Excessive glutamine sensitivity in Alzheimer' s disease and Down syndrome lymphocytes

Marie A. Peeters a(*) Aline Salabelle a, Nadine Attal b, Marie-Odile Rethore a, Clothilde Mircher a, Dominique Laplane b, Jerome Lejeune a

Journal of the Neurological Sciences 133 (1995) 31-41

• 1. Introduction


• 2. Material and methods
• 2.1. Patients
• 2.2. Statistical analysis


• 3. Results
• 3.1. Patients with Alzheimer's disease
• 3.2. Down syndrome patients
• 3.3. Comparison between Alzheimer's disease patients and Down syndrome demented patients
• 3.4. Comparison between normal controls and patients with Down syndrome


• 4. Discussion


• Annexes

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1. Introduction

Alzheimer's disease (AD) is a progressive, degenerative form of dementia of unknown etiology and without effective treatment. It is estimated that around 10% of the population in the Western world, over 65 years of age, is affected. Clinical features include memory loss, personality changes, decline of cognitive functions; there are no diagnostic tests, clinical or paraclinical, except neuropathology, to affirm the diagnosis which remains one of exclusion. Although etiologically Alzheimer's disease may be a heterogenous disease, the hallmark of the disease are the neuropathological changes characterised by neurofibrillary tangles and senile amyloid plaques seen in brain tissues on autopsy. Recent studies suggest that Alzheimer's disease may in fact be a systemic disease (Etcheberrigaray et al., 1993; Jabbour et al., 1992; Joachim et al., 1989).

Patients with Down syndrome over the age of 40 years have been found to have invariable premature development of characteristic AD neuropathological changes (Cork, 1990). There is however no absolute correlation between the pathological changes and the clinical manifestations of dementia (Olson and Shaw, 1969; Wisniewski and Rabe, 1986). Over the age of 40, some 15-51% of patients with Down syndrome develop dementia, conflicting reports result from problems associated with assessing dementia in mentally retarded patients (Cork, 1990; Zigman et al., 1987).

In addition to these clinical and neuropathological similarities (Ikeda et al., 1989), there are both genetic and biochemical data to suggest some common disease mechanisms. It has been suggested that nondisj unction of chromosome 21 may underlie both disorders (Potter, 1991). Amyloid beta (A4) precursor protein is encoded by a gene found on chromosome 21 and it is expressed in the brain (Goldgaber et al., 1987; Robakis et al., 1987; Tanzi et al., 1988). Some point mutation of APP have been described in familial cases of early-onset Alzheimer's disease (Goate et al., 1991). Furthermore, the possibility that the gene for at least some familial Alzheimer disease may be found on chromosome 21 has been suggested (Steele et al., 1989). Increased incidence of Alzheimer's disease have been reported in mothers of patients with Down syndrome (Schupf et al., 1994).

Biochemical similarities include: (1) similar neurotransmitter deficits: cholinergic, serotonin and adrenergic (Cork, 1990; Mann et al., 1985; McGeer et al., 1984; Sacks and Smith. 1989); (2) microtubule assembly defects (Khatoon and Iqbal, 1989; Krawczun et al., 1990; Matsuyama and Jarvik, 1989); (3) reduced levels of biopterin (Kay et al., 1986); (4) elevation of brain interleukin 1 and S100 im-munoreactivity (Griffin et al., 1989; Jorgensen et al., 1990); (5) reduction of D(3H) aspartate binding (Simpson et al., 1989); (6) loss of cortical glutaminergic neurons (Reynolds and Godridge, 1985; Reynolds and Warner, 1988), (7) elevation of brain aluminium (Edwardson and Candy, 1989; Farrar et al.. 1990).

In this study we expanded a preliminary report on eight patients with Alzheimer's disease demonstrating excessive in vitro glutamine toxicity (Peeters et al., 1992). We conducted the present study to test the hypothesis that a common metabolic defect could be found in Alzheimer's disease patients with or without Down syndrome.

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2. Material and methods

Peripheral blood samples were cultured for 72 hours in TC199 medium (Seromed), supplemented with 25% human AB serum, phytohaemagglutinine C (IBF France), penicillin and streptomycin. Culture technique, harvesting and microscopic examination were carried out according to standard, previously described methods (Peeters and Lejeune, 1989).

The following products were added on initiation of culture (all products were Sigma products unless otherwise specified); the entire panel could not always be performed for each patient: (1) inosine (INO): 125 mg/l; (2) alanosine (ALA): 0.0625 mg/l. kindly provided for by the Laboratory of Biochemical Pharmacology (National Institute of Health, Bethesda, Maryland, USA); (3) azaserine (AZA): 0.0156 mg/l; (4) adenosine (ADE): 16 mg/l; (5) cytidine (CYT): 6.25 mg/l; (6) guanosine (GUA): 3.1 mg/l; (7) putrescine (PUT): 6.25 mg/l; (8) methotrexate (MET): 1.2 X 10-8 M; (9) glutamine (GLN): 95 mg/l; (10) uridine (URI): 3.1 mg/l; (11) HAT medium: 62.5 µM hypoxanthine; 0.25 µM aminopterin; 10 µM thymidine.

The following products were added 12-16 h prior to termination of the culture: (12) mycophenolic acid (MYC): 0.6 X 10-6 M; (13) theophylline (THE): 750 mg/l; (14) rT3: 5 mg/l.

The lymphocytes cultures were harvested with standard techniques used for chromosomal analysis. Slides were stained with Giemsa and coded. A minimum of 2500 cells were read to calculate the mitotic index, expressed as the ratio of mitosis over the total number of nuclei. Each patient was compared to itself and paired t-tests were performed on the differences.

Fig. 1. Percentage increment or decrease of mitotic index in patients with Alzheimer's disease compared to normal controls. Abbreviations: rT3: reverse triiodothyronine; MYC: mycophenolic acid; 23D: 2,3-diphosphoglycerate; GLN: glutamine: INO: inosine; ALA: L-alanosine; AZA: azaserine; ADE adenosine; THE: theophylline; HAT: HAT medium; URI: uridine; CYT: cytidine; GUA: guanosine; PUT: putrescine; MET: methotrexate. For each group of patients tested, the first horizontal line indicates the number of patients who were tested with a given product; the second line gives the name of the product which was added to the culture; the third line indicates the mean percentage increment or decrease of the mitotic index; on the fourth line, the value of the standard deviation is indicated. STUD: Student's t-test (two-tailed). * Student's Mest > 2, p <0.05; **o Student's t-test > 2.8, p < 0.01*** Student's t-test > 3.8, p < 0.001

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2.1. Patients

Sixteen patients with Down syndrome and a regressive Alzheimer type dementia were studied (only 14 had glutamine added to their lymphocyte cultures). There were 4 men and 12 women in this study with a mean age of 44.8 years (range 20-53 years). Hypothyroidism was excluded in all these patients. Patients suffered from progessive loss of cognitive skills, desorientation, neurological degradation (functional decline in areas such as orientation, memory, verbal and motor skills and self-care abilities) and loss of sphincter control. A therapeutic trial of folic acid and of vitamine B12 therapy was ineffective. Controls used for analysis were 171 patients with Down syndrome without dementia (59 of whom had glutamine added to their lymphocyte culture). All the patients with Down syndrome had documented trisomy 21 on chromosomal analysis. Results were also compared to 156 normal controls (74 of whom had glutamine added to their lymphocyte cultures). Normal controls were healthy volonteers (85 patients) or parents of a miscarriage referred to the genetics laboratory who on chromosomal analysis had a normal caryotype (71 patients).

Nineteen patients with the clinical diagnosis of advanced Alzheimer's disease were studied (16 of whom had glutamine added to their lymphocyte culture). There were 5 men and 14 women; mean age at the time of this study 77 years (range 46-97 years). Diagnostic criteria were the following (McKhann et al., 1986): dementia as evidenced by clinical examination documented in most patients by the "mini-mental state" (Folstein et al., 1975) (MMS score ranged from 0/30 to 19/30); deficits in two or more areas of cognition; progressive deterioration in specific cognitive functions such as language (aphasia), motor skills (apraxia) and perception (agnosia); impaired acitivities of daily living; the absence of documented underlying neurological, psychiatric or systemic pathology (thyroid and hematological diseases were exclude); a brain scan which was normal for age. There were 156 normal control patients of whom 26 age-matched control patients aged more than 60 years and 11 patients over 70 years were examined with the same method. In the age-matched control group, ten patients presented dementia of known etiology (head trauma, vascular disease, etc.). Patients with Alzheimer's disease and age-matched controls were on the same hospital ward and received the same diet, they had good nutrition status and were not given any protein supplementation. All patients received medication: antidepressants and/or neuroleptic medications which had been interrupted at least 12 hours prior to blood sampling for this investigation. Other controls used were 156 normal controls and 171 patients with Down syndrome without Alzheimer's type dementia.

Fig. 2. Percentage increment or decrease of mitotic index in patients witn Alzheimer's disease compared to normal age-matched controls. Abbreviations as in Fig. 1.

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2.2. Statistical analysis

Results were analyzed by comparing the mitotic index of each experiment (each additif) to the patient's own control culture and were expressed as the percentage incre ment or decrease in mitotic index. Statistical comparison between groups were based on the Student's Mest (two tailed test). Given the complexity of the metabolic path ways which were examined, we only analyzed result which were significant at a p value of less than 0.01.

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3. Results

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3.1. Patients with Alzheimer's disease

When patients with Alzheimer's disease were compared to normal controls, there was a highly significant difference (p < 0.001) in the in vitro toxicity to glutamine (Fig 1). The same in vitro toxicity was observed when age matched controls were analyzed (Fig. 2).

It is of interest to note that 9 normal controls (14%) mean age 38.4 years, decreased their mitotic index in the presence of glutamine (more than 15% decrease in the mitotic index). When controls of more than 70 years of age were examined 4/11 demonstrated in vitro toxicity to glutamine, two of whom were demented (but thought not to have Alzheimer's disease). No age-related differences were observed between controls aged less than 70 years and those aged more than 70 years.

When Alzheimer disease patients were compared to normal controls who also demonstrated in vitro sensitivity to glutamine no differences in reaction to different additives were observed. However significant differences in reactions were noted (in the presence ofinosine(p < 0.01), adenosine (p < 0.001) and guanosine (p < 0.01) when all patients were examined (normal controls and patients with Alzheimer's disease) based on their different sensitivity to exogenous glutamine (Fig. 3).

Fig. 3. Percentage increment or decrease of mitotic index in patients (normal controls and patients with Alzheimer's disease) with different sensitivity to exogenous glutamine (increase over 0% or decrease of more than 15%). Abbreviations as in Fig. 1.

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3.2. Down syndrome patients

When patients with Down syndrome and Alzheimer type dementia were compared to Down syndrome patients without dementia (Fig. 4) or to normal controls, a highly significant difference (p< 0.001) in glutamine toxicity was noted.

In vitro toxicity to glutamine was frequently observed in Down syndrome controls. Fourty-seven percent of DS aged more than twenty years showed in vitro toxicity to glutamine.

When the metabolic patterns between Down syndrome patients who increased their mitotic index in the presence of glutamine was compared to those who demonstrated toxicity (control patients with Down syndrome and those manifesting Alzheimer type dementia) there were significant differences as shown in Fig. 5.

Fig. 4. Percentage increment or decrease of mitotic index in patients with Down syndrome with or without Alzheimer type dementia. Abbreviations as in Fig. 1.

Fig. 5. Percentage increment or decrease of mitotic index in patients with Down syndrome (with or without dementia) and different sensitivity to exogenous glutamine (increase over 0% or decrease of more than 15%). Abbreviations as in Fig. 1.

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3.3. Comparison between Alzheimer's disease patients and Down syndrome demented patients

There was no highly significant difference observed in the in vitro response to different additives between patients with Alzheimer's disease and those with Down syndrome suffering from dementia (Fig. 6).

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3.4. Comparison between normal controls and patients with Down syndrome

No differences in the different reactions to various metabolites were observed between normal controls and patients with Down syndrome (results not shown).

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4. Discussion

Our findings suggest that both patients with Alzheimer's disease and Down syndrome patients suffering from dementia have excessive in vitro lymphocyte toxcicity to glutamine. In the presence of exogenous glutamine there is a highly significant decrease in the mitotic index in lymphocyte cultures. There was no difference in the toxic response to glutamine observed between patients suffering from Alzheimer's disease and Down syndrome patients suffering from dementia.

Although glutamine is not neurotoxic it converts to glutamate (Huang et al., 1994; O' Driscoll et al., 1993; Piani and Fontana, 1994). Glutamine/glutamate metabolism is complex and regulated by several enzymes, purine nucleotides and a number of amino acids (Erecinska and Silver, 1990). Glutamine can produce glutamate not only through the action of phosphate-activated glutaminase but also through glutamine transaminase. Given the rapid interchange between glutamine and glutamate, the problems of compartmentation of the various enzymes intervening in glutamine/glutamate metabolism in the brain and in other tissues, the fluxes between the compartments and the fact that this work could not address the precise concentrations of one or the other, we will use speak of glutamine/glutamate dysregulation.

Glutamate is one of the major excitatory transmitters in the brain. Fast excitatory transmission in the central nervous system is mediated mainly by L-glutamate and glutamate receptors are found throughout the mammalian brain (Hayashi, 1954, Monaghan et al., 1989). Glutamate is released by an estimated 40% of synapses (Fonnum, 1984), The glutamate receptor system is thought to be involved in the first steps of learning and memory acquisition (Morris, 1989), as well as neuronal survival and dendritic outgrowth (Jorgensen et al., 1990). Excess exposure to glutamate can destroy neurons, a process Olney calls excitotoxicity. Glutamate is toxic to neurons in culture and in vivo; immature brains are especially susceptible to the toxic effects of glutamate. Various patterns of brain damage can be observed depending on the developmental stage in which the neurotoxic process is triggered.

Certain glutamate analogs known to share the neuroexcitatory properties of glutamate mimic its neurotoxic effects (Olney, 1991) whereas glutamate antagonists have a neuroprotective effect (Kahn et al., 1993). Recent experiments implicate the glutamate receptor as a mediator of the toxic effect of glutamate (Heinemann et al., 1991). At least 7 receptor subunits, different glutamate receptors are expressed in specific brain regions. It is of interest that human receptor gene GluR5 has been assigned to chromosome 21 (Potier et al., 1993) and maps in the vicinity of the gene for familial amyotrophic lateral sclerosis (Eubanks et al., 1993; Gregor et al., 1993).

The possible link between anomalies in glutamine/glutamate metabolism and Alzheimer's disease is not new. Glutamate is one of the major excitatory neurotransmkters in the mammalian brain and has been implicated in the pathogenesis of acute and chronic neurological diseases by virtue of its excitotoxic property. It has been proposed that degenerative diseases (Alzheimer's disease, Huntington's chorea, amyotrophic lateral sclerosis) may involve neuronal death by excessive activation of the glutamate receptor system and/or genetic or acquired abnormalities in glutamate sensitivity of certain neurons (Greenamyre and Young, 1989; Lipton and Rosenberg, 1994; Meldrum and Garthwaite, 1990).

It has been reported that early in the course of Alzheimer's disease, there is a decrease in CSF glutamine content (Procter et al., 1988) and lack of glutaminase activity. Neurochemical changes precede cellular pathology. Later in the course of the disease, there is anatomical and biochemical evidence suggesting both pre and post synaptic disruption of glutaminergic pathways (Cowburn et al., 1988; Hardy et al., 1987). It is known that in Alzheimer's disease there is severe loss of glutamataminase positive (pyramidal) cortical neurons as well as loss of glutamate terminals in the hippocampal region (Akiyama et al., 1989; Cross et al., 1989; McGeer et al., 1989) and of NMDA receptor (one of the glutamate receptors). Beta amyloid inactivates oxidation-sensitive glutamine synthetase (Hensley et al., 1994). Recently it has been suggested that the detection of glutamine synthetase in the cerebrospinal fluid of Alzheimer's disease patients may be a potential diagnostic biochemical marker (Gunnersen and Haley, 1992). Glutamine synthetase plays a key role in converting the neurotransmitter and excitotoxic amino acid glutamate to the non neurotoxic glutamine. Aluminium which has been found to be elevated in the brains of patients with Alzheimer's disease disrupts normal glutamine/glutamate homeostasis (Zielke et al., 1993). These findings all point to some abnormality in the glutamine/glutamate pathway in Alzheimer's disease which is further supported by our study.

Patients with Down syndrome have well documented biochemical anomalies of some of the major regulators of the glutamine/glutamate metabolic pathway as shown in Table 1. In patients with Down syndrome the dynamic equilibrium among multiple facilitative and inhibitory factors of glutamine/glutamate metabolism might create an imbalance rendering the system prone to an expression of excitotoxicity. It is of interest that, when we compared the in vitro metabolic responses observed in patients with Down syndrome who increased their mitotic index in the presence of glutamine to those for whom glutamine was toxic (decrease in the mitotic index greater than - 15%), other significant differences appeared. Significant opposite effects in the presence of inosine (p< 0.001), cytidine (p< 0.01) and guanosine (p< 0.001) were noted. In Down syndrome patients who demonstrated in vitro toxicity to glutamine. these nucleotides were also toxic, (whereas they slightly increased mitotic index in cultures where glutamine was beneficial). This raises the hypothesis that perhaps underlying imbalances in purine metabolisme may play an important role in the glutamine toxicity observed in patients with Down syndrome (finding which is of interest given the role purine metabolites play in glutamine/glutamate metabolism).

Fig. 6. Percentage increment or decrease of mitotic index comparing patients with Alzheimer's disease and Down syndrome demented patients. Abbreviations as in Fig. 1.

Lymphocyte toxicity to glutamine seemed to increase with age. Sixteen out of 46 (34%) Down syndrome patients aged less than 30 years demonstrated in vitro glutamine toxicity whereas in those over 30 years 10 out of 17 (58%) Down syndrome patients demonstrated toxicity. Our findings suggest that patients with Alzheimer's disease have a primary or secondary defect in glutamine/ glutamate metabolism. Alzheimer' s disease would in fact be a "metabolic disease" as has already been suggested by other authors (Etcheberrigaray et al., 1993; Iwatsuji et al., 1989; Joachim et al., 1989). This is supported by the fact that:

amyloid B protein deposits have been observed in multiple non-neuronal tissues (Joachim et al., 1989);

a decrease in heat-stable lymphocyte glutamate dehydrogenase activity has been reported (Iwatsuji et al., 1989);

similar anomalies in actin expression in lymphocytes from patients with Down syndrome and Alzheimer's disease have been found (Jabbour et al., 1992);

a potassium channel dysfunction in fibroblasts may identify patients with Alzheimer's disease (Etcheberrigaray et al., 1993).

Our findings would be in keeping with the suggestic that a generalized metabolic anomaly exists in Alzheimer disease. It is of interest that in our control sample, age less than 70 years of age, 14% decreased their mitotic index in the presence of exogenous glutamine. This finding is in keeping with the incidence of Alzheimer' s disease in the general population. However 30% of controls over the age of 70 demonstrated in vitro sensitivity to glutamine suggesting possible dysregulation with increasing age (perhaps also in relationship with purine imbalances as suggested by the toxicity of inosine, adenosine and guanosine in the cultures of control patients in which glutamine was toxic). The increased toxicity of exogenous glutamine t lymphocyte culture was more frequent and of earlier onse in patients with Down syndrome. Indeed, in the Down syndrome control group 34% of patients aged less that thirty years of age decreased their mitotic index more than 20% and excessive in vitro sensitivity to glutamine was observed in 58% of patients with Down syndrome over in age of thirty.

We hypothesize that Alzheimer's disease may be du to:

1. a primary genetic or acquired defect of glutamine/ glutamate metabolism;

2. complex purine and/or amino acid dysregulation responsable for secondary alterations in glutamine/ glutamate metabolism.

It must be remembered that monosodium glutamate one of the world's most widely and heavily used for additives. Certain people may be genetically unable metabolize exogenous glutamine/glutamate and even normal doses will eventually produce toxic effects. It is interest that in populations where glutamine/glutamate used frequently as a food additive, one might expected increased incidence of Alzheimer' s disease. In fact the incidence is lower than in the West (Treves et al., 1993) This would be in keeping with genetic metabolic differences. Prolonged exposure to low dose glutamate (due to a genetic or acquired abnormality of glutamate metabolism or sensitivity of certain neurons) is as toxic as the acute form of neurotoxicity which causes neuronal degeneration (Rothstein et al., 1993). If this hypothesis is true, it has important preventive medical implications. Indeed genetically susceptible persons should restrict their diet with regards to glutamine/glutamate intake. In patients in whom purine synthesis dysregulation exists it would be of importance to examine whether these changes are amendable to modulation as we suggested in patients with Down syndrome (Peeters et al., 1993). Further studies are needed both to confirm our findings and to expand this observation by using glutamate agonists and antagonists. Patients in the early stages of the disease should also be examined.

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