Ornithine Decarboxylase in Human Brain: Influence of Aging, Regional Distribution, and Alzheimer's Disease

Abstract: Although experimental animal data have implicated ornithine decarboxylase, a key regulatory enzyme of polyamine biosynthesis, in brain development and function, little information is available on this enzyme in normal or abnormal human brain. We examined the influence, in autopsied human brain, of postnatal development and aging, regional distribution, and Alzheimer's disease on the activity of ornithine decarboxylase. Consistent with animal data, human brain ornithine decarboxylase activity was highest in the perinatal period, declining sharply (by ∼60%) during the first year of life to values that remained generally unchanged up to senescence. In adult brain, a moderately heterogeneous regional distribution of enzyme activity was observed, with high levels in the thalamus and occipital cortex and low levels in cerebellar cortex and putamen. In the Alzheimer's disease group, mean ornithine decarboxylase activity was significantly increased in the temporal cortex (+76%), reduced in occipital cortex (−70%), and unchanged in hippocampus and putamen. In contrast, brain enzyme activity was normal in patients with the neurodegenerative disorder spinocerebellar ataxia type I. Our demonstration of ornithine decarboxylase activity in neonatal and adult human brain suggests roles for ornithine decarboxylase in both developing and mature brain function, and we provide further evidence for the involvement of abnormal polyamine system activity in Alzheimer's disease.     

Brain Quinolinic Acid in Huntington''s Disease

Concentrations of the endogenous neurotoxic tryptophan metabolite, quinolinic acid (QA), were measured in postmortem brain tissue obtained from patients with Huntington's disease (HD) and matched controls, using a gas chromatography/mass spectrometry method. There was no significant difference in either the putamen or the frontal cortex between the HD and control groups. These results do not support the hypothesis that increased QA is responsible for neuronal degeneration in HD.     

Changes in β-Adrenergic Receptor Subtypes in Alzheimer-Type Dementia

Using ligand binding techniques, we studied beta-adrenergic receptor subtypes in brains obtained at autopsy from seven histologically normal controls and seven histopathologically verified cases with Alzheimer-type dementia (ATD). Inhibition of [3H]dihydroalprenolol [( 3H]DHA) binding by the selective beta 1 antagonist, metoprolol, results in nonlinear Hofstee plots, suggesting the presence of the two receptor subtypes in the human brain. The calculated ratios of beta 1/beta 2-adrenergic receptors in control brains are as follows: frontal cortex, 49:51; temporal cortex, 31:69; hippocampus, 66:34; thalamus, 23:77; putamen, 70:30; caudate, 48:52; nucleus basalis of Meynert (NbM), 43:57; cerebellar hemisphere, 25:75. Compared with the controls, total concentrations of beta-adrenergic receptors were significantly reduced only in the thalamus of the ATD brains. beta 1-Adrenergic receptor concentrations were significantly reduced in the hippocampus and increased in the NbM and cerebellar hemisphere, whereas beta 2-adrenergic receptor concentrations were significantly reduced in the thalamus, NbM, and cerebellar hemisphere and increased in the hippocampus and putamen of the ATD brains. These results suggest that beta 1- and beta 2-adrenergic receptors are present in the human brain and that there are significant changes in both receptor subtypes in selected brain regions in patients with ATD.     

Metabolic changes in the basal ganglia of patients with Huntington's disease: an in situ hybridization study of cytochrome oxidase subunit I mRNA

On the basis of the functional model of the basal ganglia developed in the 1980s and the neuropathological findings in Huntington's disease (HD), changes in the neuronal activity of the basal ganglia have previously been proposed to explain the abnormal movements observed in this pathology. In particular, it has been stated that the neurodegenerative process affecting the basal ganglia in the disease should provoke a hypoactivity in the internal segment of the pallidum (GPi) that could explain choreic movements observed in the disease. To test this functional hypothesis, we performed an in situ hybridization study on control and HD brains postmortem, taking cytochrome oxidase subunit I (COI) mRNAs expression as index of neuronal activity. As most of the HD patients studied were under chronic neuroleptic (NL) treatment, we also studied the brains of non-HD patients under chronic NL treatment. Our results show that in HD brain the number of neurons expressing COI mRNA tends to be lower in the striatum, GPe and GPi, suggesting a severe involvement of these structures during the neurodegenerative process. Moreover, COI mRNA level of expression was markedly reduced within neurons of the putamen and GPe. Surprisingly, COI mRNA expression was not modified in the GPi in HD brains compared with controls. This paradoxical result in the GPi may be explained by the antagonistic effect of GPe hypoactivity and the degenerative process involving neurons of GPi. Our results indicate that the functional modifications, and consequently the pathophysiology of abnormal movements, observed in HD basal ganglia are more complex than expected from the currently accepted model of the basal ganglia organization.     

Alterations in dopaminergic receptors in Huntington's disease.

To detect variations in dopaminergic receptors and cholinergic activity in regions of postmortem Huntington's diseased brains, ³H-spiroperidol binding assays and choline acetyltransferase (ChAc) activities were carried out. A significant reduction in ³H-spiroperidol binding in the caudate nucleus, putamen and frontal cortex of choreic brains was detected which appeared to be due to a decrease in the total number of binding sites rather than to a decrease in affinity of ³H-spiroperidol for the dopaminergic receptor. In choreic brains, there were also significant reductions in ChAc activity in the caudate nucleus and putamen. The decreases of both ³H-spiroperidol binding and ChAc activity in the neostriatum suggest that the dopaminergic receptors are localized postsynaptically on cholinergic interneurons. Dopaminergic receptor alterations in the basal ganglia may be one of the causes of the abnormal motor movements found in HD while alterations of these receptors in the frontal cortex may be associated with the neuronal degeneration found in that area of choreic brains.     

Abnormalities of neurotransmitter enzymes in Huntington's chorea

The activities ofl-glutamate decarboxylase (GAD), GABA-transaminase (GABA-T), choline acetyltransferase (CAT), and cysteic and cysteinesulfinic acids decarboxylase (CAD/CSAD) in putamen and frontal cortex in both Huntington's chorea and normal tissues were measured. The greatest difference between Huntington's and normal tissues occurred in putamen, in which the apparent CSAD activity was reduced by 85%, while no difference was observed in frontal cortex. GAD, CAD, and CAT activities were also reduced in putamen by 65%, 63%, and 42%, respectively (P<0.05). Slight reduction in the enzyme activities was also observed in frontal cortex. However, these reductions appeared to be statistically insignificant (P>0.05 in all cases). GABA-T showed little difference in both putamen and frontal cortex in Huntington's chorea and normal tissues. GAD and GABA-T from Huntington's tissues were indistinguishable from those obtained from normal tissues by double diffusion test and by microcomplement fixation test, which is capable of distinguishing proteins with a single amino acid substitution. Furthermore, the similarity of the complement fixation curves for GAD from Huntington's and normal tissues suggests that the decrease in GAD activity is probably due to the reduction in the number of GAD molecules, presumably through the loss of neurons, and not due to the inhibition or inactivation of GAD activity by toxic substances which might be present in Huntington's chorea.     

Inhibition of Histamine Synthesis in Brain by α-Fluoromethylhistidine, a New Irreversible Inhibitor: In Vitro and In Vivo Studies

a-Fluoromethylhistidine (α-FMH), a new potent inhibitor of histidine decarboxylase (HD), has been used for in vitro and in vivo studies of brain HD. Following a preincubation with (+)-α-FMH, brain HD activity was inhibited in a time-dependent and concentration-dependent manner. The enzyme activity was not restored by overnight dialysis against standard buffer. The (–) antimer of α-FMH was ineffective. When injected intraperitoneally in a single dose of 20 mg/kg, (±)-α-FMH induced a complete loss in HD activity in cerebral cortex and hypothalamus as well as in peripheral tissues, such as stomach. At a dosage of 100 mg/kg (±)-α-FMH did not alter histamine-N-methyltransferase, DOPA decarboxylase, and glutamate decarboxylase activities. The maximal decrease of HD activity occurred after 2 h in both cerebral cortex and hypothalamus, but the time course of the recovery of enzyme activity was slower in the cerebral cortex. The enzyme activity reached control value within 3 days in hypothalamus and was not fully restored after 4 days in cerebral cortex. Contrasting with the diminished HD activity, a substantial concentration of histamine remained present in five regions of mouse brain. Thus, α-FMH is a highly specific irreversible inhibitor of brain HD activity and its efficacy makes it useful to study the physiological role of brain histamine.     

Changes of Biogenic Amines and Their Metabolites in Postmortem Brains from Patients with Alzheimer-Type Dementia

Noradrenaline (NA), 3,4-dihydroxyphenylethylamine (dopamine, DA), 5-hydroxytryptamine (serotonin, 5-HT), homovanillic acid (HVA), and 5-hydroxyindoleacetic acid (5-HIAA) were measured in 22 regions of postmortem brains from four histologically verified cases with Alzheimer-type dementia (ATD) and nine histologically normal controls. Compared with the controls, concentrations of 5-HT and 5-HIAA in the ATD brains were significantly reduced in nine regions (superior frontal gyrus, insula, cingulate gyrus, amygdala, putamen, medial and lateral segments of globus pallidus, substantia nigra, lateral nucleus of thalamus) and in eight regions (amygdala, substantia innominata, caudate, putamen, medial and lateral segments of globus pallidus, medial and lateral nuclei of thalamus), respectively. NA concentrations of the ATD brains were significantly reduced in six regions (cingulate gyrus, substantia innominata, putamen, hypothalamus, medial nucleus of thalamus, raphe area). In contrast, significant reductions of DA and HVA concentrations in the ATD brains were found only in putamen and amygdala, respectively. The 5-HIAA/5-HT ratio in the ATD brains decreased significantly in locus coeruleus, while the HVA/DA ratio increased significantly in putamen and medial segment of globus pallidus. These findings suggest that the serotonergic and noradrenergic systems are affected, while the dopaminergic system is relatively unaffected in ATD brains.     

Glucose Transporter Isoform Expression in Huntington's Disease Brain

Abstract: Several reports have suggested a characteristic decrease in glucose use in the striatum of patients with Huntington's disease (HD) may contribute to the cellular atrophy of the caudate and putamen. We examined the expression of the two major glucose transporter isoforms of brain, GLUT1 and GLUT3. GLUT1 is found largely in capillary endothelial cells and to a lesser extent in the brain parenchyma, whereas GLUT3 is localized primarily in neurons. Membranes prepared from postmortem samples of HD caudate and cortex and non-HD caudate and cortex were separated on 10% sodium dodecyl sulfate-polyacrylamide gels and probed with antisera to GLUT1 and GLUT3 by western blotting. Compared with controls, GLUT1 and GLUT3 transporter expression in caudate was decreased by three- and fourfold, respectively, in grade 3 of the disease. At earlier stages (grade 1), there was no significant difference in the expression of the two transporter isoforms compared with nondiseased controls. It is surprising that despite a substantial increase in glial fibrillary acidic protein immunoreactivity (an indicator of the extent of gliosis), glucose transporter expression was diminished significantly in HD caudate. The results suggest in the absence of a significant number of neurons, as in grade 3, glial cell GLUT1 and GLUT3 expression is down-regulated, perhaps reflecting the decreased metabolic demand of this brain region in HD.     

Serotonin Receptor Changes in Dementia of the Alzheimer Type

Serotonin receptors were assessed in post-mortem brains of control and Alzheimer-type dementia (ATD) patients using ligand binding techniques. Differential losses of serotonin S1 and S2 receptors were present in neocortex, hippocampus, and amygdala of ATD patients, whereas no significant changes were observed in basal forebrain and basal ganglia. Losses of S1 receptors were significantly age-related in the ATD group, suggesting they occurred at a later stage of the disease process. Losses of S2 receptors were considerably greater (with a reduction to 35% of control in temporal cortex) and were not age-related in ATD. Significant correlations were observed within the ATD group between S2 receptor binding and somatostatin immunoreactivity in temporal and frontal cortices. Thus the loss of S2 receptors in ATD may be a relatively early change in the disease process, and may precede the changes in ascending serotonergic neurones.     

Kynurenine Pathway Measurements in Huntington''s Disease Striatum: Evidence for Reduced Formation of Kynurenic Acid

Recent evidence suggests that there may be overactivation of the N-methyl-D-aspartate (NMDA) subtype of excitatory amino acid receptors in Huntington's disease (HD). Tryptophan metabolism by the kynurenine pathway produces both quinolinic acid, an NMDA receptor agonist, and kynurenic acid, an NMDA receptor antagonist. In the present study, multiple components of the tyrosine and tryptophan metabolic pathways were quantified in postmortem putamen of 35 control and 30 HD patients, using HPLC with 16-sensor electrochemical detection. Consistent with previous reports in HD putamen, there were significant increases in 5-hydroxyindoleacetic acid, 5-hydroxytryptophan, and serotonin concentrations. Within the kynurenine pathway, the ratio of kynurenine to kynurenic acid was significantly (p less than 0.01) increased twofold in HD patients as compared with controls, consistent with reduced formation of kynurenic acid in HD. CSF concentrations of kynurenic acid were significantly reduced in HD patients as compared with controls and patients with other neurologic diseases. Because kynurenic acid is an endogenous inhibitor of excitatory neurotransmission and can block excitotoxic degeneration in vivo, a relative deficiency of this compound could directly contribute to neuronal degeneration in HD.     

Histology and cytochemistry of human skin. XI. The distribution of beta-glucuronidase

1. Various amounts of beta-glucuronidase activity may be found in all of the cutaneous appendages. 2. In the epidermis, the basal layer and the Malpighian layer contain a moderate amount of it, but a band of cells, including the stratum granulosum and the cells immediately above it, is rich in beta-glucuronidase. 3. The cells of the duct of eccrine sweat glands have moderately strong enzyme activity, but those in the secretory coil are strongly reactive; small and large reactive granules are crowded in the reactive cytoplasm. 4. The cells of the secretory coil of the apocrine glands contain more beta-glucuronidase than any other cutaneous appendage. 5. In the sebaceous glands, a very strong concentration of enzyme activity is found in the undifferentiated peripheral cells, a smaller amount of it is found in the differentiating cells. 6. In active hair follicles, the largest amount of beta-glucuronidase is found in the outer root sheath and in the bulb. In the outer sheath, the strongest concentration is found around the level of the keratogenous zone of the cortex. The dermal papilla is strongly reactive. In quiescent hair follicles, the outer root sheath has a moderate amount of enzyme concentration, but the dermal papilla is unreactive. 7. In the dermis, the fibroblasts in the papillary layer, the smooth muscle cells of the arrectores pilorum and the tunica media of arteries, and the fat cells all exhibit enzyme activity. Mast cells show a great concentration of beta-glucuronidase.     

Cholinergic Muscarinic Receptors in Human Fetal Brain: Ontogeny of [3H]Quinuclidinyl Benzilate Binding Sites in Corpus Striatum, Brainstem, and Cerebellum

This study investigated mitochondrial respiratory activity in Huntington's disease (HD) brain. Mitochondrial membranes from caudate and cortex of HD and non-HD autopsied brains were assayed for succinate oxidation, cytochrome oxidase activity, and cytochromes b, cc1, and aa3. There was a significant decrease in HD caudate mitochondrial respiration, cytochrome oxidase activity, and cytochrome aa3, whereas cytochromes b and cc1 were normal. These findings are consistent with the hypothesis that mitochondrial dysfunction may contribute to the localized hypometabolism and progressive atrophy of the HD caudate.     

Altered Muscarinic and Nicotinic Receptor Densities in Cortical and Subcortical Brain Regions in Parkinson's Disease

Abstract: Muscarinic and nicotinic cholinergic receptors and choline acetyltransferase activity were studied in postmortem brain tissue from patients with histopathologically confirmed Parkinson's disease and matched control subjects. Using washed membrane homogenates from the frontal cortex, hippocampus, caudate nucleus, and putamen, saturation analysis of specific receptor binding was performed for the total number of muscarinic receptors with [³H]quinuclidinyl benzilate, for muscarinic M₁ receptors with [³H]pirenzepine, for muscarinic M₂ receptors with [³H]oxotremorine-M, and for nicotinic receptors with (–)-[³H]nicotine. In comparison with control tissues, choline acetyltransferase activity was reduced in the frontal cortex and hippocampus and unchanged in the caudate nucleus and putamen of parkinsonian patients. In Parkinson's disease the maximal binding site density for [³H]quinuclidinyl benzilate was increased in the frontal cortex and unaltered in the hippocampus, caudate nucleus, and putamen. Specific [³H]pirenzepine binding was increased in the frontal cortex, unaltered in the hippocampus, and decreased in the caudate nucleus and putamen. In parkinsonian patients B_max values for specific [³H]oxotremorine-M binding were reduced in the cortex and unchanged in the hippocampus and striatum compared with controls. Maximal (–)-[³H]nicotine binding was reduced in both the cortex and hippocampus and unaltered in both the caudate nucleus and putamen. Alterations of the equilibrium dissociation constant were not observed for any ligand in any of the brain areas examined. The present results suggest that both the innominatocortical and the septohippocampal cholinergic systems degenerate in Parkinson's disease. The reduction of cortical [³H]oxotremorine-M and (–)-[³H]nicotine binding is compatible with the concept that significant numbers of the binding sites labelled by these ligands are located on presynaptic cholinergic nerve terminals, whereas the increased [³H]pirenzepine binding in the cortex may reflect postsynaptic denervation supersensitivity.     

Neurotransmitter receptor alterations in Parkinson's disease.

Neurotransmitter receptor binding for GABA, serotonin, cholinergic muscarinic and dopamine receptors and choline acetyltransferase (ChAc) activity were measured in the frontal cortex, caudate nucleus, putamen and globus pallidus from postmortem brains of 10 Parkinsonian patients and 10 controls. No changes in any of these systems were observed in the frontal cortex. In the caudaye nucleus, only the apparent dopamine receptor binding was altered with a significant 30% decrease in the Parkinsonian brain. Both cholinergic muscarinic and serotonin receptor binding were significantly altered in the putamen, the former increasing and the latter decreasing with respect to controls. In addition, ChAc activity was decreased in the putamen. In the globus pallidus, only ChAc activity was significantly changed, decreasing about 60%, with no change in neurotransmitter receptor binding. The results suggest that a progressive loss of dopaminergic receptors in the caudate nucleus may contribute to the decreased response of Parkinsonian patients to L-dopa and dopamine agonist therapy.     

Biochemical characterization of alpha-adrenergic receptors in human brain and changes in Alzheimer-type dementia

Using ligand binding techniques, we studied alpha-adrenergic receptors in brains obtained at autopsy from seven histologically normal controls and seven patients with histopathologically verified Alzheimer-type dementia (ATD). Binding of the alpha-adrenergic antagonists [3H]prazosin and [3H]yohimbine to membranes of human brains exhibited characteristics compatible with alpha 1- and alpha 2-adrenergic receptors, respectively. Binding of both ligands was saturable and reversible, with dissociation constants of 0.15 nM for [3H]prazosin and 5.5 nM for [3H]yohimbine. [3H]Prazosin binding was highest in the hippocampus and frontal cortex and lowest in the caudate and putamen in the control brains. [3H]Yohimbine binding was highest in the nucleus basalis of Meynert (NbM) and frontal cortex and lowest in the caudate and cerebellar hemisphere in the control brains. Compared with values for the controls, [3H]prazosin binding sites were significantly reduced in number in the hippocampus and cerebellar hemisphere, and [3H]yohimbine binding sites were significantly reduced in number in the NbM in the ATD brains. These results suggest that alpha 1- and alpha 2-adrenergic receptors are present in the human brain and that there are significant changes in numbers of both receptors in selected regions in patients with ATD.     

Glutamine synthetase activity in Huntington's disease

Glutamine synthetase activity was measured in seven brain areas post-mortem from control patients, and those with Huntington's disease. The activity of the enzyme was reduced in the frontal and temporal cortex, putamen and cerebellum, but not in the hippocampus, thalamus or olivary nucleus. The results do not suggest a generalised deficiency of glutamine synthetase in Huntington's disease. However, as this enzyme is localised to astrocytic cells, the reduction in activity in areas of neuronal devastation, where the ration of astrocytes to neurones is increased, may reflect a greater functional deficit. The enzyme plays a crucial role in cerebral ammonia assimilation and its inhibition in laboratory animals is known to produce neuronal toxicity. A reduction in its activity in Huntington's disease may well contribute to the neuronal pathology in certain areas.     

Lysosomal enzyme release in hypothermically perfused dog kidneys

This study investigated lysosomal disruption during hypothermic perfusion preservation of kidneys and its possible relationship to viability. The percentage of free and bound enzyme activity was analyzed for three lysosomal enzymes in homogenates made from perfused canine kidney cortex tissue, including beta-glucuronidase, cathepsin-D, and aryl sulfatase. All three enzymes displayed characteristic increases in free enzyme activity (47-68%) throughout 5 days of perfusion preservation. The increased activity obtained at 5 days of preservation was found to indicate "severe" tissue damage, as shown by a similar increase obtained in renal cortex tissue exposed to warm ischemia (37 degrees C) for 4 hr or longer. Aryl sulfatase was found to be the most sensitive indicator of severe damage. Pretreatment of kidney donors with methylprednisolone, a lysosomal stabilizer, was also studied in kidneys exposed to 5 days of perfusion. Pretreatment was found to reduce the percentage of free lysosomal enzyme activity following 5 days (nonviable) of perfusion to those levels normally obtained following 3-day (viable) perfusion. This indicates that methylprednisolone may be useful in modulating the severe disruption of lysosomes induced by long-term preservation. It is concluded that extensive disruption of lysosomes occurs during hypothermic perfusion preservation and may represent one cause for loss of organ viability.     

Impaired mitochondrial function results in increased tissue transglutaminase activity in situ

Tissue transglutaminase (tTG) is a transamidating enzyme that is elevated in Huntington's disease (HD) brain and may be involved in the etiology of the disease. Further, there is evidence of impaired mitochondrial function in HD. Therefore, in this study, we examined the effects of mitochondrial dysfunction on the transamidating activity of tTG. Neuroblastoma SH-SY5Y cells stably overexpressing human tTG or mutated inactive tTG were treated with 3-nitropropionic acid (3-NP), an irreversible inhibitor of succinate dehydrogenase. 3-NP treatment of tTG-expressing cells resulted in a significant increase of TG activity in situ. In vitro measurements demonstrated that 3-NP had no direct effect on tTG activity. However, 3-NP treatment resulted in a significant decrease of the levels of GTP and ATP, two potent inhibitors of the transamidating activity of tTG. No significant changes in the intracellular levels of calcium were observed in 3-NP-treated cells. Treatment with 3-NP in combination with antioxidants significantly reduced the 3-NP-induced increase in in situ TG activity, demonstrating that oxidative stress is a contributing factor to the increase of TG activity. This study demonstrates for the first time that impairment of mitochondrial function significantly increases TG activity in situ, a finding that may have important relevance to the etiology of HD.     

Systemic and central nervous system correction of lysosomal storage in mucopolysaccharidosis type VII mice

Mucopolysaccharidosis (MPS) type VII patients lack functional beta-glucuronidase, leading to systemic and central nervous system dysfunction. In this study we tested whether recombinant adenovirus that encodes beta-glucuronidase (Adbetagluc), delivered intravenously and into the brain parenchyma of MPS type VII mice, could provide long-term transgene expression and correction of lysosomal distension. We also tested whether systemic treatment with the immunosuppressive anti-CD40 ligand antibody, MR-1, affected transgene expression. We found substantial plasma beta-glucuronidase activity for over 9 weeks after gene transfer in the MR-1- treated group, with subsequent decline in activity corresponding to a delayed anti-beta-glucuronidase antibody response. At 16 weeks, near wild-type amounts of beta-glucuronidase activity and striking reduction of lysosomal pathology were detected in livers from mice that had received either MR-1 cotreatment or control antibody. In the lung and kidney, beta-glucuronidase activity was markedly higher for the MR-1-treated group. beta-Glucuronidase activity in the brain persisted independently of MR-1 treatment. Activity was intense in the injected hemisphere and was also evident in the noninjected cortex and striatum, with dramatic improvements in storage deposits in areas of both hemispheres. These results indicate that prolonged enzyme expression from transgenes delivered to deficient liver and brain can mediate pervasive correction and illustrate the potential for gene therapy of MPS and other lysosomal storage diseases.