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.

Positron tomography and nuclear magnetic resonance imaging.

Noninvasive imaging methods for medical diagnosis and biological investigations, have evolved from qualitative radiological techniques to quantitative methods of measuring biochemical and physiological processes in human body. In particular, new developments in emission tomography, nuclear magnetic resonance imaging, and in vivo spectroscopy offer new horizons for medical research and clinical activities. These methods and their potentials are reviewed and contrasted.

Tauopathies with parkinsonism: clinical spectrum, neuropathologic basis, biological markers, and treatment options.

Tauopathies with parkinsonism represent a spectrum of disease entities unified by the pathologic accumulation of hyperphosphorylated tau protein fragments within the central nervous system. These pathologic characteristics suggest shared pathogenetic pathways and possible molecular targets for disease-modifying therapeutic interventions. Natural history studies, for instance, in progressive supranuclear palsy, frontotemporal dementia with parkinsonism linked to chromosome 17, corticobasal degeneration, and Niemann-Pick disease type C as well as in amyotrophic lateral sclerosis/Parkinson-dementia complex permit clinical characterization of the disease phenotypes and are crucial to the development and validation of biological markers for differential diagnostics and disease monitoring, for example, by use of neuroimaging or proteomic approaches. The wide pathologic and clinical spectrum of the tauopathies with parkinsonism is reviewed in this article, and perspectives on future advances in the understanding of the pathogenesis are given, together with potential therapeutic strategies.

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.

Interaction of various intracellular signaling mechanisms involved in mononuclear phagocyte toxicity toward neuronal cells.

Microglia become activated in a wide range of neurodegenerative disorders, including Alzheimer's disease. Such activation may lead to autodestruction of neurons. It is demonstrated here that activation of both human microglia and monocytic THP-1 cells by a combination of lipopolysaccharide and interferon-gamma results in secretion of neurotoxins that kill human neuronal SH-SY5Y cells. This neurotoxicity can be partially blocked by inhibitors of cytosolic phospholipase A2, cGMP-selective phosphodiesterases, or protein kinase C. When combinations of these inhibitors, or combinations of an inhibitor plus nordihydroguaiaretic acid, or the nonsteroidal anti-inflammatory drug diclofenac were tried, additive reductions in neurotoxicity were observed. It is concluded that the stimulants activated multiple intracellular pathways, and that combination therapies inhibiting these pathways might be beneficial for treating neurodegenerative disorders.

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.

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.

Tubulin immunopositive structures resembling intracellular neurofibrillary tangles.

Tau protein promotes microtubule assembly. When aberrantly phosphorylated, it forms the core of neurofibrillary tangles (NFTs). We investigated by immunohistochemistry whether microtubules might also be involved in NFT formation. We found beta-tubulin immunoreactive NFT-like structures in Alzheimer disease, and, more frequently, in the parkinsonism dementia complex of Guam (bodig disease) and Down Syndrome. The beta-tubulin immunoreactive structures were intracellular and appeared at an early stage of tangle development. This may indicate an involvement of tubulin in NFT formation.

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.

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.

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.

On the survival time of a tangled neuron in the hippocampal CA4 region in parkinsonism dementia complex of Guam.

In diseases such as the Parkinson dementia complex of Guam (PDC) or Alzheimer's disease, susceptible neurons develop intracellular tangles (iNFTs) and then die, leaving behind extracellular tangles (eNFTs). We performed counts of healthy neurons, iNFTs, and eNFTs in the hippocampus of Guamanian Chamorros who were neurologically normal or who suffered from PDC. The total of surviving and dead neurons in the CA4 region was remarkably constant from case to case, indicating that eNFTs are not phagocytosed. Since cases of recent PDC showed only marginal tangle formation in CA4, we concluded that tangle development in CA4 commenced close to the onset of the disease. Based on this assumption, as well as the further assumption that the average rate of tangle development and the average lifetime of a tangled neuron do not alter as the disease progresses, we derived equations to determine the average lifetime of tangled neurons. The results varied from 0.13 years for the most rapidly progressing case to 7.98 years for the most slowly developing case. The average for 8 cases was 2.51 years.

Casein kinase 1 delta is associated with pathological accumulation of tau in several neurodegenerative diseases.

The distribution of casein kinase 1 delta (Cki delta) was studied by immunohistochemistry and correlated with other pathological hallmarks in Alzheimer's disease (AD), Down syndrome (DS), progressive supranuclear palsy (PSP), parkinsonism dementia complex of Guam (PDC), Pick's disease (PiD), pallido-ponto-nigral degeneration (PPND), Parkinson's disease (PD), dementia with Lewy bodies (DLB), amyotrophic lateral sclerosis (ALS), and elderly controls. Cki delta was found to be associated generally with granulovacuolar bodies and tau-containing neurofibrillary tangles in AD, DS, PSP, PDC, PPND, and controls, and Pick bodies and ballooned neurons in PiD. It was not associated with tau-containing inclusions in astroglia and oligodendroglia in PPND, PSP, and PDC. It was also not associated with tau-negative Lewy bodies in PD and DLB, Hirano bodies in PDC, Marinesco bodies in PD, AD, and controls and "skein"-like inclusions in anterior motor neurons in ALS. The colocalization of the kinase Cki delta and its apparent substrate tau suggests a function for Cki delta in the abnormal processing of tau.

Molecular and cellular characterization of the membrane attack complex, C5b-9, in Alzheimer's disease.

The membrane attack complex, C5b-9, is of considerable importance in many inflammatory reactions. It is the terminal, cytolytic component of both classical and alternative pathway activation, and its presence presupposes other potentially destructive complement constituents, including anaphylotoxins and opsonins. We have characterized C5b-9 and its C9 constituent in the Alzheimer's disease (AD) and nondemented elderly (ND) brain using immunohistochemistry at the light and electron microscopic levels, Western blot analysis, and the reverse transcriptase polymerase chain reaction. We have also conducted in vitro ELISA assays of amyloid beta-peptide-stimulated SC5b-9 production. C5b-9 is abundantly present in Alzheimer's disease cortex, associated with neurofibrillary tangle containing neurons, dystrophic neurites within neuritic plaques, and neuropil threads, but is weakly detected, if at all, in nondemented elderly cortex under the same conditions. Staining of Alzheimer's disease sections is abolished both by deletion of primary antibody or preabsorption with purified SC5b-9.

Inflammation and Alzheimer's disease.

Inflammation clearly occurs in pathologically vulnerable regions of the Alzheimer's disease (AD) brain, and it does so with the full complexity of local peripheral inflammatory responses. In the periphery, degenerating tissue and the deposition of highly insoluble abnormal materials are classical stimulants of inflammation. Likewise, in the AD brain damaged neurons and neurites and highly insoluble amyloid beta peptide deposits and neurofibrillary tangles provide obvious stimuli for inflammation. Because these stimuli are discrete, microlocalized, and present from early preclinical to terminal stages of AD, local upregulation of complement, cytokines, acute phase reactants, and other inflammatory mediators is also discrete, microlocalized, and chronic. Cumulated over many years, direct and bystander damage from AD inflammatory mechanisms is likely to significantly exacerbate the very pathogenic processes that gave rise to it. Thus, animal models and clinical studies, although still in their infancy, strongly suggest that AD inflammation significantly contributes to AD pathogenesis. By better understanding AD inflammatory and immunoregulatory processes, it should be possible to develop anti-inflammatory approaches that may not cure AD but will likely help slow the progression or delay the onset of this devastating disorder.

Demonstration of a pallido-nigral projection innervating dopaminergic neurons.

Afferents to the substantia nigra from the neostriatum and globus pallidus were studied in the rat by means of the autoradiographic tracing technique. 3H-leucine was injected stereotaxically into either the globus pallidus or neostriatum. Twenty-four hours later the axoplasmic transport of labelled proteins to the substantia nigra was studied by light and electron microscopic autoradiography. In animals used for electron microscopy, degeneration of dopaminergic cells in the substantia nigra was induced by intraventricular injection of 6-hydroxydopamine 72 hours before sacrifice. After neostriatal injections, light microscopic analysis revealed heavy labelling of the globus pallidus and entopeduncular nucleus, but only background labelling of the subthalamic nucleus. There was preferential labelling of the zone reticulata of the substantia nigra, with significantly less labelling of the zone compacta. After pallidal injections, light microscopic analysis showed very light labelling of those parts of the caudate-putamen in the vicinity of the injection site. There was intense labelling of the subthalamic nucleus and heavy labelling of the entopeduncular nucleus. The zona compacta of the substantia nigra was also heavily labelled. There was considerably less labelling of the zona reticulata. The electron microscopic analyses showed that after neostriatal injections, autoradiographic grains in the substantia nigra were located preferentially over boutons which terminated on normal dendritic processes. After pallidal injections, however, grains were preferentially located over boutons synapsing with degenerating dendritic processes. The degeneration produced in these dopaminergic processes by 6-hydroxydopamine was invariably of the dark type. Except for the different association with degenerating vs. non-degenerating dendrites, the subcellular distribution of autoradiographic grains in the substantia nigra was the same after injection into either the globus pallidue or caudate-putamen. Approximately 80 percent of the grains were over axons or boutons which invariably made symmetrical synaptic contacts. These observations demonstrate the existence of a pallido-nigral projection which terminates preferentially on dopaminergic cells in the pars compacta of the substantia nigra. They also confirm previous studies indicating that the strionigral projection terminates mainly in the pars reticulata. These terminations appear to be principally to non-dopaminergic cells.