Microcebus murinus: a useful primate model for human cerebral aging and Alzheimer's disease?

Age-associated dementia, in particular Alzheimer's disease (AD), will be a major concern of the 21st century. Research into normal brain aging and AD will therefore become increasingly important. As for other areas of medicine, the availability of good animal models will be a limiting factor for progress. Given the complexity of the human brain, the identification of appropriate primate models will be essential to further knowledge of the disease. In this review, we describe the features of brain aging and age-associated neurodegeneration in a small lemurian primate, the Microcebus murinus, also referred to as the mouse lemur. The mouse lemur has a relatively short life expectancy, and animals over 5 years of age are considered to be elderly. Among elderly mouse lemurs, the majority show normal brain aging, whereas approximately 20% develop neurodegeneration. This Microcebus age-associated neurodegeneration is characterized by a massive brain atrophy, abundant amyloid plaques, a cytoskeletal Tau pathology and a loss of cholinergic neurons. While elderly mouse lemurs with normal brain aging maintain memory function and social interaction, animals with age-associated neurodegeneration lose their cognitive and social capacities and demonstrate certain similarities with age-associated human AD. We conclude that M. murinus is an interesting primate model for the study of normal brain aging and the biochemical dysfunctions occurring in age-associated neurodegeneration. Mouse lemurs might also become an increasingly important model for the development of novel treatments in this domain.     

Distribution of choline acetyltransferase,acetylcholinesterase, muscarinic receptor binding,and choline uptake in discrete areas of the rat medulla oblongata

Quantitative measurements were made of choline acetyltransferase (CAT) activity, acetylcholinesterase (AChE) acitivity and cholinergic muscarinic receptor binding ([³H]QNB) in eight areas of a cross-section of the rat medulla oblongata. A fourth cholinergic parameter, high-affinity choline uptake, was measured in three groups of these areas. CAT, AChE and [³H]QNB binding were found to be highest in the hypoglossal nucleus and the dorsal motor nucleus of the vagus; the lowest value was in the area which contains the inferior olive and the corticospinal tract. The distribution of AChE and CAT acitivities varied approximately 7- to 10-fold among the eight regions examined, whereas that of the muscarinic receptor varied only about 4-fold. The Na⁺-dependent high-affinity choline uptake varied approximately 20-fold from the region with the lowest activity (inferior olivary nucleus and corticospinal tract) to that with the highest activity (tissue areas containing the dorsal motor nucleus, hypoglossal nucleus, nucleus of the solitary tract and nucleus cuneatus). The four cholinergic parameters are statistically correlated throughout all the areas of the medulla which were studied.     

Time-dependent changes in superoxide dismutase, catalase, xanthine dehydrogenase and oxidase activities in focal cerebral ischaemia

Time-dependent changes in the activities of antioxidant enzymes and an oxidant enzyme, xanthine oxidase (XO), were detected in primary and peri-ischaemic brain regions during permanent occlusion of the middle cerebral artery (MCAO) in rats. There were no changes in superoxide dismutase (SOD) and catalase (CAT) activities after 3 h of MCAO, whereas antioxidant enzyme activities decreased significantly in ischaemic brain areas following 24 h of ischaemia. After 48 h, the enzyme activities returned to the baseline but then a further increase was observed in ischaemic brain areas by 72 h post-ischaemia. Normally, XO exists as a dehydrogenase (XD), but it is converted to XO which contributes to injury in some ischaemic tissues. The XO activity increased slightly at 3 h after ischaemia, but after 24 h of ischaemia it returned to the baseline and then remained relatively unchanged in ischaemic areas. Pretreatment with allopurinol before ischaemia prevented changes in SOD and CAT activities and attenuated brain oedema during 24 h of ischaemia. Neither XO nor XD activity changed in allopurinol-treated rats at the times of ischaemia. These results indicated that ischaemic brain tissue remained vulnerable to free radical damage for as long as 48 h after ischaemia, and XO was probably not an important source of free radicals in cerebral ischaemia.     

Decreased Catalase Activity but Unchanged Superoxide Dismutase Activity in Brains of Patients with Dementia of Alzheimer Type

Abstract: "Oxidative stress" may be of significance in the etiopathogenesis of dementia of Alzheimer type (DAT). Therefore, we measured activities of the enzymes superoxide dismutase (SOD) and catalase (CAT), which detoxicate reactive oxygen species. Enzyme activities were measured postmortem in basal ganglia, cortical, and limbic brain regions of patients with DAT and age-matched controls. SOD activity increased with age in basal nucleus of Meynert. However, there was no significant difference in SOD activity between DAT and controls. CAT activity was independent of age and postmortem time. There were significant reductions in CAT activity in parietotemporal cortex, basal ganglia, and amygdala in DAT compared with controls ( p < 0.05 to 0.01). Our findings are in line with the assumption that reactive oxygen species could contribute to the pathogenesis of DAT. Absence of these changes in basal nucleus of Meynert might reflect retrograde degeneration of cholinergic fibers.     

Evidence for a cerebral cholinergic system and suggested pharmacological patterns of neural organization in the prostomium of the polychaete Nereis virens (SARS)

The histological visualization of choline acetyltransferase (CAT) and acetylcholinesterase (AChE) on frozen sections of prostomia of Nereis virens indicate a concentration of cholinergic activity in the anterior brain. Components are probably sensory epithelial cells with cholinergic axons entering the brain in cephalic nerves and efferent cholinergic axons to prostomial muscle leaving the brain in the same nerves. There are also subepidermal cholinergic cells that may be second order motor neurons serving epidermal mucous cells. The smaller, second lobe of the corpora pedunculata and its associated vertical fibre tract are CAT⁴ and appear continuous, on each side of the cerebral ganglion, with a dorsal and a ventral longitudinal bundle of AChE⁺ fibers. This system tapers to nothing at the level of the posterior eyes. There is a small AChE⁺ component to each optic nerve and AChE is present in the nuchal epithelium. These observations are discussed in relation to earlier studies on aminergic and neurosecretory activity in the same ganglion.     

Effects of altered cholinergic function on working and reference memory in the rat

Many data suggest that the brain's cholinergic neurons participate in the control of memory and it has been suggested that cholinergic systems are involved differentially in working and reference memory. To test this hypothesis the effects on memory of unilateral injections of the neurotoxins, quinolinic acid or kainic acid into the cortically projecting cholinergic cells of the nucleus basalis magnocellularis (nbm) were evaluated. In experiment 1, quinolinate-injected (n = 7) and sham-operated (n = 7) rats were tested in a T-maze alternation task that requires working memory. Lesion rats performed significantly more poorly than shams and subsequent biochemical assays of cortical choline acetyltransferase (CAT) activity revealed significant reductions in the lesion rats. In experiment 2, kainate-injected (n = 9) and sham-operated (n = 8) rats were trained in an eight-arm radial maze with only four arms baited. Lesion rats made significantly more working memory errors (entries into baited arms from which the food had already been collected) than reference memory errors (entries into never baited arms). CAT assays showed that the lesion led to a decrease in cortical CAT with no significant change in hippocampal CAT. The results of these studies support the hypothesis that cholinergic neurons of the basocortical system may be differentially involved in working and reference memory.     

Down''s Syndrome Individuals Begin Life with Normal Levels of Brain Cholinergic Markers

We measured the activities of the cholinergic marker enzymes choline acetyltransferase (ChAT) and acetylcholinesterase (AChE) in autopsied brains of seven infants (age range 3 months to 1 year) with Down's syndrome (DS), a disorder in which virtually all individuals will develop by middle age the neuropathological changes of Alzheimer's disease accompanied by a marked brain cholinergic reduction. When compared with age-matched controls cholinergic enzyme activity was normal in all brain regions of the individuals with infant DS with the exception of above-normal activity in the putamen (ChAT) and the occipital cortex (AChE). Our neurochemical observations suggest that DS individuals begin life with a normal complement of brain cholinergic neurons. This opens the possibility of early therapeutic intervention to prevent the development of brain cholinergic changes in patients with DS.     

Impact of epigallo catechin-3-gallate on acetylcholine-acetylcholine esterase cycle in aged rat brain

Neurotransmission plays an important role in communication of messages in brain. Cholinergic alterations during aging are associated with learning and memory. Neurotransmitters and enzymes that influence these neurotransmitters are significant in age-associated memory. Neurotransmitters like acetylcholine, serotonin and dopamine levels were studied. Kinetics of acetylcholine esterase was studied. There was an alteration in km and Vm values which was brought back to near-normalcy by EGCG. Behavioural changes were assessed by radial maze experiment. EGCG, a good neuroprotective drug proved to alleviate the behavioural alterations in aged rat brain. Acetylcholine esterase was partially purified from rat brain and assayed in vitro. Several modifiers like EGCG and donepezil were added in silico and the activity of the enzyme was calculated. EGCG increased the activity when compared to negative control, donepezil. Using bioinformatics tools EGCG, acetylcholine and donepezil were docked with acetylcholine esterase. EGCG formed a good docking-complex with the enzyme. Thus, it shall be hypothesized that the neuroprotective activity of EGCG might be due to its influence on cholinergic neurotransmission thereby improving the cognitive functions of the brain.     

The effect of cobrotoxin on cholinergic neurons in the mouse.

By using multiple time-point constant-rate infusions of deuterium-labeled phosphorylcholine, appropriate kinetic parameters were obtained for use in the calculation of the turnover rate of acetylcholine (TRACh) in selected mouse brain regions. After obtaining these data, the relationship between the analgesic agent cobrotoxin (CT) and the activity of central cholinergic neurons was investigated by determination of TRACh in selected mouse brain regions 3 hours following intracerebroventricular (i.c.v.) injection of CT. There were no obvious changes in the concentrations of ACh and choline (Ch) in the cortex, hippocampus, hypothalamus, midbrain, striatum, or thalamus of the mouse after injection of an analgesic dose of CT (2 micrograms, i.c.v.). TRACh in the thalamus and the striatum were significantly increased, as compared to controls. On the other hand, i.c.v. injection of CT was found to significantly reduce TRACh in the hippocampus and midbrain. These results suggest that the activity of hippocampal and midbrain cholinergic neurons is suppressed by CT, whereas the activity of striatal and thalamic cholinergic neurons is increased by CT at a time when a maximum analgesic response to CT is expressed.     

Cell interactions and the regulation of cholinergic enzymes during neural differentiation in vitro.

Seven-day-old chick embryo neural retina (NR), telencephalon (T), optic lobe (OL), and rembencephalon (Ro) were dissociated, and the resulting cell suspensions were allowed to reaggregate in vitro during 3 days either independently or in different binary combinations. Interactions could be detected by the comparison of the activity of the enzymes of the cholinergic system, choline acetyltransferase (CAT) and acetylcholinesterase (ACE), in “pure” and “combined” aggregates.The results clearly show that the activity of both enzymes in embryonic neural cells can be modified selectively by interactions between different cell populations. Thus, combined NR-OL aggregates show an increase in CAT without changes in ACE, NR-T an increase in CAT and a decrease in ACE, T-Ro a decrease in both CAT and ACE, and OL-T no changes at all. Experiments in which NR and OL cells were combined in different proportions indicate that the interactions require the presence of defined numbers of cells from each kind. Isochronous and heterochronous combinations of 7- and 10-day-old NR and OL cells show that the interactive capacities of the cells change with development.     

Effect of anti-dementia drugs on LPS induced neuroinflammation in mice

Inflammation has been recently implicated in pathogenesis of dementia disorders. Effect of anti-dementia (Acetylcholinesterase inhibitor) drugs tacrine, rivastigmine and donepezil were studied on neuroinflammation induced by intraperitoneal administration of lipopolysaccharide (LPS) in mice. Interleukin-2 (IL-2) and isoforms of acetylcholinesterase (AChE) were estimated in different brain areas as marker for neuroinflammation and cholinergic activity respectively. LPS significantly increased the level of IL-2 in all the brain areas while enhancement of AChE activity varied in brain areas. It was found that administration of tacrine, rivastigmine and donepezil in mice significantly attenuated the LPS induced increased levels of IL-2 along with the significant reduction of AChE activity predominantly in salt soluble (SS) fraction as compared to the detergent soluble (DS) fraction in a dose dependent manner. In vitro effect of LPS was also studied in different brain areas. LPS significantly increased the AChE activity in SS fractions but the significant increase was not found in DS fractions. The present study indicate that cholinesterase inhibitor anti-dementia drugs are effective against LPS induced neuroinflammation that may be linked to enhanced cholinergic activity.     

Regional distribution of choline acetyltransferase and acetylcholinesterase in rabbit brain

Regional distribution of choline acetyltransferase (CAT) and acetylcholinesterase (AChE) in seventeen regions of the rabbit CNS has been determined by means of the cation exchange radiometric method. A significant correlation has been found between the activities of AChE and CAT which allows computation of their potencies. A comparison between activities of the cholinergic enzymes in the human and rabbit brain has been discussed.     

Cholinergic deficit in Alzheimer's disease: A study based on CSF and autopsy data

Cholinesterase (ChE) activity was measured as a possible marker of cholinergic neurotransmission of the brain in CSF of 93 patients with probable Alzheimer's disease/senile dementia of the Alzheimer type (AD/SDAT) and of 29 control patients. ChE activity in CSF was decreased significantly in the AD/SDAT patients as compared to the controls. This reduction correlated significantly with the various measures of the severity of dementia. However, the reduction of ChE activity was only moderate (25–30%) even in patients with the most severe dementia and nonsignificant in patients with early symptoms of AD/SDAT. The significance of various confounding factors, which may interfere with CSF ChE measurements is discussed. Our findings seem to indicate that the deficiency of cholinergic neurons is not directly reflected in CSF and that the measurements of ChE activities in CSF are not helpful in diagnosing AD/SDAT. In the autopsy study the activities of cholineacetyltransferase (ChAT) and ChE were determined for ten brain areas of 20 AD/SDAT patients and of 14 controls. In AD/SDAT patients ChAT activity was profoundly decreased (50–85% decrease) in the cortical areas and hippocampus, but was unchanged or only mildly reduced in other subcortical brain areas. This study further confirms that the affection of cholinergic neurons is limited to projections from nucleus basalis to cortex and hippocampus, whereas other cholinergic neurons, like in striatum, seem to be relatively spared. In general, the activities of ChAT and ChE were lower in Alzheimer patients dying at younger age suggesting more severe disease process with these patients.     

Effects of post-injury hypothermia and nerve growth factor infusion on antioxidant enzyme activity in the rat: implications for clinical therapies

The pathological sequelae of traumatic brain injury (TBI) include increased oxidative stress due to the production of reactive oxygen species (ROS). Regulation of ROS levels following TBI is determined primarily by antioxidant enzyme activity that in turn can be influenced by nerve growth factor (NGF). Hypothermia is one of the current therapies designed to combat the deleterious effects of TBI. However, it has been shown to suppress post-trauma increases in NGF levels in rat brain. The present study sought to determine whether post-injury hypothermia also impairs the antioxidant response to injury, and if such an effect could be reversed by infusion of exogenous NGF. We employed a lateral controlled cortical impact injury model in rat, followed by moderate hypothermia treatment with supplemental intracerebroventricular infusion of NGF or vehicle. The time course of changes in post-injury/intervention levels of NGF and activity of three major enzymes responsible for ROS scavenging, catalase (CAT), glutathione peroxidase (GPx) and superoxide dismutase (SOD), was determined in the hippocampus. Relative to levels in injured, normothermic animals, hypothermia treatment not only suppressed NGF levels, but also attenuated CAT and GPx activity, and increased SOD activity. Infusion of NGF in injured, hypothermia-treated animals was ineffective in restoring hippocampal antioxidant enzymes activity to levels produced after injury under normothermic conditions, although it was able to increase septal cholinergic (choline acetyltransferase) enzyme activity. These results have implications for clinical treatment of TBI, demonstrating that moderate hypothermia suppresses NGF and the antioxidant response after TBI; the latter cannot be countered by exogenous NGF administration.     

Regulation of cholinergic expression in cultured spinal cord neurons

Factors regulating development of cholinergic spinal neurons were examined in cultures of dissociated embryonic rat spinal cord. Levels of choline acetyltransferase (CAT) activity in freshly dissociated cells decreased rapidly, remained low for the first week in culture, and then increased. The decrease in enzyme activity was partially prevented by increased cell density or by treatment with spinal cord membranes. CAT activity was also stimulated by treatment with MANS, a molecule solubilized from spinal cord membranes. The effects of MANS were greatest in low-density cultures and in freshly plated cells, suggesting that the molecule may substitute for the effects of elevated density and cell-cell contact. CAT activity in ventral (motor neuron-enriched) spinal cord cultures was similarly regulated by elevated density or treatment with MANS, whereas enzyme activity was largely unchanged in mediodorsal (autonomic neuron-enriched) cultures under these conditions. These observations suggest that development of cholinergic motor neurons and autonomic neurons are not regulated by the same factors. Treatment of ventral spinal cord cultures with MANS did not increase the number of cholinergic neurons detected by immunocytochemistry with a monoclonal CAT antibody, suggesting that MANS did not increase motor neuron survival but rather stimulated levels of CAT activity per neuron. These observations indicate that development of motor neurons can be regulated by cell-cell contact and that the MANS factor may mediate the stimulatory effects of cell-cell contact on cholinergic expression.     

REGIONAL CHANGES IN RAT BRAIN CHOLINE ACETYLTRANSFERASE AND ACETYLCHOLINESTERASE ACTIVITY RESULTING FROM UNDERNUTRITION IMPOSED DURING DIFFERENT PERIODS OF DEVELOPMENT1

Abstract— The severity of mental changes in malnourished children is related to both the period of development when the malnutrition occurs and the amount of environmental stimulation. In the present study the effect of imposing protein undernutrition during the period of gestation or postweaning period, and protein-energy undernutrition during the suckling period on cholinergic enzyme activity was investigated in the rat. Six different dietary treatments were given and the activity of ChAc, ChE, and AChE determined in the forebrain, brainstem, and cerebellum of male rats on day 49. Undernutrition imposed during gestation, suckling or postweaning all resulted in changes in cholinergic enzyme activity. The direction and degree of change of enzyme activity depended on the period when undernutrition was imposed as well as the brain region. In the forebrain ChE and AChE activities were altered, in the brainstem, ChAc, ChE and AChE activities were altered, and in the cerebellum ChAc activity was altered. The effect on the activity of the individual cholinergic enzymes was complex and was not the same in the different regions of the brain or even for the same brain region exposed to undernutrition during different periods of development. These results along with earlier work indicate that cholinergic enzyme activity in brain of undernourished rats can be altered by both the period of development when undernutrition is imposed and the amount of environmental stimulation.     

Human aging brain disorders: Role of antioxidant enzymes

In order to investigate the role of two free radical detoxificant enzymes in patients with aging brain disorders, superoxide dismutase (SOD) and catalase (CAT) activities have been measured in blood from male and female human patients of different ages with several types of aging brain disorders. When compared with activities in the normal population, we have detected: 1) SOD and CAT activities are decreased in patients with Parkinson disease. 2) SOD activity seems to be normal and CAT activity is decreased in patients with dementia. 3) In the patients with stroke, SOD activity is normal, while CAT activity is decreased. SOD activity was measured in red blood cells using the Minami and Yoshikawa method. CAT activity was measured in hemolysates by the method of Aebi. We can conclude that SOD and CAT activities in patients with Parkinson disease are decreased.     

Ca2+ and mitochondria as substrates for deficits in synaptic plasticity in normal brain ageing

Normal brain ageing is associated with a degree of functional impairment of neuronal activity that results in a reduction in memory and cognitive functions. One mechanism proposed to explain the age-dependent changes was the "Ca(2+) hypothesis of ageing" but data accumulated in the last decade revealed a number of inconsistencies. Two important questions were raised: (a) which are, if any, the most reliable age-associated change in neuronal Ca(2+) homeostasis and (b) are these changes primary, and thus determinant of the ageing phenotype, or are they secondary to other changes in the physiology of the aged neurones. After a brief review of the evidence accumulated for the age-induced changes in synaptic plasticity, we assess the proposal that these changes are, ultimately, determined by changes in the metabolic state of the aged neurones, that are manifest particularly after neuronal stimulation. In this context, it appears that the changes in mitochondrial status and function are of primary importance.     

Disease-related regressive alterations of forebrain cholinergic system in SOD1 mutant transgenic mice

Transgenic mice carrying the human mutated SOD1 gene with a glycine/alanine substitution at codon 93 (G93A) are a widely used model for the fatal human disease amyotrophic lateral sclerosis (ALS). In these transgenic mice, we carried out a neurochemical study not only restricted to the primarily affected regions, the cervical and lumbar segments of the spinal cord, but also to several other brain regions. At symptomatic (110 and 125 days of age), but not at pre-symptomatic (55 days of age) stages, we found significant decreases in catalytic activity of the cholinergic enzyme, choline acetyltransferase (ChAT) in the hippocampus, olfactory cortex and fronto-parietal cortex. In parallel, we observed a decreased number of basal forebrain cholinergic neurons projecting to these areas. No alterations of the cholinergic markers were noticed in the striatum and the cerebellum. A widespread marker for GABAergic neurons, glutamate decarboxylase (GAD), was unaffected in all the areas examined. Alteration of cholinergic markers in forebrain areas was paralleled by concomitant alterations in the spinal cord and brainstem, as a consequence of progressive apoptotic elimination of cholinergic motor neuron. Gestational supplementation of choline, while able to result in long-term enhancement of cholinergic activity, did not improve transgenic mice lifespan nor counteracted cholinergic impairment in brain regions and spinal cord.     

Changes in activity and expression of phosphofructokinase in different rat brain regions after basal forebrain cholinergic lesion

Selective lesion of rat basal forebrain by the cholinergic immunotoxin 192IgG-saporin was used as an animal model to address the question of whether the changes in cortical glucose metabolism observed in patients with Alzheimer's disease may be related to impaired cholinergic transmission. At different times after creating the immunolesion, the isoenzyme pattern and steady-state mRNA levels of the key glycolytic enzyme phosphofructokinase were determined in cortex, hippocampus, basal forebrain and nucleus caudatus. The loss of cholinergic input was accompanied by a persistent decrease in choline acetytransferase and acetylcholine esterase activities in the cortical target areas similar to the cholinergic malfunction seen in Alzheimer's dementia. The basal forebrain lesion induced by the immunotoxin resulted in a transient increase in phosphofructokinase activity peaking on day 7 after inducing the lesion in cortical areas. In parallel, an increased steady-state level of phosphofructokinase mRNA was determined by RT/real-time PCR and in situ hybridization. In contrast, analysis by western blotting and quantitative PCR revealed no changes in the phosphofructokinase isoenzyme pattern after immunolesion. It is concluded that common metabolic mechanisms may underlie the degenerative and repair processes in denervated rat brain and in the diseased Alzheimer's brain.