Aluminum has been recognized as a neurotoxic agent in animals for more than 100 years [1]. It is also known to be toxic to humans and represents a relevant etiological factor in pathologies related to long-term dialysis treatment [2,3]. In addition, aluminum has been proposed (with some controversy) as a cofactor in the etiopathogenesis of Alzheimer's disease (AD) as well as other neurodegenerative pathologies [4]. If aluminum indeed plays a role in the development of AD, possible individual differences in susceptibility to the metal ion would become of great relevance and concern. Preliminary experiments carried out in this laboratory suggest that there indeed exists an individual predisposition or at least an individual sensitivity to aluminium accumulation (aluminum phenotype?) [5].

Transferrin represents the main protein carrier of Al [6], Ti [7], Ga [8] and other metal ions in the plasma. The uptake of Al into cells appears to be largely determined by the distribution of transferrin receptors [9] located on the cell surface; these receptors are present at a high density in some regions of the brain that are affected by AD, as well as in a selected relevant population of neurons [10]. Interestingly, a genetic variation in transferrin has been detected in AD patients [11,12]. On the basis of cellular internalization Ga⁶⁵via the transferrin receptor after transit across the blood–brain barrier (BBB), Edwardson et al. [10], calculated that the human brain accumulates approximately 8 µg of Al³⁺ per year of life, an amount consistent with that observed after a lifetime of exposure [13]. Farrar et al. [8] reported defective Ga-transferrin binding in AD, and hypothesized a possible mechanism of Al accumulation in the brain due to a reduction in the interaction between the metal and receptor. It is noteworthy that, under certain conditions, Al(III) can modify the permeability of the BBB [14,15]; upon entrance into the brain, the metal may become differentially compartmentalized in the various subcellular organelles in a pH-dependent manner.

The most important function of mitochondria is the oxidation of substrates to produce bioenergy. Several lines of evidence indicate that mitochondria from neurons affected by degenerative disorders related to aging exhibit aberrant oxidation processes [16]. Furthermore, it is worth mentioning that there is a strict correlation between Krebs cycle and glycolysis. In this connection it has been demonstrated that Al is a strong inhibitor of some enzymes of the glycolysis pathway [17–19] as well as glucokinase and phosphofructokinase [20]. All these elements reinforce the possibility that Al could interfere the bioenergetic pathways in mitochondria.

With the aim of establishing how Al³⁺ may influence mitochondrial enzymes, we studied the effects of the metal on the activities of enzymes involved in the Krebs cycle, as well as that of glutamate dehydrogenase (GDH), which catalyzes a reaction directly connected to the Krebs cycle.

MATERIALS and METHODS

Results

Effects of Al on the activity of Krebs cycle enzymes

Aluminum shows a strong positive effect on the activity of succinate dehydrogenase from rat brain. As illustrated in Fig. 1, this stimulation is a function of the metal ion concentration. Maximal enzymatic activity is reached at around 50 µm of Al³⁺. The Lineweaver–Burk plot shown in Fig. 2 reports the kinetics parameters obtained using 10 µm aluminum: K_m = 40.81 mm in the absence of aluminum and 16.22 mm in the presence of the metal ion; V_max = 16.39 nmol·min⁻¹ both in the absence and presence of the metal. The Michaelis–Menten equation shows that the activation is of the competitive type.

As reported in Fig. 3, KGDH is also activated by aluminum as a function of the metal ion concentration. The Lineweaver–Burk plot shown in Fig. 4 reveals a K_m of 0.39 µm and 0.34 µm in the absence and presence of Al, respectively; V_max = 5.55 nmol·min⁻¹ both in the absence and presence of the metal ion. Again, the Michaelis–Menten equation shows that the activation is competitive.

In contrast to the two above reported enzymes, aconitase is markedly inhibited by Al³⁺, as shown in Fig. 5, which reports the activity of aconitase from rat brain homogenate as a function of the concentration of Al³⁺. Such an inhibitory effect is also evident in the Lineweaver–Burk plot illustrated in Fig. 6, which shows that the enzyme exhibits a K_m of 0.11 m and 0.26 m in the absence and presence of Al, respectively, while the V_max is 0.02 nmol·min⁻¹ in the absence of Al³⁺ and 0.012 nmol·min⁻¹ in the presence of the metal ion. According to the Michaelis–Menten equation, the inhibitory action of Al³⁺ is of a mixed type.

Figure 7 demonstrates that GDH, which catalyzes a reaction directly linked to the Krebs cycle, is inhibited by aluminum in a dose-dependent manner. Figure 8 reports the kinetic paremeters of GDH and the effect of aluminum on its enzymatic activity. The K_m increases from 21.27 µm to 68.63 µm in the presence of aluminum, while the V_max remains unchanged at 18.69 nmol·min⁻¹. The Michaelis–Menten equation shows that this inhibition is of the competitive type. All the other enzymes of the Krebs cycle were found to be unaffected by aluminum (data not shown).

Discussion

In spite of an abundant number of published studies on the topic, the molecular mechanisms that could explain the neurotoxicological action of aluminum remain to be fully understood. The Krebs cycle is the final pathway for the oxidation of fuel molecules, and represents a link between glycolysis and the oxidative decarboxylation of pyruvate to form acetylCoA. It is important to note that mitochondria, which represent the site of all major reactions of oxidative metabolism, also play an important role in aging and aging-related diseases.

Abundant data confirm abnormal cerebral metabolism in several neurodegenerative diseases including Alzheimer's disease. Alterations in mitochondrial enzymes underline these deficits. According to some authors, impaired oxidative and energy metabolism are relevant features in Alzheimer's disease due to compromised mitochondrial oxidative metabolism in brain cells. Therefore, defects in energy metabolism may play a role in the pathogenesis of neurodegenerative diseases in general and in AD in particular. The demonstration of a link between alterations in mitochondrial enzymes and neurodegeneration is of paramount importance in that some of these enzymes are related to certain neurotransmitter systems.

Recent data raise the possibility of a genetically determined alteration in some enzymes of the Krebs cycle in a subgroup of patients affected by Alzheimer's disease [16,31]. Abnormalities in oxidative metabolism have consistently been detected in biochemical assays of AD brain autopsies, with a functional defect at the level of the Krebs cycle [32–37]. This is of great importance with regard to AD, as impairment of oxidative processes is known to lead to an abnormal increase in production of amyloid precursor protein a hallmark of this disease [38–39]. Furthermore, cytoskeletal disorganization [40–41] with appearance of epitopes cross-reacting with antibodies to AD neurofibrillary tangles [42] has also been described.

Several authors reported a high and focal accumulation of aluminum in specific brain areas affected by relevant neurodegenerative diseases such as AD and PD [4,43]. Experimental data reported in this paper indicate that Al³⁺ markedly activates only two Krebs cycle enzymes, i.e. KGDH and SDH (Fig. 9). On the other hand, aconitase is the only Krebs cycle enzyme to be inhibited by the metal ion.

AD is a multifactorial disease whose etiology remains to be understood, in spite of an abundance of information obtained in the last three decades. A correlation between Al and AD pathogenesis remains also elusive, due in part to the intricate biochemistry of Al³⁺ existing data remain mostly at the phenomenological level, and need to be reinforced by further investigations.

The present study demonstrated that KGDH is activated by Al, an observation in apparent opposition with the reported reduction in KGDH activity in AD brain tissue [35]. However, in this connection, it is important to consider that KGDH represents only a part of a rather complex enzymatic system that also includes transsuccinylase and dihydrolipoyl dehydrogenase, with transsuccinylase representing the core of the complex catalyzing oxidative decarboxylation of α-ketoglutarate. In addition, to further complicate the scenario, recent studies reported an important association between polymorphisms in dihydrolipoyl-succinyltransferase (DLST) and AD, suggesting a relationship between Apolipoprotein E4, a well recognized significant risk factor in AD [44], and the DLST locus in the pathogenesis of AD [45]. Further studies are therefore needed in order to reach a final interpretative conclusion, to better understand not only the real action of Al on each of these three enzymes, but also their behavior in AD.

Aconitase is a 4Fe−4S cluster-containing protein that binds citrate and catalyzes its isomerization to isocitrate via the intermediate cis-aconitate. Aconitase also participates in cellular iron regulation and mitochondrial energy production [46] by regulating the stability and translation of mRNA coding for proteins controlling as a function of iron availability [47]. Aconitase activity is severely depleted in the brains of patients with certain severe neurological disorders such as Huntington's disease [48]. Both iron and aluminum accumulate in the human brain with ageing [4,10] either via interaction with the transferrin receptor or by transferrin-independent pathways [49,50]. Al has a strong affinity for citrate (log K_7.4 = 14.7) [6], a property that could explain the observed reduction in the ability of aconitase to catalyze isocitrate production in the presence of the metal ion.

Aside from its activity as a neurotransmitter glutamate may have nontransmitter functions in many different neuronal and glial cell types. Glutamate can be produced from α-ketoglutarate via transamination by glutaminase, by the reaction catalyzed by GDH (Fig. 9), or by other pathways [51]. The precise role of glutamate formed by each of these routes remains unclear. GDH activity is allosterically regulated and can catalyze the following reaction in both sites: