A panel of 148 monoclonal antibodies directed against Drosophila neural antigens has been prepared by using mice immunized with homogenates ofDrosophila tissue. Antibodies were screened immunohistochemically on cryostat sections of fly heads. A large diversity of staining patterns was observed. Some antigens were broadly distributed among tissues; others were highly specific to nerve fibers, neuropil, muscle, the tracheal system, cell nuclei, photoreceptors, or other structures. The antigens for many of the antibodies have been identified on immunoblots. Monoclonal antibodies that identify specific molecules within the nervous system should prove useful in the study of the molecular genetics of neural development.The development and function of the nervous system involves the participation of various genetically encoded macromolecules whose nature and identities are largely unknown. An incisive approach toward identifying them is offered by the hybridoma technique (1), which can produce monoclonal antibodies (MAbs) against specific molecules singled out from a complex mixture of immunogens. For example, by immunization with leech segmental ganglia, Zipser and McKay (2) found MAbs that recognized antigens expressed in different subsets of neurons. Trisler et al. (3) used immunization with chick retinal segments and identified a molecule that occurs in a gradient across the developing retina.As a step toward elucidating, at the molecular level, the relationship between the genome and the nervous system, we have produced a battery of MAbs showing specificity for the Drosophila nervous system and related tissues. Drosophila offers the advantage that both classical genetic and recombinant DNA techniques can be used to identify the gene that encodes the antigen. Mutations in the gene that alter or delete the antigen can provide insight into its role in development, physiology, and behavior.MATERIALS AND METHODS Generation of MAbs. Hybridomas were obtained from five fusions. Immunogens were homogenates of heads, brains, or retinas dissected from Drosophila melanogaster (C-S strain) adult flies previously frozen at -90°C and dehydrated at -200C in acetone. Each BALB/c mouse received, over a period of 4-20 months, five injections (four intraperitoneal and one intravenous). Each injection contained material dissected from 20-50 flies. Standard procedures using NS-1 myeloma cells (4) were followed to generate hybridomas, which were cloned by limiting dilution. The MAbs described here are IgGs, except 3F12 which is IgM and 3H6 and 4E9 for which the class is undetermined.Immunohistochemistry. Cryostat sections (2-10 ,um) of fly heads were prepared and stored on coverslips at -20'C over silica gel. To eliminate eye pigments, which can cause fluorescent background, cinnabar brown mutant flies were used. Staining was done at room temperature. The sections were fixed with 2% formalin in 75 mM Na phosphate buffer (pH 7.0) for 30 min and then rinsed for 5 min in 10 mM Tris HCl, pH 7.5/ 130 mM NaCl/5 mM KCI/5 mM NaN3/1 mM...

We have reported previously (Michikawa, M., Fan, Q.-W., Isobe, I., and Yanagisawa, K. (2000) J. Neurochem. 74, 1008 -1016) that exogenously added recombinant human apolipoprotein E (apoE) promotes cholesterol release in an isoform-dependent manner. However, the molecular mechanism underlying this isoform-dependent promotion of cholesterol release remains undetermined. In this study, we demonstrate that the cholesterol release is mediated by endogenously synthesized and secreted apoE isoforms and clarify the mechanism underlying this apoE isoform-dependent cholesterol release using cultured astrocytes prepared from human apoE3 and apoE4 knock-in mice. Cholesterol and phospholipids were released into the culture media, resulting in the generation of two types of high density lipoprotein (HDL)-like particles; one was associated with apoE and the other with apoJ. The amount of cholesterol released into the culture media from the apoE3-expressing astrocytes was ϳ2.5-fold greater than that from apoE4-expressing astrocytes. In contrast, the amount of apoE3 released in association with the HDL-like particles was similar to that of apoE4, and the sizes of the HDL-like particles released from apoE3-and apoE4-expressing astrocytes were similar. The molar ratios of cholesterol to apoE in the HDL fraction of the culture media of apoE3-and apoE4-expressing astrocytes were 250 ؎ 6.0 and 119 ؎ 5.1, respectively. These data indicate that apoE3 has an ability to generate similarly sized lipid particles with less number of apoE molecules than apoE4, suggesting that apoE3-expressing astrocytes can supply more cholesterol to neurons than apoE4-expressing astrocytes. These findings provide a new insight into the issue concerning the putative alteration of apoE-related cholesterol metabolism in Alzheimer's disease.Previous epidemiological studies show that an elevated serum cholesterol level is a risk factor for the development of Alzheimer's disease (AD) 1 (1-3) and that statin therapy reduces the frequency of AD (4) and dementia (5). The decreased levels of cellular cholesterol have been shown to reduce A␤ production in vitro (6) and in vivo (7). Previous studies have also shown the association of cholesterol accumulation with mature senile plaques (8) and neurofibrillary tangle-bearing neurons (9). Additionally, a recent study (10) has suggested that an increased cholesterol level in the membrane facilitates amyloid fibril formation through formation of GM1 gangliosidebound A␤, a putative endogenous seed. These findings suggest that increased cellular cholesterol levels induce high amyloid ␤-protein (A␤) production and subsequent AD development. However, several studies (11-14) have shown opposing evidence indicating that cholesterol levels in serum, cell membranes of brains, and cerebrospinal fluid are decreased in AD patients compared with those in controls. Previous studies have shown that increased dietary cholesterol levels reduce A␤ secretion (15) and that increased cellular cholesterol levels inhibit the A␤-mediated cell tox...

Hyperphosphorylated tau is the major component of paired helical filaments in neurofibrillary tangles found in Alzheimer's disease (AD) brain. Starvation of adult mice induces tau hyperphosphorylation at many paired helical filaments sites and with a similar regional selectivity as those in AD, suggesting that a common mechanism may be mobilized. Here we investigated the mechanism of starvation-induced tau hyperphosphorylation in terms of tau kinases and Ser/Thr protein phosphatases (PP), and the results were compared with those reported in AD brain. During starvation, tau hyperphosphorylation at specific epitopes was accompanied by decreases in tau protein kinase I/glycogen synthase kinase 3␤ (TPKI/ GSK3␤), cyclin-dependent kinase 5 (cdk5), and PP2A activities toward tau. These results demonstrate that the activation of TPKI/GSK3␤ and cdk5 is not necessary to obtain hyperphosphorylated tau in vivo, and indicate that inhibition of PP2A is likely the dominant factor in inducing tau hyperphosphorylation in the starved mouse, overriding the inhibition of key tau kinases such as TPKI/GSK3␤ and cdk5. Furthermore, these data give strong support to the hypothesis that PP2A is important for the regulation of tau phosphorylation in the adult brain, and provide in vivo evidence in support of a central role of PP2A in tau hyperphosphorylation in AD. Alzheimer's disease (AD)1 is a neurodegenerative disorder characterized by the presence of two histopathological hallmarks called senile plaques and neurofibrillary tangles. The former are deposits of the ␤-amyloid peptide (A␤) (1), whereas neurofibrillary tangles consist of hyperphosphorylated tau protein assembled in paired helical filaments (PHF) (2). Hyperphosphorylation refers to the state that tau is phosphorylated at more sites than tau from adult brain and that, for a given site, a higher than normal percentage of tau molecules is phosphorylated (3). Tau is a microtubule-associated protein, and its normal physiological function is to bind and stabilize microtubules. In vitro studies have shown that PHF-tau fails to promote microtubule assembly (4 -9), and thus it has been proposed to lead to microtubule destabilization, appearance of neurofibrillary tangles, and neurodegeneration in AD brain (10). Phosphorylation of tau can be regulated by many protein kinases and phosphatases in vitro (11-13). These findings, and the changes in kinases and phosphatases observed in AD, suggest that tau hyperphosphorylation in AD brain is likely to be due to an imbalance of the protein phosphorylation and dephosphorylation systems. But to date the mechanism of conversion from normal adult tau to hyperphosphorylated tau, the significance of tau hyperphosphorylation in PHF formation, and its relationship to A␤ deposition remain largely elusive.Tau hyperphosphorylation is a physiological reversible response of the brain to stressful conditions like cold water stress 2 (14), heat-shock (15), or starvation (16). Starvation induces decreases in circulating glucose, insulin, and leptin, and inc...

Glycogen synthase kinase 3 (GSK3) plays important roles in Wnt and insulin signaling, cell fate determination, and Alzheimer-like tau phosphorylation. We discovered an isoform of tau protein kinase I (TPKI) / GSK3b with a 13 amino acid insert in the catalytic domain owing to alternative splicing. The alternative transcripts were found in the brains of the mouse, rat and human, with highly conserved sequences. The variant protein, named TPKI2 / GSK3b2, was abundant in the brain. Immunohistochemistry indicated differential distribution of the conventional and the new TPKI / GSK3b isoforms within young neurons. TPKI2 / GSK3b2 showed decreased kinase activities towards two phosphorylation sites on tau compared with the conventional isoform. Immunohistochemistry indicated that TPKI2 / GSK3b2 occurs predominantly in the neuronal soma, while TPKI1 / GSK3b1 is found both in the soma and processes. These results indicate that the new splice isoform has a different function. Because the amino acid insert occurs in the domain implicated in interaction with a protein phosphatase in a homologous kinase cdk-2, the alternative splicing can regulate multiprotein complex formation and function involving TPKI / GSK3b. Keywords: glycogen synthase kinase 3, protein kinase subdomain, splice variant, tau phosphorylation, tau protein kinase.

τ protein kinase I (TPKI) purified from bovine brain extract has been shown to phosphorylate τ and to form paired helical filament (PHF) epitopes and was found recently to be identical to glycogen synthase kinase‐3β (GSK‐3β). Before elucidating a role of TPKI/GSK‐3β in PHF formation, it is necessary to investigate the normal function of the enzyme. To study the distribution and developmental changes of the enzyme, specific polyclonal antibodies were prepared against TPKI and GSK‐3α. Immunoblot analysis demonstrated that TPKI was nearly specifically localized in the brain of adult rats. The level of TPKI in the rat brain was high at gestational day 18, peaked on postnatal day 8, and then decreased rapidly to a low level, which was sustained up to 2 years. Immunohistochemistry indicated primarily neuronal localization of TPKI. Growing axons were stained most intensely in the developing cerebellum, but the immunoreactivity became restricted to the gray matter in the mature tissue. Parallel fibers had a high level of TPKI and also stained intensely for τ. These findings indicate that τ is one of the physiological substrates of TPKI and suggest that the enzyme plays an important role in the growth of axons during development of the brain.

Hyperphosphorylated tau is the major component of paired helical filaments in neurofibrillary tangles found in Alzheimer's disease brains, and tau hyperphosphorylation is thought to be a critical event in the pathogenesis of this disease. The objective of this study was to reproduce tau hyperphosphorylation in an animal model by inducing hypoglycemia. Food deprivation of mice for 1 to 3 days progressively enhanced tau hyperphosphorylation in the hippocampus, to a lesser extent in the cerebral cortex, but the effect was least in the cerebellum, in correspondence with the regional selectivity of tauopathy in Alzheimer's disease. This hyperphosphorylation was reversible by refeeding for 1 day. We discuss possible mechanisms of this phenomenon, and propose the starved mouse as a simple model to study in vivo tau phosphorylation and dephosphorylation which are altered in Alzheimer's disease.z 1999 Federation of European Biochemical Societies.

Intracellular recordings ofaction potentials were made from the cervical giant axon in Shaker (Sh) mutants and normal Drosophila. The mutants showed abnormally long delays in repolarization. The defect is not due to abnormal Ca2+ channels, because it persists in the presence of Co2+, blocker. On the other hand, the K+-channel blocker 4-aminopyridine causes a similar effect in normal animals, suggesting that the

Aging and apolipoprotein E4 (apoE4) expression are strong risk factors for the development of Alzheimer's disease (AD); however, their pathological roles remain to be clarified. In the process of AD development, the conversion of the nontoxic amyloid -protein (A) monomer to its toxic aggregates is a fundamental process. We previously hypothesized that A aggregation is accelerated through the generation of GM1 ganglioside (GM1)-bound A which acts as a seed for A fibril formation. Here we report that GM1 level in detergent-resistant membrane microdomains (DRMs) of synaptosomes increased with age and that this increase was significantly pronounced in the apoE4-than the apoE3-knock-in mouse brain. Furthermore, we show that A aggregation is markedly accelerated in the presence of the synaptosomes of the aged apoE4-knock-in mouse brain. These observations suggest that aging and apoE4 expression cooperatively accelerate A aggregation in the brain through an increase in the level of GM1 in neuronal membranes.