involved in the etiology of neurodegenerative diseases in humans. Zinc is redox-inactive and, as a result of efficient homeostatic control, does not accumulate in excess. However, adverse symptoms in humans are observed on inhalation of zinc fumes, or accidental ingestion of unusually large amounts of zinc. Also, high concentrations of zinc have been found to kill bacteria, viruses, and cultured cells. Some of the possible mechanisms for cell death are reviewed.

Aluminium(III) as a promoter of cellular oxidation

Aluminium has been known as a neurotoxic agent to experimental animals since the last century (Arch. Exp. Pharmacol. 40 (1897) 98). However, great interest arose in it bioinorganic chemistry as well biology when it was demonstrated to be the causative agent in pathologies related to the long-term dialysis treatment of uremic subjects with renal failure (Life Chem. 11 (1994) 197), and as a potential etiopathogenic cofactor for several neurodegenerative diseases. The inorganic biochemistry of aluminium is still largely to be discovered. In this review the pro-oxidative property of aluminium toward biological membrane will be presented and its implications in involvement in human pathology will be discussed in an interdisciplinary frame from the bioinorganic point of view.

In vivo and in vitro effects of aluminum on the activity of mouse brain acetylcholinesterase

Cholinesterases are a large family of enzymatic proteins widely distributed throughout both neuronal and non-neuronal tissues. In Alzheimers disease (AD), analytical as well as epidemiological studies suggest an implication of an abnormal focal accumulation of aluminum in the brain. In this devastating disease, aluminum may interfere with various biochemical processes including acetylcholine metabolism, and can thus act as a possible etiopathogenic cofactor. Acetylcholinesterase (AChE) exists in several molecular forms that differ in solubility and mode of membrane attachment rather than in catalytic activity. Mice were treated orally with aluminum chloride or aluminum lactate (Al(lac)(3)), and AChE activity in their brain homogenates was then assayed. Results showed that this in vivo treatment augmented the activity of the enzyme. An activating effect was also observed in vitro, when the aluminum compounds were added directly to mouse brain homogenates. However, the activating effect observed in vivo was much more marked than that observed in vitro. In addition, the activation produced by Al(lac)(3) was higher than that obtained after aluminum chloride treatment. Kinetics measurements of AChE activity in the absence and presence of treatment with aluminum both in vivo and in vitro are reported. The influence of the metal speciation on enzymatic activity is discussed in relation to a possible implication of aluminum in some neurodegenerative diseases.

Metal Ion Physiopathology in Neurodegenerative Disorders

Metal dyshomeostasis in the brain (BMD) has often been proposed as a possible cause for several neurodegenerative disorders (NDs). Nevertheless, the precise nature of the biochemical mechanisms of metal involvement in NDs is still largely unknown. Mounting evidence suggests that normal aging itself is characterized by, among other features, a significant degree of metal ion dysmetabolism in the brain. This is probably the result of a progressive deterioration of the metal regulatory systems and, at least in some cases, of life-long metal exposure and brain accumulation. Although alterations of metal metabolism do occur to some extent in normal aging, they appear to be highly enhanced under various neuropathological conditions, causing increased oxidative stress and favoring abnormal metal–protein interactions. Intriguingly, despite the fact that most common NDs have a distinct etiological basis, they share striking similarities as they are all characterized by a documented brain metal impairment. This review will primarily focus on the alterations of metal homeostasis that are observed in normal aging and in Alzheimer’s disease. We also present a brief survey on BMD in other NDs (Amyotrophic Lateral Sclerosis, Parkinson’s, and Prion Protein disease) in order to highlight what represents the most reliable evidence supporting a crucial involvement of metals in neurodegeneration. The opportunities for metal-targeted pharmacological strategies in the major NDs are briefly outlined as well.

Metallothioneins are highly expressed in astrocytes and microcapillaries in Alzheimer's disease

One of the neuropathological characteristics of Alzheimers disease is the presence of a large number of reactive astrocytes, often, but not always, associated with senile plaques. The factors responsible for such an activation are as yet totally unknown. Other characteristic features of this disease such as betaA4 amyloid accumulation, senile plaques and neurofibrillary tangles represent well known pathological phenomena. Some studies suggest that betaA4 plays a major role in the reactive astrocytosis characteristic of Alzheimers disease. In the normal human brain, metallothionein isoforms I and II are expressed in astrocytes but not in neurons. In the present study, we used anti-metallothionein antibodies to detect cells expressing metallothioneins isoforms I and II in normal and Alzheimers disease (AD) brain sections. Results showed that expression of these proteins in the cortex, cerebral white matter and cerebellum is a relevant anatomopathological characteristic of Alzheimers disease. Analysis of Alzheimers disease brain sections revealed high expression of metallothioneins I/II in astrocytes and microcapillaries, and in the granular but not the molecular layer of the cerebellum. Furthermore, metallothionein expression can be used as a marker to identify subtypes of astrocytes.

Role of Metal Ions in the Aβ Oligomerization in Alzheimers Disease and in Other Neurological Disorders

Neurodegeneration is a complex and multifaceted process leading to many chronic diseased states. Neurodegenerative disorders include a number of different pathological conditions, like Alzheimers and Parkinsons diseases, which share similar critical metabolic processes, such as protein aggregation, which could be affected by some metal ions. A huge number of reports indicate that, among putative aggravating factors, metal ions (Al, Zn, Cu, Fe) could specifically impair protein aggregation of Abeta, prion protein, ataxin, huntingtin, etc. and their oligomeric toxicity. While studying the molecular basis of these diseases, it has become clear that protein conformation plays a critical role in the pathogenic process. In this review, we will focus on Alzheimers disease and on the role of metal ions, specifically aluminium, in affecting amyloid aggregation, oligomerization and toxicity.

Aluminum complexing enhances amyloid β protein penetration of blood-brain barrier

A significant co-morbidity of Alzheimers disease and cerebrovascular impairment suggests that cerebrovascular dysregulation is an important feature of dementia. Amyloid beta protein (Abeta), a relevant risk factor in Alzheimers disease, has neurotoxic properties and is thought to play a critical role in the cognitive impairments. Previously, we demonstrated that the 42mer of Abeta (Abeta42) complexed with aluminum (Al-Abeta42) is much more cytotoxic than non-complexed Abeta42. The level of Abeta in the brain is a balance between synthesis, degradation, and fluxes across the blood-brain barrier (BBB). In the present paper, we determined whether complexing with aluminum affected the ability of radioactively iodinated Abeta to cross the in vivo BBB. We found that the rates of uptake of Al-Abeta42 and Abeta42 were similar, but that Al-Abeta42 was sequestered by brain endothelial cells much less than Abeta42 and so more readily entered the parenchymal space of the brain. Al-Abeta42 also had a longer half-life in blood and had increased permeation at the striatum and thalamus. Brain-to-blood transport was similar for Al-Abeta42 and Abeta42. In conclusion, complexing with aluminum affects some aspects of blood-to-brain permeability so that Al-Abeta42 would have more ready access to brain cells than Abeta42.

Aluminium toxicity and metal speciation: established data and open questions

Histopathological findings in humans and extensive toxicological investigation in vivo and in vitro point to an unambiguous neurotoxic potency of AI(III). Experimental toxicology with aqueous AI(III) is very difficult owing to the complex and somewhat uncontrollable aqueous chemistry of the metal centre in the neutral range. The choice of neutral, hydrolytically stable synthetic toxins makes possible both the control of metal speciation and of analytical metal concentration down to about 1 mM in neutral buffered solutions. The employment of less stable complexes like Al2(citrate)2(H2O)6 and AI(lactate)3 or of ordinary salts is unavoidably complicated by the precipitation of Al(OH)3 under the same conditions. In spite of this, the choice of a carefully designed protocol, based on ensuring well defined steps, enables one to successfully control the analytical metal concentration down to 10 μM AI(III). The control of the metal speciation at these concentration levels remains an open question.

Aluminum Modulates Effects of βAmyloid1–42 on Neuronal Calcium Homeostasis and Mitochondria Functioning and Is Altered in a Triple Transgenic Mouse Model of Alzheimer's Disease

Recent findings suggest that beta-amyloid (A beta) is more neurotoxic when present in its oligomeric configuration rather than as monomers or fibrils. Previous work from our laboratories has shown that A beta aggregation is strongly influenced by the conjugation of the peptide with metal ions (aluminum A, copper [Cu], zinc [Zn], and iron [Fe]) that are found in high concentrations in the core of senile plaques. Disruption of Ca++ signaling and mitochondrial dysfunction are potent triggers of neuronal death and have been implicated in the neuronal loss that is associated with Alzheimers disease (AD). In this study, we explored whether A beta-metal complexes can have detrimental effects on intraneuronal Ca++ ([Ca++]i) homeostasis and mitochondrial function in vitro. Results from our experiments indicate that, when conjugated with Al, A beta perturbs neuronal [Ca++]i homeostasis and inhibits mitochondrial respiration. Finally, we analyzed the content of the four metals in the brain of a triple transgenic animal model of AD and found that Al is the only one to be increased in the cortex of these mice.