Lrrk2 interaction with alpha-synuclein in diffuse Lewy body disease.

Mutations of the leucine-rich repeat kinase 2 (LRRK2) gene are the leading cause of genetically inherited Parkinson's disease (PD) and its more severe variant diffuse Lewy body disease (DLB). Pathological mutations in Lrrk2 are autosomal dominant, suggesting a gain of function. Mutations in alpha-synuclein also produce autosomal dominant disease. Here we report an interaction between Lrrk2 and alpha-synuclein in a series of diffuse Lewy body (DLB) cases and in an oxidative stress cell based assay. All five cases of DLB, but none of five controls, showed co-immunoprecipitation of Lrrk2 and alpha-synuclein in soluble brain extracts. Colocalization was also found in pathological deposits in DLB postmortem brains by double immunostaining. In HEK cells transfected simultaneously with plasmids expressing Lrrk2 and alpha-synuclein, co-immunoprecipitation of Lrrk2 and alpha-synuclein was detected when they were exposed to oxidative stress by H(2)O(2). Taken together, these results suggest the possibility that in PD and related synucleinopathies, oxidative stress upregulates alpha-syn and Lrrk2 expression, paving the way for pathological interactions. New therapeutic approaches to PD and the synucleinopathies may result from limiting the interaction between Lrrk2 and alpha-synuclein.

Circadian rhythm in pineal tyrosine hydroxylase.

Tyrosine hydroxylase is the rate-limiting enzyme in catecholamine synthesis. The rat pineal gland is richly innervated by sympathetic nerves from the superior cervical ganglia. The activity of tyrosine hydroxylase was measured in rat pineal gland at 4-hour intervals over a daily cycle of 12 hours of light (7 a.m. to 7 p.m.) and 12 hours of darkness. The results indicate a circadian rhythm with the maximum activity, at 11 p.m. to 3 a.m., about triple the low values observed at 3 p.m. The pattern is similar in phase to that previously reported for melatonin and hydroxyindole-O-methyl transferase activity.

Choline acetyltransferase-containing neurons in rodent brain demonstrated by immunohistochemistry.

Choline acetyltransferase was demonstrated in neuronal structures of the rodent central nervous system by immunohistochemistry through the application of Fab fragments obtained from monospecific antiserums to human choline acetyltransferase. The specificity of the antiserum for the enzyme was confirmed by the staining of both the ventral horn motor neurons in the rat spinal cord and the neuromuscular junction of the guinea pig diaphragm. Enzyme-containing cell bodies were observed in frontal sections of rat and guinea pig brain in the neostriatum, accumbens, nucleus of the diagonal band, medial septum, and olfactory tubercle. Positively staining fibers and probable nerve terminals were also found in the olfactory tubercle field and other areas of the basal forebrain. The results provide information on the distribution of the cholinergic systems in the rostral forebrain of the rodent.

Possible changes in striatal and limbic cholinergic systems in schizophrenia.

Enzymes concerned with neurotransmitter metabolism were measured postmortem in 50 regions from the brains of 11 chronic schizophrenics, 2 patients with senile dementia, 1 depressive, and 18 controls. Enzymes studied were tyrosine hydroxylase, dopa decarboxylase, glutamic decarboxylase, choline acetyltransferase (CAT), and acetylcholinesterase. The schizophrenic group had high CAT activities in the hippocampus, caudate, putamen, and nucleus accumbens; the other patients from the same hospital did not. A compensatory response to long- or short-term drug usage is considered, but correlations are hard to establish in the group studied. An alternative hypothesis proposes that the high levels are a compensatory response to defective cholinergic receptors in the affected areas. On this hypothesis, and by analogy with chorea, dopaminergic antagonists would act in schizophrenia by helping to reestablish cholinergic-dopaminergic balance.

Complement components, but not complement inhibitors, are upregulated in atherosclerotic plaques.

Complement activation occurs in atherosclerotic plaques. The capacity of arterial tissue to inhibit this activation through generation of the complement regulators C1 inhibitor, decay accelerating factor, membrane cofactor protein (CD46), C4 binding protein (C4BP), and protectin (CD59) was evaluated in pairs of aortic atherosclerotic plaques and nearby normal artery from 11 human postmortem specimens. All 22 samples produced mRNAs for each of these proteins. The ratios of plaque versus normal artery pairs was not significantly different from unity for any of these inhibitors. However, in plaques, the mRNAs for C1r and C1s, the substrates for the C1 inhibitor, were increased 2.35- and 4.96-fold, respectively, compared with normal artery; mRNA for C4, the target for C4BP, was elevated l.34-fold; and mRNAs for C7 and C8, the targets for CD59, were elevated 2.61- and 3.25-fold, respectively. By Western blotting and immunohistochemistry, fraction Bb of factor B, a marker of alternative pathway activation, was barely detectable in plaque and normal arterial tissue. These data indicate that it is primarily the classical, not the alternative pathway, that is activated in plaques and that key inhibitors are not upregulated to defend against this activation.

Inflammation of the brain in Alzheimer's disease: implications for therapy.

We briefly describe the similarities and differences between a systemic and a local immune reaction and review the evidence that the latter occurs in Alzheimer's disease (AD) brains. The evidence comes mainly from studies on the complement system, microglia, and cytokines, all of which are important actors in the inflammatory process. The evidence is now overwhelming that the complement proteins and many of the mediators of inflammation are produced locally by brain cells. We will mention briefly the many epidemiological studies indicating that the use of anti-inflammatory drugs reduces the incidence and slows the progress of AD. Mention will also be made of some recent work on animal models of possible relevance to AD and inflammation.

Extracellular neurofibrillary tangles are immunopositive for the 40 carboxy-terminal sequence of beta-amyloid protein.

Neurofibrillary tangles (NFTs) form in a number of neurodegenerative disorders. In Alzheimer disease (AD), intracellular NFTs (iNFTs) develop along with extracellular beta-amyloid (Abeta) deposits. Reports on whether NFTs have Abeta associated with them are inconsistent. Here we study NFTs and their direct relationship with Abeta-like fragments in cases of AD, Down Syndrome, and the parkinsonism-dementia complex of Guam, using a panel of antibodies which recognize different epitopes of Abeta. In all diseases, as well as in the aged controls, the majority of extracellular NFTs (eNFTs) are stained with antibodies recognizing the 40 carboxy-terminal of Abeta, but not other epitopes. Such staining is morphologically distinguishable from the previously described Abeta positive 'tangle associated amyloid deposits' (TAADs), which surround some eNFTs, and are immunopositive for all epitopes of the Abeta molecule. Some iNFTs are immunoreactive with antibodies to the 42 carboxy-terminal epitope, and, to a lesser extent, with antibodies to midportions and more N-terminal epitopes of Abeta. These results may indicate a direct interaction between Abeta and NFTs, although secondary deposition or crossreactivity with other epitopes associated with NFTs cannot be ruled out.

Evaluation of dopaminergic presynaptic integrity: 6-[18F]fluoro-L-dopa versus 6-[18F]fluoro-L-m-tyrosine.

The effectiveness of 6-[18F]fluoro-L-m-tyrosine (6FMT) to evaluate dopamine presynaptic integrity was compared to that of 6-[18F]fluoro-L-dopa (6FDOPA) in vivo by positron emission tomography (PET). Six normal and six 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-lesioned monkeys received 6FDOPA and 6FMT PET scans on separate occasions with identical scanning protocols. Four measures, the rate of uptake of tracer into striatum using either the arterial input function (Ki) or the activity in the occipital cortex as the input function (Kc), the rate of loss of striatal radioactivity (k(loss)), and an index of "effective turnover" of dopamine (k(loss)/Ki), were obtained for both tracers during extended PET studies. 6-[18F]Fluoro-L-m-tyrosine was as effective as 6FDOPA in separating normals from MPTP-lesioned subjects on the basis of the uptake rate constants Ki and Kc. However, in contrast to 6FDOPA, it was not possible to differentiate the normal from the lesioned animal using k(loss) or k(loss)/Ki for 6FMT. Thus, FMT appears to be a reasonable, highly specific tracer for studying the activity of aromatic dopa decarboxylase enzyme as an index of presynaptic integrity. However, if one is interested in investigating further the metabolic pathway and obtaining an in vivo estimate of the effective turnover of dopamine (after pharmacologic manipulation, for example), 6FDOPA remains the tracer of choice.

Excitatory amino acids and Alzheimer's disease: idle thoughts on an exciting subject.

The reported loss of cortical glutaminase activity in Alzheimer's disease is another possible indicator of loss of glutamate neurons. A diversity of excitatory amino acids (EAA) and NMDA receptor subtypes might explain selective neuronal losses of neurons in various diseases. Weaknesses in the arguments presented are the multiple actions of THA and that 2-amino-3-(methylamino)propionate is probably not an EAA.

Inflammation, autotoxicity and Alzheimer disease.

Neuroinflammation is a central feature of Alzheimer disease (AD). It involves an innate immune reaction of sufficient intensity that self attack on neurons occurs. This phenomenon is best described as autotoxicity to distinguish it from classical autoimmunity which involves cloning of peripheral lymphocytes. Many compounds have been identified in AD brain which are known to promote and sustain inflammatory responses. They include beta-amyloid protein; the pentraxins C-reactive protein and amyloid P; complement proteins; the inflammatory cytokines interleukin-1, interleukin-6 and tumor necrosis factor-alpha; the protease inhibitors alpha-2-macroglobulin and alpha-1-antichymotrypsin; and the prostaglandin generating cyclooxygenases COX-1 and COX-2. Orally effective agents which can counteract the influence of these inflammatory stimulators should be effective in treating AD. Epidemiological evidence, coupled with results from pilot clinical trials, suggest there is great promise for traditional COX-1 inhibiting NSAIDs. Inhibitors of mediators closer to the core processes might offer even greater therapeutic promise. Some theoretical opportunities are suggested, based on intervention in the action of the above mentioned mediators.

Lipid peroxides in brain during aging and vitamin E deficiency: possible relations to changes in neurotransmitter indices.

Lipid peroxide levels, were found to be significantly higher in brains of 18 month old as compared to 4 month old rats, with particularly large increases occurring in the olfactory bulb, globus pallidus, cerebral cortex and caudate-putamen (CP). Eighteen month old rats fed a vitamin E deficient diet for 9 months before sacrifice had lipid peroxide levels significantly higher than age-matched controls in the cerebral cortex, hippocampus and hypothalamus. Age-related decreases were seen in choline acetyltransferase, acetylcholinesterase and 3H-QNB binding in some but not all brain regions, while GABA transaminase and MAO showed age-related increases. No age-related change was seen in tyrosine hydroxylase in the CP or in 3H-dihydroalprenolol (DHA) or 3H-spiroperidol binding in the cortex. As compared with controls, vitamin E deficient rats showed decreases of 38% in cortical 3H-DHA binding, of 33% in 3H-QNB binding in the CP and of 23% and 12% in choline acetyltransferase in the CP and cerebellum, respectively. There were no completely consistent regional correlations between significant changes in lipid peroxidase levels and any neurotransmitter indices studied except for MAO which was only measured in the caudate-putamen.

Sodium-dependent glutamate binding in senile dementia.

Sodium-dependent glutamate binding was studied as a possible index of the integrity of glutamate/aspartate (Glu/Asp) nerve endings in seven cortical areas from postmortem brains of 15 persons with senile dementia of the Alzheimer type (SD), 10 controls matched for age, sex and postmortem delay (PMD), and single cases of Down's syndrome and Parkinson-dementia. Binding affinities (Kd) were quite variable from brain to brain but showed no relation to sex, age, PMD or disease. Specific binding site densities did not vary with age or sex but showed overall a significant negative correlation with PMD, a significant decrease in SD, and a significant correlation in the SD--but not the control--samples with choline acetyltransferase (ChAT) activities. The binding data on individual brain regions, however, showed no significant difference between SD cases and controls despite highly significant differences in the ChAT activities. The overall results support but do not confirm a defect in cortical Glu/Asp systems in SD. Reported and obtained data on lesioned rats are summarized to suggest that sodium-dependent glutamate binding may be an ineffective index of Glu/Asp nerve endings.

The pentraxins: possible role in Alzheimer's disease and other innate inflammatory diseases.

Two short pentraxins, C-reactive protein and amyloid P, are found in association with the senile plaques and neurofibrillary tangles of Alzheimer disease (AD). Formerly thought to be made primarily if not solely in liver, recent work has shown that they are made not only in the brain but in other tissues such as heart and arteries. Their synthesis is markedly upregulated in affected brain regions in AD. Since they are known to activate the complement cascade in an antibody-independent fashion and chronic activation can cause destruction of host tissue, these pentraxins may be important initiators of an autodestructive process. As such, they may be prime targets for therapeutic intervention.

Distribution of GABA-T-intensive neurons in the rat forebrain and midbrain.

The neuronal distribution of gamma-aminobutyric acid (GABA) transaminase (GABA-T), the enzyme which metabolizes GABA, has been mapped in rat brain. The method involves staining for newly synthesized GABA-T by the previously established nitro blue tetrazolium technique in animals killed 8-48 hours after administration of gabaculine, an irreversible inhibitor of GABA-T. Neuronal staining is obscured by staining of other elements if initial suppression is inadequate or survival times postgabaculine are too long. With appropriate conditions, GABA-T-positive neuronal somata can be widely detected. The stained cells include neuronal groups previously reported to be GABAergic on the basis of glutamate decarboxylase (GAD)-colchicine immunocytochemistry and other methods, i.e.: Purkinje, basket, Golgi, and stellate neurons of the cerebellum; basket and stellate neurons of the hippocampus; granule and periglomerular cells of the olfactory bulb; magnocellular neurons of the hypothalamus; and neurons of the striatum, pallidum, entopeduncular nucleus, cortex, medial septal area, diagonal band, substantia innominata, reticular nucleus of the thalamus, substantia nigra, and dorsal raphe. Other cells that stain intensely for GABA-T and may be GABAergic include neurons in the midlateral septal area, accumbens, the central medial and basal nuclei of the amygdala, zona incerta, the brainstem reticular formation, central gray, interstitial nucleus of Cajal, and various thalamic nuclei including the periventricular, intralaminar, rhomboid, and subparafascicular. Known non-GABA neuronal groups are negative for GABA-T staining under these conditions, reinforcing the hypothesis that GABA neurons are far more GABA-T intensive than other neurons.

Fine structural analysis of the cortico-striatal pathway.

Considerable evidence has accumulated indicating that one neurotransmitter in the excitatory cortico-striatal tract is glutamate. Lesions of the tract result in reductions in the striatum of glutamate levels as well as high affinity uptake of glutamate into synaptosomes. Furthermore, such lesions eliminate the neurotoxicity of the glutamate analog kainic acid when injected into the striatum. The fine structure of the cortico-striatal pathway was studied to provide evidence regarding the morphology of glutamate nerve endings. Seven days after injection of 3H-proline (20-25 mu Ci) into the rat frontal cortex, axonally transported label appeared in the striatum with uniform distribution in a single type of nerve ending. The labeled boutons had common round vesicles and made asymmetrical contacts, mostly with dendritic spines. This morphology is typical of excitatory synapses, and similar to that previously shown for cholinergic boutons in the striatum. In four animals similarly injected with 3H-proline, kainic acid was administered directly into the striatum to induce degeneration of postsynaptic elements eight to ten hours before sacrifice. In areas affected by these injections, grains appear in patches, possibly resulting from glial swelling. Labeled boutons were seen almost four times as often in synaptic contact with degenerating dendritic elements as with normal ones. The data provide morphological evidence as to the nature of the probable glutamatergic boutons in the striatum, and show the close relationship of such boutons with the neurotoxic effects of kainic acid. This would be anticipated in view of the dependency of kainic acid neurotoxicity on the integrity of the cortico-striatal pathway.

Organization of substance P fibers within the hippocampal formation demonstrated with a biotin-avidin immunoperoxidase technique.

The distribution of substance P-containing fibers within the hippocampal formation of the cat was examined using an immunohistochemical approach. A new, indirect immunoperoxidase method based on the high affinity binding of vitamin H (biotin) by avidin was developed to demonstrate substance P-like immunoreactive fibers. The neocortex contained only occasional single substance P fibers. In contrast, the archicortical structures contained a well organized substance P innervation. The entorhinal area of the parahippocampal gyrus contained a delicate network of varicose axons which appeared to ascend from the subcortical white matter to terminate predominantly in layer II. The subiculum contained a network of positive varicosities throughout the neuropil of both the pyramidal and the molecular layers. These substance P terminals appeared to arise, at least in part, from fibers entering laterally across the angular bundle. Within the ventral hippocampus the substance P innervation was predominantly in the pyramidal cell layer, and was heaviest in field CA3 and weakest in CA1. Some fibers were also observed in strata oriens, lucidum, and radiatum. Occasional varicose fibers could be seen entering the hippocampus from the fimbria. Within the dentate gyrus the substance P fibers were most concentrated in the supragranular layer and among the more superficial of the granule cells. A moderately dense plexus of fibers also occurred in the neuropil of the hilus among the polymorphic cells. In addition, a distinct, narrow band of varicosities was found at the outer edge of the inner molecular layer, while the outer molecular layer did not contain any positive elements. It is concluded that the hippocampal formation receives a distinct, well organized substance P innervation. Substance P fibers appear to enter the hippocampus both laterally across the angular bundle and ventrally through the fimbria. From this detailed examination of the distribution of the substance P fibers within the hippocampus in relation to the other known inputs to this area it is suggested that many of the substance P fibers arise in or, more probably, pass through the septal area to innervate the hippocampus.

Ontogeny of histidine-decarboxylase-immunoreactive neurons in the tuberomammillary nucleus of the rat hypothalamus: time of origin and development of transmitter phenotype.

The ontogeny of the histidine decarboxylase (HDC)-immunoreactive neurons of the tuberomammillary (TM) nucleus was studied in the rat brain. The time of origin of TM neurons was studied by counting the percentage of HDC-immunopositive neurons double labelled by autoradiography in adult progeny of dams injected with [3H]-thymidine at various times during gestation. Neurogenesis began on embryonic day (E) 13, peaked on E16, and was complete by E18. HDC immunoreactivity was first detected in the fetal rat brain on E16. Experiments utilizing short-survival [3H]-thymidine autoradiography combined with HDC immunohistochemistry demonstrated that TM neurons undergo their final mitotic division prior to expression of their transmitter phenotype.

Brainstem afferents to the magnocellular basal forebrain studied by axonal transport, immunohistochemistry, and electrophysiology in the rat.

Brainstem afferents to the magnocellular basal forebrain were studied by using tract tracing, immunohistochemistry and extracellular recordings in the rat. WGA-HRP injections into the horizontal limb of the diagonal band (HDB) and the magnocellular preoptic area (MgPA) retrogradely labelled many neurons in the pedunculopontine and laterodorsal tegmental nuclei, dorsal raphe nucleus, and ventral tegmental area. Areas with moderate numbers of retrogradely labelled neurons included the median raphe nucleus, and area lateral to the medial longitudinal fasciculus in the pons, the locus ceruleus, and the medial parabrachial nucleus. A few labelled neurons were seen in the substantia nigra pars compacta, mesencephalic and pontine reticular formation, a midline area in the pontine central gray, lateral parabrachial nucleus, raphe magnus, prepositus hypoglossal nucleus, nucleus of the solitary tract, and ventrolateral medulla. A similar but not identical distribution of labelled neurons was seen following WGA-HRP injections into the nucleus basalis magnocellularis. The possible neurotransmitter content of some of these afferents to the HDB/MgPA was examined by combining retrograde Fluoro-Gold labelling and immunofluorescence. In the mesopontine tegmentum, many retrogradely labelled neurons were immunoreactive for choline acetyltransferase. In the dorsal raphe nucleus, some retrogradely labelled neurons were positive for serotonin and some for tyrosine hydroxylase (TH); however, the majority of retrogradely labelled neurons in this region were not immunoreactive for either marker. The ventral tegmental area, substantia nigra pars compacta, and locus ceruleus contained retrogradely labelled neurons which were also immunoreactive for TH. Of the retrogradely labelled neurons occasionally observed in the nucleus of the solitary tract, prepositus hypoglossal nucleus, and ventrolateral medulla, some were immunoreactive for either TH or phenylethanolamine-N-methyltransferase. To characterize functionally some of these brainstem afferents, extracellular recordings were made from antidromically identified cortically projecting neurons, mostly located in the HDB and MgPA. In agreement with most previous studies, about half (48%) of these neurons were spontaneously active. Electrical stimulation in the vicinity of the pedunculopontine tegmental and dorsal raphe nuclei elicited either excitatory or inhibitory responses in 21% (13/62) of the cortically projecting neurons.(ABSTRACT TRUNCATED AT 400 WORDS)

The central cholinergic system studied by choline acetyltransferase immunohistochemistry in the cat.

An atlas of the distribution of cholinergic cell bodies, fibers, and terminals, as well as cholinoceptive cells, in the central nervous system of the cat (excluding the cerebellum) is presented from results obtained in immunohistochemical work on choline acetyltransferase. Cholinergic cell bodies are observed in more than forty areas, and cholinoceptive cells in sixty discrete areas of brain sections from the spinal cord to the olfactory bulb. The atlas is presented in seventy cross-sectional drawings of cat brain extending from the olfactory bulb to the upper cervical spinal cord.

Distribution of reduced-nicotinamide-adenine-dinucleotide-phosphate diaphorase-positive cells and fibers in the cat central nervous system.

Previous histochemical studies have suggested that reduced nicotinamide adenine dinucleotide phosphate (NADPH) diaphorase exists in distinct subsets of neurons that neither belong to a single transmitter type nor embrace all the neurons using a single transmitter. As a step toward establishing the role of this enzyme, the distribution of NADPH-diaphorase-positive neurons and fibers in the cat central nervous system was mapped by using a direct histochemical method. Heavily stained NADPH-diaphorase-positive neurons with many prominent cell processes were observed in the cerebral cortex, white matter, caudate nucleus, putamen, nucleus accumbens, septal nucleus, amygdala, anterior, lateral and posterior hypothalamic areas, dorsolateral part of the periaqueductal gray, superior colliculus, central tegmental field (Berman) (pedunculopontine tegmental area), dorsal tegmental nucleus, nucleus coeruleus, mesencephalic and pontine reticular formation, gigantocellular and magnocellular tegmental fields, nucleus facialis, and motor nucleus of the vagus. Moderately stained neurons with two or three prominent cell processes were observed in the nucleus of the diagonal band of Broca, globus pallidus, and substantia innominata. Medium-size, moderately stained neurons that had round large nuclei and no visible cell processes were found in the subthalamic nucleus, pontine gray, trapezoid body, and infratrigeminal, cochlear, and vestibular nuclei. Very dense NADPH-diaphorase-positive nerve terminal fields were seen in the olfactory tubercle, cortex, caudate nucleus, putamen, dentate gyrus, and interpeduncular nucleus. Intensely stained NADPH-diaphorase-positive nerve fibers were found in the stria terminalis, marginal region of the central tegmental field, dorsal tegmental nucleus, and spinal trigeminal tract as well as around the brachium conjunctivum. Although the staining of neurons and tracts was highly selective, they did not correspond to any single known neuronal or neurotransmitter type. Positive staining occurred in discrete subsets of neurons known to be associated with a variety of peptides and classical neurotransmitters. The functional significance of high NADPH diaphorase activity is unknown.