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In exploring the causative role of the most common Pro(301)-to-Leu (TauP301L) tau missense mutation associated with neurodegenerative tauopathies, we examined TauP301L-mediated apoptotic cell death and the expression of a cluster of genes involved in the inhibition of apoptosis (IAPs) in human neuroblastoma SH-SY5Y cells. Our research found that the expression of TauP301L, but not wild-type tau, down regulated the expression of IAPs, including survivin, which plays a role in the mitotic spindle checkpoint. The inhibition of IAPs coincided with the activation of the pro-apoptotic caspase 3, but preceded apoptotic cell death by TUNEL. Furthermore, TauP301L altered the expression of the cell cycle regulatory proteins and induced the cell cycle arrest at G(2)/M phase. Our studies demonstrate that TauP301L downregulates the expression of genes that protect against apoptosis and regulate cell cycle progression. These results suggest a novel mechanism of apoptotic cell death in TauP301L-expressing cells that involves survivin-mediated activation of cell cycle checkpoint.

Protein kinase C (PKC) has been implicated in the pathophysiology of Alzheimer's disease (AD). The levels of particular isoforms and the activation of PKC are reduced in postmortem brain cortex of AD subjects. Receptors for activated C kinase (RACK) are a family of proteins involved in anchoring activated PKCs to relevant subcellular compartments. Recent evidence has indicated that the impaired activation (translocation) of PKC in the aging brain is associated with a deficit in RACK1, the most well-characterized member of this family. The present study was conducted to determine whether alterations in RACK1 occurred in cortical areas where an impaired translocation of PKC has been demonstrated in AD. Here we report the presence of RACK1 immunoreactivity in human brain frontal cortex for the first time and demonstrate a decrease in RACK1 content in cytosol and membrane extracts in AD when compared with non-AD controls. By comparison, the levels of the RACK1-related PKCbetaII were not modified in the same membrane extracts. These observations add a new perspective in understanding the disease-associated defective PKC signal transduction and indicate that a decrease in an anchoring protein for PKC is an additional determinant of this deficit., (Copyright 1999 Academic Press.)

Prior studies indicated that glycoprotein 330 (gp330)/megalin mediates transcytosis of apolipoprotein J (apoJ) with Alzheimer's amyloide-peptide (Abeta) across the vascular membranes of the central nervous system (CNS). Here we show the presence of gp330/megalin mRNA and gp330-like immunoepitopes in brain capillaries and choroid plexus and their absence from brain parenchyma. By polymerase chain reaction (PCR) we estimated 1.2 x 10(5) molecules (1 pg) of gp330/megalin mRNA/microg total brain capillary RNA, which is 3% of that in kidney RNA. However, gp330 mRNA was not detected by in situ hybridization in vascular CNS tissue, presumably because of low transcript prevalence. The ratio of gp330 protein:RNA was 17-fold higher in choroid plexus vs brain capillaries, which implies tissue specific regulation of the protein and mRNA prevalence. We conclude that gp330/megalin mRNA and protein are expressed in brain capillaries and choroid plexus in small amounts that are consistent with the observed activities of this endocytosing receptor in the regulation of apoJ and Abeta uptake by the CNS., (Copyright 1999 Academic Press.)

We explored the constitutive expression, maturational regulation, and relation to kainic-acid-induced apoptosis of cyclooxygenase (COX)-2 mRNA in rat brain. In adult rats, COX-2 mRNA was expressed primarily in limbic structures. Constitutive COX-2 mRNA expression increased markedly between Postnatal Day 7 (P7) and P14, reaching adult levels by P21. Despite intense KA-induced seizures, no COX-2 mRNA induction was found before P14 in any brain region examined. During response to KA-induced seizures in adult brain, COX-2 mRNA induction paralleled temporally and overlapped anatomically the appearance of cellular morphological features of apoptosis in subsets of cells of the pyramidal neuron layer of the hippocampal formation, amygdaloid complex, and pyriform cortex. While COX-2 mRNA showed KA-induced elevation in the granule cell layer of the dentate gyrus, no detectable morphological features of apoptosis were found in this region. Finally, monotypic culture of rat corticohippocampal neurons confirmed the neuronal expression of COX-2 in vitro and revealed that COX-2 is induced during response to glutamate treatment, leading to neuron death. These studies may provide novel insights into the role of COX-2 and mechanisms of action of nonsteroidal anti-inflammatory drugs in Alzheimer's disease.

Possible roles of the complement (C) system in the normal and injured brain were explored with inbred mice that carried a frameshift mutation in the C5 gene. A congenic pair was used: the C5-sufficient (C5+) B10.D2/nSnJ strain with the functional allele (Hc1) from the C57BL/10J donor strain was compared with the C5-deficient (C5-) B10.D2/oSnJ with the Hc0 allele from the C5-deficient DBA/2J donor strain. In response to the excitotoxin kainic acid (KA), C5- mice had more hippocampal pyramidal neuron death and greater induction of astrocyte mRNAs (GFAP, apoE, apoJ). In primary astrocyte cultures from unlesioned mice, an inflammatory stimulus (LPS) caused greater production of IL-6 and TNF production in C5- mice. These enhanced responses to KA and LPS suggest that hereditary C5 deficits modify responses to neurodegenerative stimuli of neurons and astrocytes. Moreover, unlesioned C5- mice had smaller input-output slopes for the NMDA component of the EPSP amplitude, but enhanced the Ca(+2)-dependent AMPA binding. Thus, C5 deficits also modify basal properties of glutamatergic neurotransmission that pertain to synaptic plasticity. These findings are also discussed in relation to roles of the C-system in Alzheimer disease (AD). C5 deficiencies may also be considered in the choice of strains as transgene hosts and for genetic analysis of normal and pathological brain functions. In recent transgenic studies for AD, C5- hosts showed greater neurodegeneration, consistent with the present data. These pleiotropic associations of C5 deficiency indicate roles for the C-system in neurodegeneration, but also in normal neural functions.

Clusterin (apoJ), a multifunctional apolipoprotein made by cells in the brain and many other locations, is associated with aggregated amyloid beta-peptide (A beta) in senile and diffuse plaques of Alzheimer's disease (AD). We observed that purified human serum clusterin partially blocked the aggregation of synthetic A beta 1-42, as shown by centrifugal assays (14,000g x 10 min) and by atomic force (scanning probe) microscopy. Slowly sedimenting A beta complexes were formed in the presence of clusterin, which included aggregates > 200 kDa that resist dissociation by low concentrations of SDS. Clusterin enhanced the oxidative stress caused by A beta, as assayed by oxidative stress in PC12 cells with MTT, which is widely used to estimate neurotoxicity. These indications of enhanced neurotoxicity by the MTT assay were observed in the highly aggregated rapidly sedimenting fraction, but also in more slowly sedimenting "soluble" forms. This novel activity of slowly sedimenting A beta may enhance the neurotoxicity of A beta deposits in AD brains, because soluble complexes have a potential for diffusing to damage distal neurons.

This study examined TGF-beta 1 mRNA levels and cellular localization in the F344 rat hippocampus following deafferentation or kainic acid (KA)-induced neurodegeneration. By RNA solution hybridization, TGF-beta 1 transcripts were at low prevalence in intact adult rat hippocampus (0.02 pg/microgram total RNA). Four days after unilateral entorhinal cortex lesioning (ECL), TGF-beta 1 mRNA increased threefold in the ipsilateral hippocampus. This increase was localized to the outer molecular layer of the dentate gyrus, where gliosis, synapse loss, and synaptic reorganization occur. TGF-beta 1 mRNA also increased in the hippocampus after KA-induced limbic seizures, particularly in the areas of the hippocampus undergoing neurodegeneration. Microglia [OX-42 immunoreactive (IR) cells] responded to these two lesions with distinct morphological changes. Combined immunocytochemistry-in situ hybridization showed that TGF-beta 1 mRNA was localized to reactive microglia (OX-42-IR, with blunt processes), but not to resting ramified microglia (OX-42-IR, with numerous fine processes) or to astrocytes (GFAP-IR). After ECL, round macrophage-like cells (OX-42-IR with TGF-beta 1 mRNA) were seen at the wound site. Thus, brain macrophage/microglial cells produce TGF-beta 1 mRNA in the hippocampus in response to deafferentation and neurodegeneration.

A 168-nucleotide exon, found in alternatively spliced amyloid precursor protein (APP) mRNAs, encodes a Kunitz protease inhibitor (KPI) domain. Kainic acid (ip) caused a selective increase of KPI mRNA in rat hippocampus. By in situ hybridization, KPI mRNA was induced in the neuronal layers of the hippocampus 11-12 h after the onset of kainate-induced seizures. The kainate-induced elevation of the KPI-containing APP-770 mRNA was blocked by pretreatment with the anticonvulsant pentobarbital. These data suggest that kainate-induced seizures cause alterations in APP RNA stability and/or processing in rat hippocampal neurons.

These data show the presence of mRNAs for two complement components (C) in the adult rat brain and describe their responses to experimental lesions. Cortical deafferentation caused elevations in striatal C1qB and C4 mRNAs that coincided temporally and overlapped anatomically with the course of degeneration of corticostriatal afferent fibers. By in situ hybridization, C1qB mRNA in the lesioned striatum was colocalized to cells immunoreactive for CR3, a complement receptor found on microglia-macrophages. The mRNA for SGP-2, a putative C inhibitor in rat, showed parallel changes. Similarly, in hippocampus and other brain regions, kainic acid lesions increased C1qB mRNA. The data suggest that microglia-macrophages and possibly other cells in rat brain rapidly up-regulate C-mRNAs in response to deafferentation and local neuron injury. These experimental responses provide models to analyze changes in C components during Alzheimer's disease and other chronic neurodegenerative conditions.

Transneuronal regression in substantia nigra reticulata (SNR) and substantia nigra compacta (SNC) neurons was studied in Fischer 344 male rats by immunocytochemistry and by in situ hybridization. Three months after striatal lesioning by ibotenic acid, there was a shrinkage (30%) of the SNR region cross-sectional area and a 50% disappearance of neurons that contain glutamic acid decarboxylase (GAD)-mRNA, but only in the ventromedial portion of this nucleus. Loss of dopaminergic neurons, as recognized by tyrosine hydroxylase immunoreactivity, occurred only in caudal portions of the SNC and SNR. These findings suggest that lesions in reciprocally connected pathways, like the nigrostriatal and striatonigral systems, may produce a vicious cycle (feedforward cascade) of neurodegeneration due to interference with retrograde ana anterograde influences.

Immunocytochemical and electrophysiological studies indicate the existence of a functional relationship between Cholecystokinin (CCK) and dopaminergic transmission. In order to gain more information on this relationship, the effect of Caerulein, a CCK stable analogue, on rat spontaneous locomotor activity and on biochemical markers of dopaminergic transmission were measured simultaneously. The concentrations of 3,4-dihydroxyphenylacetic acid (DOPAC) and the spontaneous or K+ evoked release of dopamine were studied in rat striatum and nucleus accumbens immediately after testing for motor activity. An almost complete reduction in locomotor activity but not significant changes in DOPAC content and dopamine release were observed in rats injected with the peptide (0.25/microgram/Kg, intraperitoneally). DOPAC concentrations were slightly (30%) decreased by increasing 200 folds caerulein dose. In addition, a very minute dose of haloperidol (25 /microgram/Kg) potentiated the caerulein (0.25/microgram/Kg) induced hypomotility, while the parameters of dopaminergic metabolism were unaffected. Our results indicate the existence of a relevant pharmacological interaction between caerulein and dopamine antagonists, although it is not clear whether this interaction takes place at the dopamine terminals level.

Plasma and CSF met-enkephalin immunoreactive material (ME-IR) concentrations were measured in 5 subjects at different hours of the day. Within the 24 hrs CSF ME-IR concentrations ranged in the same patient from 20 to 5000 pg/ml. Plasma ME-IR concentrations presented a circadian rhythm with lower values between 4:00 and 8:00 p.m.

In poly(A) RNA from cerebral cortex obtained postmortem from victims of Alzheimer's disease (AD), an alternatively spliced mRNA for the amyloid precursor protein (APP-695 mRNA) was shown to be decreased by 65%. Another form (APP-751 mRNA) with an additional exon encoding a Kunitz-type (serine) protease inhibitor motif did not change appreciably (less than 30% decrease) in AD cortex. If this twofold increase in the APP-751 mRNA/APP-695 mRNA ratio results in a corresponding increase in the APP-751/APP-695 protein ratio, this would support the hypothesis that impaired proteolysis promotes the accumulation of abnormal proteins in the brain during AD. In the two previously known, major alternatively spliced forms of ca. 3.3 and 3.5 kb, we resolved doublet RNAs for each form that are consistent with sequence data showing multiple polyadenylation sites (J. Kang et al., 1987, Nature (London) 325, 733-736.). Smaller APP-related transcripts were also found (1.1, 1.0, 0.8, and 0.3 kb), some of which are selectively altered in AD.