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For the past 20 years the scientific community and the pharmaceutical industry have been searching for treatments to neutralize the devastating effects of Alzheimer disease (AD). During this period important changes in the etiopathogenic concept of AD have occurred and, as a consequence, the pharmacological approach for treating AD has also changed. During the past 2 decades only 3 drugs for AD have been formally approved by the FDA, although in many countries there are several drugs which are currently used as neuroprotecting agents in dementia alone or in combination with cholinesterase inhibitors. The interest of the pharmaceutical industry has also shifted from the cholinergic hypothesis which led to the development of cholinesterase inhibitors to enhance the bioavailability of acetylcholine at the synaptic cleft to a more "molecular approach" based on new data on the pathogenic events underlying neurodegeneration in AD. In our opinion, the pharmacological treatment of AD should rely on a better understanding of AD etiopathogenesis in order to use current drugs that protect the AD brain against deleterious events and/or to develop new drugs specifically designed to inhibit and/or regulate those factors responsible for premature neuronal death in AD. The most relevant pathogenic events in AD can be classified into main categories: primary events (genetic factors, neuronal apoptosis), secondary events (beta-amyloid deposition in senile plaques and brain vessels, neurofibrillary tangles due to hyperphosphorylation of tau proteins, synaptic loss), tertiary events (neurotransmitter deficits, neurotrophic alterations, neuroimmune dysfunction, neuroinflammatory reactions) and quaternary events (excitotoxic reactions, calcium homeostasis miscarriage, free radical formation, primary and/or reactive cerebrovascular dysfunction). All of these pathogenic events are potential targets for treatment in AD. Potential therapeutic strategies for AD treatment include palliative treatment with nonspecific neuroprotecting agents, symptomatic treatment with psychotropic drugs for noncognitive symptoms, cognitive treatment with cognition enhancers, substitutive treatment with cholinergic enhancers to improve memory deficits, multifactorial treatment using several drugs in combination and etiopathogenic treatment designed to regulate molecular factors potentially associated with AD pathogenesis. This review discusses the conventional cholinergic enhancers (cholinesterase inhibitors, muscarinic agonists), noncholinergic strategies that have been developed with other compounds, novel combination drug strategies and future trends in drug development for AD treatment. Stem-cell activation, genetically manipulated cell transplantation, gene therapy and antisense oligonucleotide technology constitute novel approaches for the treatment of gene-related brain damage and neuroregeneration. The identification of an increasing number of genes associated with neuronal dysfunction along the human genome together with the influence of specific allelic associations and polymorphisms indicate that pharmacogenomics will become a preferential procedure for drug development in polygenic complex disorders. Furthermore, genetic screening of the population at risk will help to identify candidates for prevention among first-degree relatives in families with transgenerational dementia.

Scientists from the EuroEspes Biomedical Research Center, La Coruna, Spain, presented the conclusions of a pharmacogenomics study in Alzheimer's disease at the Sixth International Stockholm/Springfield Symposium on Advances in Alzheimer Therapy, held in Stockholm, April 5-8, 2000. The authors carried out a genomic screening to search for potential genes associated with neuronal death and found that in practically all 23 human chromosomes, except chromosome 16, there is at least one gene whose dysfunction is associated with neuronal death and/or dementia-related syndromes. This distribution of genomic loci related to neurodegeneration could indicate that is unlikely that a single drug would be capable of preventing neuronal death in subjects with a multifactorial, polygenic disease and that a genotype-related differential response should be expected in AD patients treated with any potential neuroprotective drug. It is suggested that AD patients be genotyped prior to the administration of novel drugs and that a pharmacogenomics approach to AD treatment would help in the development of more effective drugs and in the prediction of therapeutic responses.

Neurotrophins, such as NGF, BDNF and NT-3 play a regulatory role on the function of microglial cells in vivo and in vitro, and the identification of new compounds with neurotrophic properties is becoming a new strategy for the prevention and/or treatment of neurodegenerative disorders. In this study we describe the use of two different models to demonstrate the ability of Cerebrolysin to reduce microglial activation. The results of these in vitro and in vivo studies indicate that Cerebrolysin might exert a neuroimmunotrophic activity reducing the extent of inflammation and accelerated neuronal death under pathological conditions such as those observed in neurodegenerative diseases.

Cerebrolysin is a porcine brain derived peptide preparation with potential neurotrophic activity. The effects of a single oral dose of the Cerebrolysin solution (30 ml) on brain bioelectrical activity and on cognitive performance were investigated in healthy elderly people. A single oral dose of Cerebrolysin induced a progressive increase in relative alpha activity power from 1 to 6 hours after treatment in almost all the brain electrodes in elderly control subjects. As compared with baseline alpha power (45.8+/-9.5%), the increase in relative alpha activity in the left occipital electrode (O1) reached significant values at 1 hour (57.2+/-8.5%; p < 0.05), 3 hours (59.4+/-7.6%; p < 0.05) and 6 hours (63.4+/-9.8%; p < 0.05) after Cerebrolysin administration. Enhancement in relative alpha power was accompanied by a generalized decrease in slow delta activity that was maximum at 6 hours after Cerebrolysin intake. A significant improvement in memory performance, evaluated with items of the ADAS cog, was also found in elderly people taken a single dose of oral Cerebrolysin (6.9+/-1.0 errors at baseline versus 4.9+/-1.0 errors after treatment; p < 0.01). This memory improvement was more evident in recognition (2.8+/-0.6 errors vs. 1.5+/-0.7 errors; p < 0.05) than in recall tasks (4.1+/-0.5 errors versus 3.4+/-0.5 errors; ns). These data indicate that Cerebrolysin potentiates brain alpha activity, reduces slow EEG delta frequencies and improves memory performance in healthy elderly humans, suggesting that this compound activates cerebral mechanisms related to attention and memory processes. According to the present results, it seems that oral Cerebrolysin might be useful for the treatment of memory impairment and brain damage in eldely subjects with or without neurodegenerative disorders.

Single nucleotide polymorphisms (susceptibility genetics) and genomic point mutations (mendelian genetics) can be used in Alzheimer's disease (AD) for diagnostic, predictive and therapeutic purposes. Using a matrix genetic model, including APOE, PS1 and PS2 allelic variants, we have studied the distribution of 36 different genotypes in the AD population (N= 479) and the genotype-related cognitive response to a multifactorial therapy in AD patients with mild-to-moderate dementia. The 10 most frequent AD genotypes are the following: 1) E33P112P2 + (17.75%), 2) E33P112P2- (15.55%), 3) E33P111P2+ (10.85%), 4) E34P112P2+ (9.60%), 5) E34P112P2- (7.56%), 6) E33P111P2- (7.10%), 7) E34P111P2+ (4.80%), 8) E33P122P2+ (4.38%), 9) E34P111P2- (4.18%), and 10) E34P122P2+ (3.55%). APOE-4/4-related genotypes represent less than 3% in the following order: E44P112P2 + > E44P111P2+ = E44P111P2- > E44P112P2+ > E44P122P2+ = E44P122P2. Multifactorial therapy with CDP-choline (1,000 mg/day) + piracetam (2,400 mg/day) + anapsos (360 mg/day) did improve mental performance during the first 6-15 months in a genotype-specific fashion. The best responders in the APOE series were patients with APOE-3/4 genotype (r= +0.013), while the worst responders were APOE-4/4 patients (r= -0.93). PS1-related genotypes responded in a similar manner; and patients with a defective PS2 gene exon 5 (PS2+) always showed a poorer therapeutic response than PS2- patients. All these data suggest that the therapeutic outcome in AD exhibits a genotype-specific pattern, and that a pharmacogenomic approach to AD might be a valuable strategy for drug development and monitoring.

Cytidine 5'-diphosphocholine (citicoline) is a an endogenous intermediate in the biosynthesis of structural membrane phospholipids and brain acetylcholine. Citicoline has been extensively used for the treatment of neurodegenerative disorders associated with head trauma, stroke, brain aging, cerebrovascular pathology and Alzheimer's disease. In this study we have investigated the efficacy and safety of the treatment with citicoline versus placebo in patients with Alzheimer disease. Thirty patients (age = 73.0 +/- 8.5 years; range = 57-87 years) with mild to moderate senile dementia (GDS: stages 3-6) of the Alzheimer type were included in a double-blind, randomized and placebo-controlled clinical trial. After a 2-week period of drug washout, patients were treated with i) placebo (n = 17; age = 73 +/- 5 years) or ii) 1,000 mg/day of citicoline (n = 13; age = 76 +/- 9 years) for 12 weeks (84 days). Examinations were done at baseline (T0) and after the 12 weeks of treatment (T12). As compared to placebo, citicoline improved cognitive performance in Alzheimer's disease patients with APOE E4 (ADAS: difference between groups = -3.2 +/- 1.8 scores, p < 0.05; ADAS-cog: difference between groups = -2.3 +/- 1.5, ns); and this improvement on cognition was more pronounced (ADAS, p < 0.01; ADAS-cog: difference between groups = -2.8 +/- 1.3, p < 0.06) in patients with mild dementia (GDS < 5). Citicoline also increased cerebral blood flow velocities in comparison with placebo (p < 0.05) when transcranial Doppler recordings from both hemispheres were considered together, as well as diastolic velocity in the left middle cerebral artery (p < 0.05). Patients treated with citicoline showed an increase in the percentage of brain bioelectrical activity of alpha (occipital electrodes) and theta type (left side electrodes), accompanied by a decrease in relative delta activity particularly marked in the left temporal lobe. Significant differences with respect to placebo (p < 0.05) were observed for theta activity in several fronto-parieto-temporal electrodes of the left hemisphere. Treatment with citicoline tended to reduce serum IL-1 beta levels, mainly after 4 weeks of administration, with no modified blood histamine content. In addition, neither adverse side effects nor alterations in biological and hematological parameters were induced by citicoline. The present data indicate that citicoline (1,000 mg/day) is well tolerated and improves cognitive performance, cerebral blood perfusion and the brain bioelectrical activity pattern in AD patients. According to our results, it seems that citicoline might be a useful treatment in Alzheimer's disease, and that the efficacy of this compound is greater in patients with mild mental deterioration and/or bearing the epsilon 4 allele of the APOE.

Citicoline is an endogenous intermediate involved in the biosynthesis of brain phospholipids and acetylcholine which has been extensively used for the treatment of several neurodegenerative conditions. The effects of citicoline on neurodegeneration, apoptosis and learning were investigated in male Sprague Dawley rats subjected to implants of the beta-amyloid fragment 1-40 (A beta 4: 3 Mmol) into the right hippocampus and to permanent unilateral occlusion of the carotid artery. Citicoline (CDP; 0, 62.5, 125 and 250 mg/kg/day i.p.) was given during 2 days before and for 5 days after surgery, and the extension of the degeneration and the number of apoptotic figures (TUNEL technique) were evaluated in the dentate gyrus (DG) and the CA1 area of the hippocampus. Citicoline, at 125 and 250 mg/kg, reduced the number of apoptotic neurons in the hippocampus of rats with A beta 4/hypoperfusion-induced neurodegeneration (CDP0 = 105.3 +/- 32.8 apoptotic figures; CDP125 = 39.2 +/- 7.4** apoptotic figures; CDP250 = 34.5 +/- 14.4** apoptotic figures; **p < 0.01 vs. CDP0). CDP also reduced neuronal degeneration in the CA1 area in a dose-dependent manner (CDP0 = 450.5 +/- 130.1 microns; CDP62.5 = 280.6 +/- 76.3 microns; CDP125 = 86.6 +/- 37.3* microns; CDP250 = 121.7 +/- 85.3* microns; p < 0.05 vs. CDP0). Variability of results was very high in the DG, where a significant reduction in the extent of neurodegeneration was only observed in the group of rats receiving 62.5 mg/kg of citicoline. Finally, citicoline improved retention of a passive avoidance learning task, increasing the number of avoidances (Av) (CDP0 = 4.2 +/- 0.7 Av; CDP62.5 = 6.9 +/- 1.0 Av; CDP125 = 7.9 +/- 0.7** Av; CDP250 = 8.5 +/- 0.6** Av; **p < 0.01 vs. CDP0) in a dose-related manner. Based on these results, it was concluded that citicoline exerts antiapoptotic, neuroprotective and antiamnesic effects in conditions of neurodegeneration induced by A beta 4 plus hypoperfusion.

In recent years the potential use of neurotrophic factors in the prevention and/or treatment of neurodegenerative diseases has received much attention. To determine whether Cerebrolysin, a porcine brain-derived peptide preparation, was able to modulate in vitro lipopolysaccharide (LPS)-induced microglial activation and to test the direct effect of Cerebrolysin on astrocyte morphology, survival and proliferation, rat glial and astrocyte cell culture experiments were carried out. The morphology of microglia, ameboid/activated and flat/resting, was examined under contrast microscopy and cell counts obtained. In addition, the release of interleukin (IL)-1 beta and brain-derived neurotrophic factor (BDNF) was measured from cell culture supernatant using an enzyme-linked-immunoassay (ELISA). The results obtained in this study clearly suggest a protective effect of Cerebrolysin as revealed by downregulation of microglial activation after LPS treatment as well as by the control of IL-1 beta expression. No significant differences were observed on astrocyte morphology, survival or the production and/or release of BDNF. In conclusion, these in vitro studies indicate that Cerebrolysin might exert a neuroimmunotrophic function which can in turn reduce the extent of inflammation and accelerate neuronal death under pathological conditions such as human neurodegenerative disorders.

In order to investigate the possibility of whether or not the lymphocytes of patients with Alzheimer's Disease (AD) are in an activated state, blood mononuclear cells from 45 AD patients and 45 healthy age matched controls were immunophenotyped by measuring the expression of CD3, CD4, CD7, CD8, CD25, CD28, CD56 and HLA-DR by flow cytometry. Circulating and in-vitro-produced cytokines were also measured by ELISA tests. CD7 and CD8 were significantly decreased in AD patients (48.3% and 18.2%, respectively) when compared to healthy subjects (63.2% and 28.3%, respectively). A significant increase in the CD4, CD25 and CD28 antigen expression was also observed in the AD group (55.3% 24.8% and 65.1%) with respect to healthy subjects (44.5%, 10.3% and 54.3%). In addition there was a significant difference in the extent of apoptosis in lymphocyte culture, as measured by mean fluorescence intensity (MFI) of Fas antigen (CD95) expression on CD4+ T cells in 6 AD patients (MFI = 36% and 43%, by anti-CD3 and hyperthermia mediated-apoptosis, respectively) with respect to 6 healthy individuals (MFI = 24% and 31%, by anti-CD3 and hyperthermia mediated-apoptosis, respectively), as well as in T-cell proliferation assay. A decline of Fas antigen expression on CD8+ subset was observed in the AD group with both stimuli (19% and 28%) comparing to the control group (29% and 39%). No differences were observed on circulating cytokines and spontaneous in vitro production of proinflammatory interleukin 1beta (IL-1beta), Tumor Necrosis Factor-alpha (TNF-alpha), IL-6 and IL-10 cytokines. Lipopolysaccharide (LPS)-stimulated in vitro production of IL-1beta, TNF-alpha, IL-6 and IL-10 measured by a whole blood culture system was significantly higher in AD patients comparing to controls. Furthermore, the observed differences were more evident at late stages of disease. These findings suggest that immunological tests, based on lymphocyte immunophenotyping combined with pro-inflammatory cytokine determinations and measurement of apoptosis in peripheral blood might represent a useful tool to obtain more insight into the pathogenesis of AD and into the level of immune activation which could characterize the pathological state of lymphocytes from individual AD patients.

Several factors that increase the likelihood of developing Alzheimer's disease (AD) have already been identified. A correct evaluation of these may contribute to a better understanding of the etiology of the disease. The risk of developing AD definitely increases with (a) age, (b) head injuries, (c) family history of AD or Down syndrome, (d) sex (higher prevalence of AD in women), (e) vascular disease, (f) exposure to environmental toxins, (g) infectious processes, or (h) changes in immune function, and recent advances in molecular genetics have suggested that genetic predisposition (i) can be considered one of the most important risk factors in the development of AD. A significant increase in the number of amyloid plaques in AD patients with an apolipoprotein E4 (ApoE) allele has been observed and the results of several genetic studies indicate that the etiology of this neurodegenerative disease is associated with the presence of the allele E4 of ApoE. A potential source of damage in the AD brain is an altered response triggered by microglial activation, which is associated with amyloid plaques. It has become evident that a dysregulation of cytokine release appears within lesions of many types of brain disorders including infection, trauma, stroke, and neurodegenerative diseases. Many studies have shown that microglia secrete both cytokines and cytotoxins and since reactive microglia appears in nearly every type of brain damage, it is likely that their secreted products ultimately help to determine the rate of damaged brain tissue. In this study, in vitro cell cultures were established to investigate the effect of different concentrations of human sera (2.5% and 10%) with specific ApoE genotypes from Alzheimer's and non-Alzheimer's subjects on ameboid and flat microglial cells obtained from neonatal rat hippocampi. Results show that a modulation in the proliferation and activation of microglial cells was obtained and that AD sera, mainly in the ApoE 3/4 and 4/4 genotype contain factor(s) which are able to induce morphological changes, as measured by an increase in the ameboid cell type. In addition, major histocompatibility complex (MHC) class II antigen expression, as measured by flow cytometric analysis, and interleukin-1beta (IL-1beta) release as measured by enzyme linked immunoadsorbent assay (ELISA), in comparison with control groups and lipopolysaccharide (LPS)-treated cells, clearly demonstrate a direct effect of ApoE 3/4 and 4/4 and/or an indirect effect mediated by the release of IL-1beta on microglia activation. These results strongly suggest that primary in vitro microglial cell cultures can be used as a screening model to test human sera as well as the effect of new potential drugs aimed at down-regulating microglia activation.

Mutations in the presenilin-2 (PS-2) gene are less frequent than mutations in the PS-1 gene. All mutations described in the PS-1 gene were found in early-onset Alzheimer's disease (AD) patients. At present, there are two missense mutations described for the PS-2 gene in some AD pedigrees. We have therefore analyzed transmembrane 2 (TM2) and TM5 domains of the PS-2 gene in AD patients and in a group of age-matched healthy controls. In a patient who was clinically diagnosed as having late-onset AD, we found a novel missense mutation consisting of a G->A substitution on exon 5 of the PS-2 gene, which results in a Val to Ile substitution at codon 148 within the predicted TM2 domain of the PS-2 protein. This is the third mutation described in the PS-2 gene and the first presenilin mutation detected in a Spanish AD patient. Both, the N141I mutation and the V148I mutation described here are located within the predicted TM2 domain and both were found in late-onset AD kindreds, whereas the mutation within the predicted TM5 domain was found in an early-onset AD pedigree. Carriers of mutations within TM2 of PS-1 have a mean age at onset of 40 years, while the other mutations in PS-1 occur in families with a mean age at onset of 47 years. In summary, we report here the first mutation in a presenilin gene in a Spanish AD case, which is the third mutation detected for the PS-2 gene.

We document the presence of subpopulations of neurons within the rat central nervous system that are labelled with a new Congo red staining technique. These neurons (CR neurons) show shrunken somata, and smaller and darker nuclei than Congo red-negative cells (non-CR cells). With the Bielschowsky and the cresyl violet Nissl staining methods, two comparable subpopulations of cells can be distinguished by the same morphometrical criteria as those used for CR and non-CR cells. CR neurons are located preferentially in some brain regions while in others they are virtually absent. Their distribution and proportion varied greatly from animal to animal and after particular treatments. Injections of water that damaged the hippocampal dentate gyrus, cortical lesions or eye enucleation decreased the number of CR-cells in the CA1 subfield, reflected in a shift from the CR-staining subclass to the non-CR subclass. Treatment with 200 mg/kg of CDP-choline also significantly reduced the number of CR cells observed in CA1. In the red nucleus, CR neurons showed a characteristic distribution of beta-amyloid precursor protein (APP) immunoreactivity. The population of dendrites immunolabelled for microtubule-associated protein 2 was markedly decreased in the areas of the hippocampus with high numbers of CR cells. Therefore, it is proposed that neurons labelled with the present Congo red technique might be in a reversible degenerative state or represent a particular physiological state in some areas of the central nervous system.

Small volumes of solutions injected into the hippocampus produce dramatic degeneration in dentate gyrus neurons, but not in neurons of the CA1 subfield. The aim of the present study was to ascertain whether solutions with different fragments of the beta-amyloid protein (Abeta) could produce further degeneration in areas beyond the dentate gyrus. It was found that 5 days after injection of an aqueous solution containing the Abeta 1-40 fragment into the hippocampus, long stretches of the CA1 subfield were either deprived of neurons or most of the neurons were degenerating. By contrast, in animals with deposits containing Abeta 1-28, Abeta 1-42 or water, neuronal degeneration or depletion only occurred in a reduced area around the place where the implant needle penetrated the CA1 subfield. In animals injected with Abeta 1-40, many profiles in the CA1 subfield and dentate gyrus were undergoing apoptosis, as seen using preparations processed by routine histology or the TUNEL technique for detection of fragmented DNA. In addition, there was higher infiltration by ED1-positive, activated microglia-macrophagic cells in Abeta 1-42 deposits than in deposits of Abeta 1-40. The present results suggest that the intrahippocampal injection of toxic Abeta fragments produces neuronal degeneration in the rat CA1 subfield when using the appropriate protocol, and, thus, can provide an in vivo model to investigate the neurotoxic effects of Abeta and for the evaluation of drugs with potential anti-neurodegenerative activity.

beta-amyloid (beta A) deposition is a key event in the etiopathogenesis of Alzheimer's disease (AD), contributing to neuronal degeneration and cognitive impairment in AD patients. Both neurotrophic and neurotoxic actions of beta A have been demonstrated in experimental conditions. In order to further characterize the effects of brain beta A deposits on behavioral processes, we evaluated psychomotor activity (PMA), psychomotor coordination (PMC) and learning in a passive avoidance task (PAL) in rats with unilateral or bilateral 2 microliters injections of beta-amyloid (1-28) protein (beta A; 1.5 nmol/microliter) or vehicle (water; W) into the hippocampus, 1 and 4 weeks after neurosurgery. The extent of neuronal loss in the lateral blade of the gyrus dentatus (LBGD) and the area percentage occupied by APP immunoreactivity in neurons of the CA3c subfield of the hippocampus were also measured in animals with unilateral beta A implants. PMA levels were similar in water- and beta A-injected animals 1 and 4 weeks after recovery. As compared to water-injected rats, beta A animals showed reduced PMC values 1 week, but not 4 weeks, after injections. beta A also impaired learning acquisition in a passive avoidance task, reducing the number of avoidances and mean latency per trial at both 1 and 4 weeks postsurgery in rats with unilateral or bilateral beta A implants. The extent of neuronal loss in the LBGD) was not different in rats receiving water or beta A injections. Hippocampal APP expression tended to increase in beta A-implanted rats and showed a negative correlation with cognitive performance at the 4-week period. According to these results it seems that beta A implants into the hippocampus reduce psychomotor coordination performance in a transient manner, with no effect on psychomotor activity, and induce durable learning impairment in rats, and that changes in cognitive performance correlate with histochemical parameters such as APP expression. In conclusion, the present results contribute to a better understanding of beta A-induced behavioral alterations and to the identification of potential molecular mechanisms involved in cognitive dysfunctions in this animal model of neurodegeneration.

Rats with neurotoxic lesions, induced by single or double bilateral injections of ibotenic acid into different parts of the nucleus basalis of Meynert (nbM), showed increased psychomotor activity (PMA) and impaired learning in a passive avoidance task. Behavioral deficits were similar in all the groups of lesioned animals, suggesting that the lesion site was not relevant for the ibotenic effects under testing procedures used here. In another experiment, nbM-lesioned rats received acute or daily (5 days) i.p. injections of vehicle or anapsos (100 mg/kg) from the 7th day after surgery. Data indicated that nbM lesions induced an increased production of interleukin-1 beta (IL-1 beta), motor hyperactivity and learning impairment, and that anapsos, a vegetal extract with immunomodulatory activity, reversed brain IL-1 beta overexpression and behavioral alterations in lesioned rats. These results confirm the involvement of IL-1 beta in neurodegeneration associated with cholinergic deficits and the potential utility of compounds with neuroimmunotrophic activity as a new therapeutic strategy in neurodegenerative disorders.

Citicoline is a choline donor involved in the biosynthesis of brain phospholipids and acetylcholine extensively used in the treatment of neurodegenerative diseases. In this study we investigated the effects of the oral administration of citicoline alone (C1000:1000 mg/day; C500:500 mg/day) or in combination with nimodipine (C +NI:300 + 90 mg/day) during 4 weeks on memory performance in elderly subjects with memory deficits and without dementia (N = 24; age = 66.12 +/- 10.78 years; MMS score = 31.69 +/- 2.76). Results indicated that citicoline in comparison with placebo improves memory in free recall tasks, but not in recognition tests. A significant improvement in word recall (5.17 +/- 1.1 vs. 3.95 +/- 1.2 omissions; p < 0.005), immediate object recall (6.5 +/- 1.6 vs. 5.5 +/- 1.2 omission; p < 0.05) and delayed object recall (8.5 +/- 2.1 vs. 6.7 +/- 2.4 omissions; p < 0.005) was observed after citicoline treatment. Similar results were found in the three subgroups of treatment (8 subjects per group), suggesting that citicoline possesses memory-enhancing activity at doses of 300-1000 mg/day. A decrease in systolic blood pressure and minor changes in lymphocyte cell counting were also observed in old subjects after receiving citicoline. These effects are consistent with the vasoregulatory and neuroimmune actions of citicoline and suggest that this compound may improve memory by acting on mechanisms of brain neurotropism and cerebrovascular regulation. According to the present results, showing that citicoline improves memory performance in elderly subjects, we concluded that this molecule is suitable for the treatment of memory deficits in old people.

A general methodology for the analysis of human diseases at the nucleic acid level has been described. This method consists of a semiautomated, nontoxic analytic procedure, staining DNA bands with silver nitrate instead of ethidium bromide after DNA amplification by PCR. This technique provides a sensitive assay with enhanced accuracy and offers the possibility of carrying out the molecular analysis of several polymorphisms in a single day simplifying molecular genetics with diagnostic purposes.

The enzyme Cu-Zn-SOD is a metalloenzyme that catalyzes the dismutation of the superoxide radical into hydrogen peroxide and molecular oxygen, being a defense system against free radical formation. Free radical reactions are implicated in a variety of physiological and pathological processes as aging, apoptosis and neurodegenerative diseases, and abnormalities associated with SOD have been recently documented in several neurodegenerative processes. In this study, we have evaluated the effect of anapsos on Cu-Zn-SOD activity in rats with injections of beta-amyloid protein or water bilaterally into the hippocampus. These injections caused severe cell depletion in the gyrus dentatus. Anapsos is a biological extract obtained from the fern Polypodium leucotomos with immunomodulatory and anti-neoplastic effects tested in animals and humans. Cu-Zn-SOD activity was measured in the hypothalamus, hippocampus, cerebral cortex, liver and spleen of rats treated i.p. with three doses of anapsos for 7 days (4, 20 and 100 mg/kg/day). Control animals were treated with saline solution under the same conditions. Anapsos significantly modified enzyme activity in all the areas tested. Lower doses of anapsos produced decreased SOD activity in the hypothalamus, hippocampus, liver and spleen, while in the cerebral cortex, a significant dose-dependent increase in SOD activity was observed. These results indicate that anapos was able to modify Cu-Zn-SOD activity in this animal model of neuronal degeneration, which may indicate the participation of anapsos in mechanisms of tissue repair after brain damage.

Behavioral dysfunction is a problem in patients with Alzheimer disease (AD), and is apparent in up to 67% of individuals. Such changes are a primary cause of individual institutionalization and often lead to their functional disability. As AD progresses, the worsening of behavioral dysfunction becomes increasingly evident and is linked with decreased patient survival. Unfortunately, some of the more common drug therapies used in AD patients to stabilize other facets of their disease worsen behavioral dysfunction. Behavioral changes are associated with endogenous and exogenous factors such as disease stage, environmental factors, other medical conditions, drug regimen, and AD genotype. The most commonly examined and important genotype in AD is the apolipoprotein E (APO E) series, and APO E genotyping is also a useful diagnostic tool. The most frequent APO E genotypes encountered in AD are APO E-4/4, APO E-3/4, and APO E-3/3. In the current study, AD behavioral dysfunction, anxiety, and psychoses were commonly associated with the APO E-3/3 genotype, whereas disorientation, agitation, depression and motor disorders were common among patients with the APO E-4/4 and APO E-3/4 genotypes. These differences were not statistically significant but they suggest that different APO E genotypes influence the phenotypic expression of specific noncognitive symptoms, including behavioral function, in AD.

Lactate dehydrogenase (LDH) enzyme activity was measured in the supernatant of rat aortic endothelial cell cultures to evaluate the cytotoxic effects of two proinflammatory mediators such as LPS and histamine, as well as beta-amyloid protein (fragment 1-28) on endothelial cells. In the same culture we also studied the influence of different sera from patients with Alzheimer's disease (AD) or healthy elderly subjects. The results indicate that very low concentrations (1 microgram/ml) of beta-amyloid or histamine (1 microM) were able to produce cell damage after an incubation time of 4 h. Treatment with LPS induced cell damage at the highest concentration (2.5 micrograms/ml) after 24 h of incubation. When endothelial cells were treated with serum from AD and non-AD individuals, an inhibition of endothelial cell proliferation was observed only with activated AD serum. These results indicate that rat endothelial cell cultures represent an important model to study inflammatory mediators and to evaluate the therapeutic effect of antiinflammatory molecules in AD and other neurodegenerative disorders.

Although memory disorders and the aphaso-apraxo-agnosic syndrome are the most relevant clinical symptoms in dementia, behavioral changes, mood-related disturbances and sleep disorders are the major cause of institutionalization and caregiver concern. In the present study we have investigated the frequency and progression of cognitive and noncognitive symptoms in Alzheimer's disease (AD) as well as the APOE-related frequency of clinical symptoms in dementia. Memory decline (100%), aphasia (94%), apraxia (99%), agnosia (94%) and motor dysfunction (90%) appeared in practically all cases with mild (GDS-3), moderate (GDS 3-4) and severe (GDS 6-7) dementia. The most frequent noncognitive symptoms include anxiety (76%), depression (68%), behavioral changes (67%), psychotic symptoms (43%), sleep disorders (43%), incontinence (23%) and cerebrovascular symptoms (75%). Anxiety, depression, behavioral changes, psychotic symptoms, motor dysfunction and cognitive deterioration paralleled the severity of dementia, increasing their frequency from mild to severe dementia. The most important sleep disorders were irregular sleep-wake pattern (67%) and insomnia (47%). Disorientation (90%) and drug administration (88%) appeared to be the most important factors in causing sleep disorders in dementia. Disorientation, agitation and motor disorders were slightly more frequent in patients with APOE-4/4, while anxiety and sleep disorders appeared more frequently in APOE-3/4. Behavioral changes and psychotic symptoms did not show any clear association with specific APOE subtypes. In conclusion, our results suggest that noncognitive symptoms are very important clinical events in the disease progression and in decision making for therapeutic intervention and institutionalization. Furthermore, it is likely that some brain dysfunctions leading to particular clinical symptoms might be associated with specific AD genotypes.

In this study, we have evaluated the levels of blood histamine, serum interleukin-1 beta (IL-1 beta), and plasma tumor necrosis factor-alpha (TNF-alpha) in 20 patients with mild to moderate Alzheimer disease (AD; 13 early onset and 7 late-onset AD subjects) and in 20 age-matched control subjects (C). AD patients showed higher concentrations of histamine (AD = 452.9 +/- 237.9 pmol/mL; C = 275.3 +/- 151.5 pmol/mL; p < 0.05) and IL-1 beta (AD = 211.2 +/- 31.1 pg/mL; C = 183.4 +/- 24.4 pg/mL; p < 0.01), and lower values of TNF-alpha (AD = 3.59 +/- 2.02 pg/mL; C = 9.47 +/- 2.64 pg/mL; p < 0.001) than elderly controls. Increased levels of histamine and decreased levels of TNF-alpha were observed in both early onset AD (EOAD) and late-onset AD (LOAD) patients, but only EOAD subjects had elevated serum IL-1 beta values compared with age-matched controls. Age negatively correlated with histamine (r = -0.57; p < 0.05) and positively with IL-1 beta levels (r = 0.48; p < 0.05) in healthy subjects, but not in AD, whereas a positive correlation between TNF-alpha scores and age was only found in AD patients (r = 0.46; p < 0.05). Furthermore, histamine and TNF-alpha values correlated negatively in AD (r = -0.50, p < 0.05). In addition, cognitive impairment increased in patients with lower TNF-alpha and higher histamine and IL-1 beta levels, as indicated by the correlations between mental performance scores and histamine (r = -0.37, ns), IL-1 beta (r = -0.33, ns) and TNF-alpha levels (r = 0.42, p < 0.05). Finally, histamine concentrations decreased as depression scores increased in AD (r = -0.63, p < 0.01). These data suggest a dysfunction in cytokine and histamine regulation in AD, probably indicating changes associated with inflammatory processes.

S-9977-2 is a new molecule with promnesic and antiamnesic properties. In this study, we investigated the effects of i.p. administration of S-9977-2 on histamine (HA) levels in several regions of the rat brain, and also in peripheral areas, using high performance liquid chromatography (HPLC) with fluorometric detection. S-9977-2 (0.025 mg/kg) induced an increase in the content of HA in the anterior hypothalamic region, with a significant effect 30 min after injection (1.77 +/- 0.36 nmol/g vs. 2.43 +/- 0.40 nmol/g; p < 0.01). It did not significantly modify HA levels in the posterior hypothalamic region, hippocampus, fronto-parietal cortex, blood, spleen, or adrenal glands. A clear tendency to increase HA content was observed in the hippocampus (0.16 +/- 0.03 nmol/g vs. 0.39 +/- 0.31 nmol/g, ns) and fronto-parietal cortex (0.55 +/- 0.45 nmol/g vs. 0.70 +/- 0.28 nmol/g; ns). S-9977-2 at doses of 0.005 and 0.125 mg/kg i.p. did not produce any significant change in HA levels in any of the samples tested 30 min after administration. The present results demonstrate that S-9977-2 increases HA levels in the anterior hypothalamus, which is the brain region with the highest content in histaminergic fibers. This fact suggests that the neuronal HA system might be involved directly or indirectly in the pharmacological actions of S-9977-2.

CDP-choline was given to patients with Alzheimer's disease (AD) at a daily dose of 1000 mg/day p.o. for one month. This compound slightly improved mental performance, tended to reduce theta activity in fronto-temporal regions, increasing alpha power in occipital areas, and enhanced cerebrovascular perfusion by increasing blood flow velocity and reducing pulsatility and resistance indexes. In addition, CDP-choline diminished histamine and interleukin-1 levels in blood and serum, respectively, and increased plasma TNF.

The early identification of Alzheimer's disease (AD) requires a multifactorial diagnostic approach. Components of a comprehensive assessment include a clinical examination, neuropsychological and psychometric evaluations, biochemical tests, cardiovascular and radiological examinations, neuroimaging, brain bioelectrical activity mapping, an evaluation of brain hemodynamics, and assessment of biological markers. Genetic testing should be incorporated into the diagnostic protocol only for research purposes and risk evaluation. The genetic characterization of familial AD genotypes (FAD-21, FAD-14, FAD-1, FAD-19, APP-21m) can help define the phenotypic profiles of AD clinical subtypes. The correlation of genotypic and phenotypic profiles might have a predictive value in terms of AD diagnosis and therapeutic responses to particular drugs. However, available genetic markers (APP-21m, APO-E) are not yet conclusive for diagnostic purposes. In contrast, AD-related Apo-E genotypes appear to correlate with defined AD phenotypes. The presence of an Apo-E4 allele seems to represent an important risk factor for dementia.

Besides the role of histamine (HA) as a neurotransmitter, a new concept has emerged presenting HA as an immunomodulator. Several studies have demonstrated interactions among HA, interleukin-1 beta (IL-1 beta) and tumor necrosis factor (TNF), suggesting a possible bidirectional communication among them. In this study we have investigated the effects of i.p. administrations of HA diphosphate (6 mumol/kg) and alpha-fluoromethylhistidine (FMH; 100 mg/kg) on TNF-alpha levels in the hippocampus, hypothalamus, and posterior hypothalamic region of the rat brain. The concentrations of TNF-alpha at 0 (Control, C) and 30 min after i.p. administration of HA were 0.26 +/- 0.02 pg/mg and 0.32 +/- 0.02 pg/mg in the hippocampus, 0.46 +/- 0.04 pg/mg and 0.09 +/- 0.006 pg/mg (p < 0.01) in the hypothalamus, and 0.47 +/- 0.05 pg/mg and 0.26 +/- 0.05 pg/mg in the posterior hypothalamic region. Three hours after FMH administration, an increase in the hippocampal levels of TNF-alpha was observed (0.43 +/- 0.04 pg/mg; p < 0.01), while in the hypothalamus (0.11 +/- 0.02 pg/mg; p < 0.01) and in the posterior hypothalamic region (0.21 +/- 0.04 pg/mg; p < 0.05) a decrease in TNF-alpha levels was detected. These results suggest that changes in the histaminergic system influence TNF-alpha production in the brain in an area-specific fashion.

Histamine (HA) acts as a neurotransmitter and/or neuromodulator in mammalian brain. Central HA has been found to be involved in the regulation of behavioral, cognitive, neurovegetative, neuroendocrine and neuroimmune functions. In this study we have evaluated psychomotor activity (PMA) and passive avoidance behavior (PAB) in rats with bilateral neurotoxic lesions in the posterior hypothalamic region (PHR) (L), where histaminergic neurons are located, and in sham-operated rats (S), two weeks after neurosurgery. In an open-field paradigm, lesioned rats showed higher PMA scores than sham-operated animals. However, L rats exhibited a significant decrease in PMA on consecutive days (motor habituation) similar to that found in S rats. In a maze paradigm, in which the animals had to learn to stay on a neutral platform in order to avoid a 1.5 mA electric footshock during 10 trails, no significant differences were observed between L and S rats on the task performance. According to the present results, it seems that bilateral neurotoxic lesions in the PHR induced hyperactivity with no apparent effects on PAB, suggesting that neuronal HA might be involved, directly and/or by influencing arousal/alertness-mediated mechanisms, in the regulation of PMA processes.

CDP-choline participates in brain phospholipid metabolism and acts as an endogenous intermediate in a biosynthetic pathway incorporating free choline into phosphatidylcholine and choline plasmalogens in several tissues, including the central nervous system (CNS). In patients with chronic cerebrovascular disorders, CDP-choline reduces the slow delta frequencies and increases alpha activity in spectral electroencephalogram analysis. We have studied the effect of CDP-choline (cytidine-S-diphosphate-choline; 1000 mg/day x 30 days, p.o.) on brain electrical activity mapping and mental performance in 19 Alzheimer's disease (AD) patients (10 males/9 females; age: 66.21 +/- 1.48 years; Mini-Mental State Examination (MMSE): 26.55 +/- 1.22, Spanish version max. score 35). EEG was registered from 19 electrodes placed according to the 10-20 system and digitalized online. Artefact-free 2-s periods were visually selected, submitted to a frequency analysis and averaged across periods. CDP-choline significantly decreased spectral amplitude in the theta band in F4, F8, and T4 electrodes, and did not modify relative power parameters in any of the frequency bands (delta, theta, alpha, beta) as compared to basal recordings. In patients with late-onset AD (LOAD; N = 6; age: 73.5 +/- 1.34 years; MMSE: 28.75 +/- 2.33), CDP-choline tended to increase relative alpha power in O1 and O2 electrodes. No changes were found in early-onset AD patients (EOAD; N = 13; age: 62.85 +/- 1.21 years; MMSE: 25.54 +/- 1.4). We detected a significant improvement in mental performance after 1 month of treatment with CDP-choline in patients with early-onset AD in whom brain electrical activity data correlated with cognitive parameters. It is likely that the bioelectrical changes induced by CDP-choline in AD are the result of its immunogenic and/or neurotrophic activity in the vicinity of the vascular microenvironment.

Histamine (HA) is a biogenic amine involved in the regulation of neurovegetative, cognitive, neuroendocrine and neuroimmune functions in the central nervous system (CNS). A bidirectional interaction between the CNS and the neuroimmune system has been demonstrated in recent years. However, data concerning brain HA-cytokine interactions are scarce. In this study we have evaluated tumor necrosis factor-alpha (TNF-alpha) levels in the posterior hypothalamic region (PHR) and hippocampus (HP) of rats with: (a) bilateral ibotenic acid neurotoxic lesions in histaminergic neurons located in the PHR (I); (b) saline injections in the PHR (S); and (c) sham operation (C), two weeks after neurosurgery. The bilateral disruption of PHR HA neurons with ibotenic acid decreased TNF-alpha levels in the PHR with respect to sham-operated (C), but not saline-injected (S), rats. In contrast, hippocampal TNF-alpha concentrations were higher in lesioned rats (I) than in C and S animals. Our results indicate that the neurotoxic destruction of HA neurons decreases TNF-alpha synthesis in the hypothalamus while enhancing TNF-alpha production in the hippocampus, suggesting that neuronal HA might be involved in the regulation of the brain TNF-alpha system.

Histamine (HA) is a known neurotransmitter with a wide spectrum of biological actions at the central and peripheral levels. Recently, it has been found that HA is involved in the regulation of immune cell function, acting as an immunomodulator. A hyperactivation in the histaminergic system has been demonstrated in Alzheimer's disease (AD), including increased levels of HA in brain, serum, and cerebrospinal fluid of AD patients. In addition, changes in phospholipid metabolism and neuroimmune function have been reported in AD. CDP-choline (cytidine-5-diphosphate-choline) participates in the phospholipid metabolism pathway incorporating free choline into phosphatidyl-choline and choline plasmalogens in several tissues, including the central nervous system. In this study we have measured the concentration of HA in blood from patients with early-onset AD (EOAD) and late-onset AD (LOAD) under treatment with CDP-choline (1000 mg p.o. x30 days). HA was measured by high performance liquid chromatography (HPLC) with fluorometric detection. CDP-choline reduced the basal levels of blood HA in both EOAD and LOAD by 2-fold. The reduction in blood HA content was observed 2 h after CDP-choline administration and gradually progressed for 30 days of treatment. These results confirm the potential immunogenic effects of CDP-choline and also that an excess of HA might influence some etiopathogenic events in AD.

CDP-choline (cytidine-5-diphosphate-choline) is an acetylcholine precursor frequently used in cerebrovascular disorders and psychoorganic syndromes. Furthermore, several authors have demonstrated the positive effects of CDP-choline on cognitive disorders and memory deficits. In the present study, the effects of CDP-choline (1000 mg/day, p.o. for 1 month) on cognition, evaluated by the Mini-Mental State Examination (MMSE) of Folstein et al., and on blood flow velocities, measured by transcranial Doppler ultrasonography (TCD), were investigated in patients with Alzheimer's disease: (AD, n = 20, age: 66.75 +/- 6.73 years, range: 57-78 yr). Cognitive function was measured by means of the MMSE in basal conditions (A) and after 1 month of treatment with CDP-choline (C). TCD measures were taken through the temporal window for right (MCA-R) and left (MCA-L) middle cerebral arteries with a 2 MHz pulsed transducer using a TC-2000S in basal conditions (A), 1 h after the administration of CDP-choline (B) and after 1 month of treatment with CDP-choline (C). MMSE scores were significantly increased (p < 0.005) in patients with early-onset Alzheimer's disease (EOAD) after CDP-choline treatment. Moreover, the orientation subtest significantly increased in the global group of AD patients (p < 0.01) and in EOAD patients (p < 0.02). Significant differences (p < 0.05) were also found in MCA-Land MCA-R measures between recordings. These results suggest that CDP-choline influences cognitive and cerebrovascular function in Alzheimer's disease, probably through a mechanism linked to an immunogenic and/or neurotrophic effect at the microvascular niche.(ABSTRACT TRUNCATED AT 250 WORDS)

Recent investigations indicate that a neuroimmune reaction, associated with inflammatory mechanisms, can contribute in Alzheimer's disease (AD) to cell damage and neurodegeneration. Activation of microglial cells, expression of immunohistochemical markers of brain immune function, the presence of complement proteins in brain tissue and changes in cytokine production have been reported in AD. We have studied the concentration of interleukin-1 beta (IL-1 beta) in different regions of the central nervous system (CNS) in post-mortem samples from patients with AD or vascular dementia (VD) and in age-matched control subjects (CS). IL-1 beta levels were significantly higher in AD than in VD or CS in the frontal cortex, parietal cortex, temporal cortex, hypothalamus, thalamus and hippocampus. The highest increases in IL-1 beta levels were observed in the frontal cortex (CS = 0.75 +/- 0.045; AD = 2.47 +/- 0.12, p < 0.001; VD = 1.52 +/- 0.078 pg/mg, p < 0.001) and hippocampus (CS = 0.71 +/- 0.042; AD = 2.63 +/- 0.19, p < 0.001; VD = 1.21 +/- 0.23 pg/mg, p < 0.01). No significant changes were detected in the occipital cortex and cerebellum in either AD or VD. These results clearly demonstrate that demented patients show a generalized increment of IL-1 beta production in the CNS, with maximum response in those brain regions where AD neuropathology is most prominent. This overall increase in cytokine production might represent an early event in the activation of a neuroimmune cascade leading to cell death and neurodegeneration in brain regions where a primary cause (e.g., genetic, toxic, vascular) facilitates the induction of resting microglia for firing brain immune function.

Changes in neuroimmune parameters and cytokine production have been reported in patients with Alzheimer's disease, including increased levels of interleukin-1 (IL-1) and histamine in brain tissue, cerebrospinal fluid and serum. Specific neuroimmune reactions may be responsible in part for astrogliosis and neuronal death in particular circumstances. Since IL-1 and tumor necrosis factor (TNF) tend to act synergistically in physiological conditions and in some pathological processes, we have studied the concentration of TNF-alpha in patients with Alzheimer's disease (AD) or multi-infarct dementia (MID) and in age-matched control subjects (CS) in order to evaluate possible changes in the levels of this cytokine with potential influence on the pathogenesis of AD. Serum TNF-alpha levels were significantly lower in AD (2.5 +/- 1.25 pg/ml, p < 0.01) and MID (1.64 +/- 1.17 pg/ml, p < 0.001) than in CS (10.66 +/- 8.92 pg/ml). A negative correlation between serum TNF-alpha levels and age in AD was found (r = -0.645, p < 0.01); however, no significant correlations were detected between serum TNF-alpha levels and mental performance, cerebrovascular risk, heart rate and blood pressure in either AD or MID. In conclusion, there is a marked reduction in the concentration of serum TNF-alpha in both AD and MID which seems to be poorly related to cognitive dysfunction and/or neurovascular damage, probably reflecting an endogenous immune dysregulation and/or an inhibitory reactive process in demented patients.

The effect of intracerebroventricular (icv) administration of lipopolysaccharide on histidine decarboxylase activity and histamine content in the hypothalamus were investigated in male mice of ddY strain in vivo. Two-fold increase in histidine decarboxylase activity (HDC) was observed 4 h after administration of 50 mcg lipopolysaccharide, and HDC activity returned to the basal level within 12 h after injection. Furthermore, histamine contents showed a slight decrease at 1 and 2 h and a mild increase at 12 h after administration. However, changes in histamine content were not statistically significant. These results suggest that the increase of HDC activity in the hypothalamus by lipopolysaccharide may be involved in the central neuroimmune responses.

The cholinergic dysfunction present in Alzheimer's disease (AD) might be due to a specific vulnerability of cholinergic neurons linked to neurotrophic imbalance, neuroimmune impairment, and/or direct effects of beta-amyloid deposition and NFT formation in ACh neurons. The presence of abnormal epitopes exposed on neuronal membranes may contribute to the activation of resting microglia initiating a neuroimmune cascade leading to cell destruction. According to this hypothesis, a multifactorial treatment in AD should produce: 1) inhibition of beta-amyloid and NFT formation; 2) restoration of neuronal membrane integrity; and 3) control of neuroimmune auto-aggression. Since interleukin-1 (IL-1) is an APP gene promoter showing a progressive increase in body fluids in parallel with mental deterioration in AD patients, we have studied the effects of CDP-choline on cognition, several biological parameters, and IL-1 beta production in AD and multi-infarct dementia (MID) in order to elucidate whether this compound alone or in combination with other drugs is able to restore immune function and improve mental performance in senile dementia.

Blood flow velocities were measured in right and left middle cerebral arteries (MCAs) and in basilar artery (BA) on rest conditions by transcranial Doppler ultrasonography in three groups of patients: a) Alzheimer's disease patients (AD, N = 12, age = 63.5 +/- 6.6 years), b) patients with multi-infarct dementia (MID, N = 12, age = 72.8 +/- 9.0 years), and c) control subjects (CS, N = 12, age = 57.20 +/- 7.5 years). TCD measures were taken through the temporal window for MCA recordings, and transforaminal approach for BA recordings, with a 2 MHz ultrasonic probe using a TC-2000S (EME). A significantly (p < 0.05) decrease in TCD measures was found in right and left MCA and BA of dementia patients with respect to controls. Data analysis included a significant correlation between blood flow velocities and Hachinski scores (p < 0.016) in multi-infarct dementia patients. These results appear to show a general hypoperfusion pattern in the brain of senile dementia patients.