Fibrillary beta-amyloid deposits are closely associated with atrophic nitric oxide synthase (NOS)-expressing neurons but do not upregulate the inducible NOS in transgenic Tg2576 mouse brain with Alzheimer pathology.

Transgenic mice (Tg2576) that express the Swedish double mutation of human amyloid precursor protein and develop Alzheimer-like beta-amyloid deposits in the aged brain, were used to study the effect of beta-amyloid deposition on expression of both neuronal (nNOS) and inducible nitric oxide synthase (iNOS) in cells surrounding beta-amyloid plaques. Nicotinamide adenine dinucleotide phosphate-diaphorase histochemistry and double immunofluorescent labeling revealed that most of the fibrillary, thioflavine-S-positive cortical beta-amyloid deposits in 13-, 17-, and 21-month-old transgenic animals were closely associated with dystrophic nNOS-positive neurons, while nNOS-bearing neurons located more distal to plaques appeared to be unaffected. There was no significant expression of iNOS in transgenic mouse brain. The data suggest enhanced vulnerability of nNOS-containing neocortical neurons to beta-amyloid toxicity. Alternatively, expression of nNOS may also be a response to plaque-mediated damage of neurons, consistent with a neuroprotective role of nitric oxide.

Sequential upregulation of cell adhesion molecules in degenerating rat basal forebrain cholinergic neurons and in phagocytotic microglial cells.

To study the functional role of adhesion molecules in neurodegenerative events in vivo, the basal forebrain cholinergic lesion-induced expression of the intercellular adhesion molecule (ICAM)-1 and leukocyte function-associated antigen (LFA)-1 was studied by double immunocytochemistry and Western blot analysis. A single intracerebroventricular application of the cholinergic immunotoxin, 192IgG-saporin, produced a selective cholinergic cell loss in rat basal forebrain nuclei detectable by gradual loss of choline acetyltransferase (ChAT)-immunoreactive cells starting 3 days but being nearly complete 7 days after injection of the toxin. The degeneration of cholinergic neurons was accompanied by a striking appearance of activated microglial cells in the lesioned areas. Four days following injection of 192IgG-saporin, ICAM-1 immunoreactivity was predominantly observed in ChAT-positive neurons and partly in activated microglia in the basal forebrain nuclei, while LFA-1 expression at this time point was restricted to neurons. However, 7 days after cholinergic lesion, only a few, shrunken neuronal somata were found to be immunoreactive for ICAM-1 and LFA-1, while activated microglial cells demonstrated strong immunoreactivity for ICAM-1 and LFA-1 in the lesioned forebrain areas, persisting up to 14 days after lesion while no immunoreactivity was observed in neurons at this time point. Western blot analysis demonstrated increased ICAM-1 level in the basal forebrain already detectable 4 days after surgery but being more pronounced 7 days post lesion. The data suggest that ICAM-1 and LFA-1 may act as intercellular recognition signals by which degenerating cholinergic neurons actively participate in the sequence of events leading to their targeting and elimination by phagocytotic microglia.

Rat basal forebrain cholinergic lesion affects neuronal nitric oxide synthase activity in hippocampal and neocortical target regions.

Nitric oxide (NO)-mediated mechanisms have been assigned a role in cortical perfusion, learning and memory as well as in neuronal plasticity. Dysfunction of cortical cholinergic transmission has also been associated with reduced cortical cerebral blood flow and impaired performance in learning and memory tasks suggesting a link between the basal forebrain cholinergic system and cortical NO-mediated mechanisms. The aim of this study was therefore to study the influence of cholinergic input on neuronal NO-synthase (nNOS) activity in cortical cholinoceptive target neurons. A nearly complete loss of rat basal forebrain cholinergic cells was induced by a single intracerebroventricular application of the cholinergic immunotoxin 192IgG-saporin. Basal forebrain cholinergic hypofunction resulted in reduced catalytic and substrate binding activity of nNOS in a number of hippocampal and neocortical subregions 7 days after lesion as revealed by NADPH-diaphorase enzyme histochemistry and quantitative autoradiography of [3H]L-N(G)-nitro-arginine binding, respectively. The total amount of nNOS protein assayed by Western analysis, was not affected in the cortical and hippocampal regions examined. The data indicate that cortical cholinergic deafferentation results in reduced nNOS activity in select cholinoceptive neocortical and hippocampal neurons. As the total amount of cortical nNOS protein was not affected by basal forebrain cholinergic lesion, the results suggest that the ratio of catalytically active and inactive cortical nNOS is driven by basal forebrain cholinergic input presumably via M1-muscarinic cholinergic receptors.

Interleukin-1beta, inducible nitric oxide synthase, and nuclear factor-kappaB are induced in morphologically distinct microglia after rat hippocampal lipopolysaccharide/interferon-gamma injection.

In a number of pathological states of the brain, the activation of the inducible nitric oxide synthase (iNOS) plays a major role. Interleukin (IL)-1beta is believed to be an essential factor in the induction of iNOS. However, little is known about the cascade of events culminating in iNOS expression in vivo. To identify the morphological as well as temporal relationship of lipopolysaccharide (LPS)/interferon-gamma (IFN-gamma) -induced microglial iNOS- and IL-1beta expression, a mixture of LPS and IFN-gamma was injected into the rat hippocampus. IL-1beta immunoreactivity was detected as early as 3 hr following surgery in ramified microglia in the lesioned hippocampus and in distal cortical layers adjacent to the pia mater. By 12 hr post-injection, IL-1beta immunoreactive, ramified microglia with swollen processes were widely distributed throughout hippocampal and neocortical areas, and staining was observed up to 48 hr after treatment. In contrast, iNOS immunostaining was seen in activated amoeboid microglia/macrophages in the ipsilateral hippocampus and around blood vessels but not earlier than 12 hr post-surgery. The temporal pattern of iNOS and IL-1beta expression corresponded to newly induced transcriptional activity as revealed by RT-PCR. Activation of NF-kappaB was restricted to brain regions in which IL-1beta was expressed and was detected both in microglia and astrocytes. A number of LPS/IFN-gamma-stimulated, IL-1beta-expressing microglia exhibited co-staining for activated NF-kappaB. The finding that IL-1beta precedes iNOS expression is consistant with a role of IL-1beta in the intercellular signaling events leading to microglial iNOS-induction. Co-localization of IL-1beta and NF-kappaB suggests an association between IL-1beta and NF-kappaB induction.

Differential injury-dependent glial expression of interleukins-1 alpha, beta, and interleukin-6 in rat brain.

Interleukins (IL)-1 alpha, beta and IL-6 may play essential roles in early inflammatory processes in response to degenerating cholinergic cells observed in the basal forebrain of Alzheimer patients. To address this question in vivo, two distinct lesion paradigms were used. A specific and selective basal forebrain cholinergic cell loss was achieved by a single intracerebroventricular application of the cholinergic immunotoxin, 192IgG-saporin. Intrahippocampal injection of lipopolysaccharide and interferon-gamma was used to produce an exogenously-induced acute inflammation in the brain. In order to disclose the lesion-induced temporal cascade of the expression pattern of IL-1 alpha, IL-1 beta, and IL-6, and the cell types expressing IL-1 alpha, beta/IL-6 mRNA, Western analysis, RT-PCR, and double labeling immunocytochemistry were applied. In the intact brain, IL-6, IL-1 alpha and IL-1 beta demonstrated a constitutive expression in neurons. Following cholinergic lesion neither IL-1 beta nor IL-6 expression could be detected in any of the activated glial cell types, whereas IL-1 alpha was found to be expressed in astroglial cells only. In contrast, hippocampal administration of lipopolysaccharides/interferon-gamma resulted in expression of IL-1 alpha in microglial but not astroglial cells. These in vivo studies clearly demonstrate that the cellular expression of IL-1 alpha, IL-1 beta, and IL-6 in the brain is differentially regulated depending on the kind of injury producing the inflammatory response in the brain. The data suggest that each glial cell seems to be equally capable of expressing a number of various cytokines, but it depends on the kind of stimulus which temporal and cellular cascade of cytokine expression pattern is initiated under a particular pathological condition in the brain.

Influence of mitochondrial protein synthesis inhibition on deafferentation-induced ultrastructural changes in nucleus magnocellularis of developing chicks.

Following cochlea removal in developing chicks, about 30% of the neurons in the ipsilateral second-order auditory nucleus, nucleus magnocellularis, undergo cell death. Administration of chloramphenicol, a mitochondrial protein synthesis inhibitor, results in a pronounced increase in deafferentation-induced cell death. In this study, we examined whether the chloramphenicol enhancement of deafferentation-induced cell death reveals the same ultrastructural characteristics that are seen in degenerating nucleus magnocellularis neurons after cochlea removal alone. Unilateral cochlea removal was performed on anaesthetized posthatch chicks. One group of animals was simultaneously treated with chloramphenicol. Six, twelve, or twenty-four hours following cochlea removal, n. magnocellularis neurons were studied by routine transmission electron microscopy. Particular attention was paid to the integrity of the polyribosomes and rough endoplasmic reticulum. Two ultrastructurally different types of neuronal degeneration were observed in the deafferented nucleus magnocellularis neurons: an early onset electron-lucent type that always involved ribosomal dissociation and a late-onset electron-dense type displaying nuclear pyknosis and severely damaged mitochondria. The percentage of nucleus magnocellularis neurons displaying ribosomal disintegration following cochlea removal was found to be markedly increased after chloramphenicol treatment. This finding suggests that mitochondrial function is important for the maintenance of a functional protein synthesis apparatus following deafferentation.

Protein masking of a ribosomal RNA epitope is an early event in afferent deprivation-induced neuronal death.

Cell death in the developing nervous system is regulated by both afferent synaptic activity and target-derived neurotrophic factors. Loss of afferent innervation via unilateral cochlea removal results in the death of 20-40% of neurons in the neonatal chick cochlear nucleus, nucleus magnocellularis (NM). The process of NM neuronal death involves structural and functional alterations in ribosomes, including decreased protein synthesis, loss of immunoreactivity for a monoclonal anti-ribosomal RNA (rRNA) antibody, Y10B, and eventual ribosome degradation. In the present report we confirm that the Y10B antibody binds specifically to ribosomes in chick NM neurons by electron microscopy. We then performed experiments designed to determine whether loss of rRNA immunoreactivity observed in NM neurons following cochlea removal involves induction of a protein-rRNA interaction. Brain stem tissue from animals subjected to unilateral cochlea removal was treated with protease prior to immunolabeling. Protease treatment restored rRNA immunoreactivity after 3 h of afferent deprivation, confirming that afferent deprivation induces protein-rRNA interactions which mask the Y10B epitope. Immunoprecipitation experiments confirmed that the Y10B antibody recognizes a specific rRNA sequence without posttranscriptional modification.