Irina Alafuzoff

Nerve growth factor affects11C-nicotine binding, blood flow, EEG, and verbal episodic memory in an Alzheimer patient (Case Report)

SummaryBased on animal research suggesting that nerve growth factor (NGF) can stimulate central cholinergic neurons, the known losses of cholinergic innervation of the cortices in Alzheimers disease (AD), and our experience of infusing NGF to support adrenal grafts in parkinsonian patients, we have initiated clinical trials of NGF infusions into the brain of patients with AD. Here we report a follow-up of our first case, a 69-year-old woman, with symptoms of dementia since 8 years. Intraventricular infusion of 6.6 mg NGF during three months resulted in a marked transient increase in uptake and binding of11C-nicotine in frontal and temporal cortex and a persistent increase in cortical blood flow as measured by PET as well as progressive decreases of slow wave EEG activity. After one month of NGF, tests of verbal episodic memory were improved whereas other cognitive tests were not. No adverse effects could be ascribed to the NGF infusion. Taken together, the results of this case study indicate that NGF may counteract cholinergic deficits in AD, and suggest that further clinical trials of NGF infusion in AD are warranted.

Intracranial infusion of purified nerve growth factor to an Alzheimer patient : the first attempt of a possible future treatment strategy

We report on the clinical outcome of a first case of intracranial infusion of nerve growth factor (NGF) to an Alzheimer patient. The therapeutic attempt is based on animal research showing that NGF stimulates central cholinergic neurons of the type known to be lost during the development of Alzheimers disease (AD). Furthermore, our own previous clinical experience of infusing NGF to support the survival of intracranially transplanted adrenal chromaffin cells to Parkinsonian patients indicate this approach to be technically possible and safe and clinically of significant potential. Our first case was a 69-year-old woman, with symptoms of dementia since 8 years. Intraventricular infusion of 6.6 mg NGF over three months resulted in a marked transient increase in uptake and binding of [11C]nicotine in frontal and temporal cortex and a persistent increase in cortical blood flow as measured by PET as well as progressive decreases of slow wave EEG activity. After one month of NGF infusion, tests of verbal episodic memory were improved whereas other cognitive tests were not. No adverse effects of the NGF infusion were found. The results of this single case indicate that NGF may counteract cholinergic deficits in AD, and suggest that further clinical trials of NGF infusion in AD are warranted.

A retrospective analysis of clinical diagnoses and autopsy findings in 3,042 cases during two different time periods

The accuracy of clinical diagnoses was determined and compared between the two periods of time 1977 to 1978 and 1987 to 1988 based on the analysis of 3,042 autopsies at Huddinge University Hospital. The discrepancy rates were calculated by counting the number of missed or incompletely diagnosed major diseases and their complications. Moreover, sensitivity, specificity, and clinical accuracy for positive and negative diagnoses also were calculated for all cases of acute myocardial infarction and malignant tumors. The autopsy rate decreased from 80% to 39%. The autopsy successfully addressed the clinical questions in 97% of the cases. The selection of the cases possibly could explain the significant 5% increase in the proportion of clinically undetected major underlying diseases. The discrepancy rate was higher among the older patients. There were no significant changes in the diagnosis of cardiovascular diseases apart from thrombosis of the mesenteric artery, which more than doubled. The proportion of infectious diseases increased significantly from 27% to 32%. The number of cases with clinically missed tuberculosis was twice as high in the 1987 to 1988 period as in the 1977 to 1978 period and there was a marked increase in fungal and viral infections. There was no significant change in the clinical diagnosis of malignant tumors between the two periods. Approximately 15% of all major cancers were not diagnosed before autopsy; half of these tumors were lethal. We conclude that the role of the autopsy has not diminished in spite of advanced diagnostic methods and it remains an effective tool in the assessment of medical care.

Coupling of muscarinic receptors to GTP proteins in postmortem human brain-alterations in Alzheimer's disease

The coupling of muscarinic agonist receptors to guanine nucleotide-binding (G) proteins was investigated in the frontal, temporal cortices and thalamus of control and Alzheimer brains by using carbachol in competition experiments with [3H]QNB. In the presence of GppNHp, the carbachol/[3H]QNB competition binding data showed a 6-fold increase in the high-affinity muscarinic agonist coefficient (Ki high) in the thalami of control brains and a significantly increased proportion of low-affinity agonist binding sites (Bmax low) in the temporal cortices of control brains, while no significant effect of GppNHp was observed in Alzheimer brains. The results suggest a disturbance of the muscarinic receptor-G protein coupling in Alzheimers disease.

Characterization of muscarinic receptor subtypes in Alzheimer and control brain cortices by selective muscarinic antagonists

Subtypes of muscarinic receptors were characterized in the frontal cortices of control and Alzheimer brains, with labelled quinuclidinyl benzilate [3H]QNB and the unlabelled muscarinic antagonists pirenzepine, AF-DX 116, hexahydro-sila-diphenidol (HHSiD), para-fluoro-hexahydro-sila-diphenidol (p-F-HHSiD) and himbacine. High and low affinity sites were observed for both pirenzepine and AF-DX 116 in human control frontal cortices. The majority (76%) of the pirenzepine binding sites showed high affinity to the muscarinic receptors (M1), while the rest of the binding sites had an affinity that was 40 times less. AF-DX 116 displayed two sets of binding sites where the high affinity AF-DX 116 (M2) sites constituted 27%, while the low affinity AF-DX 116 (non-M2 site) was 73%. A single class of binding sites was observed for HHSiD, p-F-HHSiD and himbacine in human frontal cortices. HHSiD showed an affinity in the frontal cortices that was comparable to that of the pirenzepine high affinity binding (M1) sites. The affinity of p-F-HHSiD was three times lower than that of HHSiD but similar to himbacine. A significant increase in the affinity (+ 40%) as well as in the Bmax (+ 99%) value was observed for the pirenzepine high affinity binding sites (M1) in the frontal cortices of Alzheimer brains compared to controls. Similarly, a significant increase was observed in the Bmax value (+ 60%) for the AF-DX 116 low affinity binding sites (non-M2), while no change was found for the high affinity binding sites (M2).(ABSTRACT TRUNCATED AT 250 WORDS)

Chapter 35 The role of nicotinic receptors in the pathophysiology ofAlzheimer's disease

Publisher Summary This chapter discusses the role of nicotinic receptors in the pathophysiology of Alzheimers disease (AD). The nicotinic receptor binding sites in human brain have been identified and characterized by various radioligands such as the nicotinic antagonists [ 3 H]α-bungarotoxin ([ 3 H]Btx), [ 3 H]tubocurarine and the agonists [ 3 H]ACh, [ 3 H]nicotine and [ 3 H]methylcarbamylcholine. In rodent brain receptor binding experiments in tissue homogenates or thin tissue slices (autoradiography) have demonstrated a different localization of the antagonist [3H]Btx sites compared to the agonist sites labeled by [3H]ACh and [3H]nicotine. The difference in antagonist/agonist sites is evident at molecular level. Subtypes of nicotinic receptors are widely distributed in the human brain. Some of the nicotinic receptors are presynaptically located. In AD/SDAT brains there is a loss of high affinity to low affinity nicotinic receptors which partly might be due to an interconversion of high affinity to low affinity nicotinic sites with a simultaneous reduction in binding affinity for the low affinity sites. By lowering the affinity of the nicotinic sites, quick desensitization phenomena can be devoided and the nicotinic receptors might influence the release of acetylcholine. The finding that cholinesterase inhibitors such as THA restore the ACh release in AD/SDAT brain tissue via interaction with the nicotinic receptors open up new therapeutic strategies in the treatment of AD/SDAT and related disorders.

Loss of Dopamine Uptake Sites Labeled with [3H]GBR-12935 in Alzheimer’s Disease

The binding of the dopamine uptake inhibitor [3H]GBR-12935 to postmortem putamen from a control group and patients with Alzheimers disease/senile dementia of Alzheimer type (AD/SDAT) or vascular dementia (VD) was studied. The binding density (Bmax) in AD/SDAT was significantly reduced to 50% of control. A reduction of Bmax in VD was also noted, but it did not reach statistical significance. No differences in apparent binding affinity (Kd) between controls and dementia groups were obtained. The concentrations of dopamine (DA), dihydroxyphenylacetic acid (DOPAC), 3-methoxytyramine (3-MT) and homovanillic acid were also determined. The concentrations of DA and DOPAC were reduced by 30-40% in AD/SDAT and VD, but the reductions did not reach statistical significance. The concentration of 3-MT was reduced by 40% in AD/SDAT and by 30% in VD. The [3H]GBR-12935-binding densities correlated significantly with corresponding concentrations of DA in control brains. It is suggested that the loss of [3H]GBR-12935-binding sites in human putamen in AD/SDAT reflects a degeneration of dopamine neurites.

Neurotransmitter-mediated inhibition of post-mortem human brain adenylyl cyclase.

The effects of a range of neurotransmitter agonists showing selectivity for receptor types inhibitorily coupled to adenylyl cyclase were compared in membrane preparations of hippocampus, frontal cortex and caudate nucleus/ striatum from previously frozen post-mortem human and rat brain. Agonists were tested against basal and forskolin stimulated activities, forskolin being a potent activator of the catalytic sub-unit of the enzyme. Of those agonists tested, only somatostatin (100 μM) and neuropeptide Y (10 μM) gave consistent inhibitions of basal and forskolin stimulated enzyme activities in all three regions of both human and rat brain. Somatostatin-mediated inhibition of human brain adenylyl cyclase was reduced in the absence of GTP and in the presence of the guanine nucleotide partial agonist, guanosine 5′-O-thiodiphosphate, consistent with a G-protein-linked receptor. No such GTP-dependence was found for the neuropeptide Y-mediated adenylyl cyclase inhibition. GTP-dependent somatostatin mediated inhibitions of human brain adenylyl cyclase activity were of highest magnitude in the thalamus, intermediate magnitude in the hippocampus and caudate nucleus and lowest magnitude in the frontal cortex. It is concluded that of a range of neurotransmitter receptor agonists tested, only somatostatin gives robust, GTP-dependent responses that are reproducible enough to be used with post-mortem tissue for the comparison of receptor function in human brain disorders.

Assay of a phosphatidylinositol bisphosphate phospholipase C activity in postmortem human brain

The activity of a phospholipase C which hydrolyses exogenous phosphatidylinositol-4,5-bisphosphate [( 3H]PtdIns(4,5)P2) in membranes prepared from frozen postmortem human brain and rat brain was investigated. Enzyme characteristics were essentially similar in membranes prepared from frozen postmortem brain and fresh or frozen rat brain. The [3H]PtdIns(4,5)P2 solubilization and assay procedure employed resulted in an efficient availability of the substrate for the enzyme. The non-hydrolysable guanosine triphosphate analogue guanosine 5-[beta gamma-imido]diphosphate (Gpp[NH]p) stimulated hydrolysis rapidly with a half maximum activity of approximately 25 microM. This stimulation was not specific for guanine nucleotides as ATP, imidodiphosphate and pyrophosphate also caused enzyme activation. However these activation effects could be distinguished by the polyanion spermine. The non-hydrolysable guanine dinucleotide analogue guanosine 5-[beta-thio]diphosphate acted as a partial agonist thereby inhibiting the stimulatory effect of Gpp[NH]p. Gpp[NH]p-stimulated enzyme activity showed a maximum response in the presence of 1 mM deoxycholate and displayed a pH optima in the range 7.0-7.5. PtdIns(4,5)P2 hydrolysis was observed in the absence of added calcium, but hydrolytic cleavage was inhibited in the presence of divalent ion chelators. Magnesium inhibited PtdIns(4,5)P2 hydrolysis in a concentration-dependent manner. Elucidation of these aspects of the phosphatidylinositol cycle in normal human postmortem brain will permit comparative studies in CNS disease states.

Isoelectric Focusing and Two-Dimensional Gel Electrophoresis in Plasma and Cerebrospinal Fluid from Patients with Dementia

IgG profiles of cerebrospinal fluid (CSF) and plasma from patients with Alzheimers disease/senile dementia of Alzheimer type, multi-infarct dementia and aged nondemented individuals were analyzed using isoelectric focusing (IEF) followed by immunofixation and high resolution two-dimensional polyacrylamide gel electrophoresis (PAGE). The IEF profiles of plasma and CSF in patients with dementia were diffuse and lacked oligoclonal bands and were identical to those of nondemented individuals. The investigation of plasma and CSF by two-dimensional PAGE did not reveal any specific protein pattern in the demented individuals compared to the controls.