Brain Cholesterol Pathology is the Cause of Alzheimer's Disease.
Koudinov AR, Koudinova NV
Clin Med Health Res (2001) Published online November 27, 2001. clinmed/2001100005

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Abstract

Several recent reports provided important knowledge on how “inhibiting cholesterol production in the brain might inhibit amyloid b(Ab) production, and reduce the accumulation of Ab that causes Alzheimers disease (AD)”. As we show and discuss here cholesterol homeostasis biological misregulation itself has a key role for synaptic plasticity impairment, neuronal degeneration and is the primary cause for several AD hallmarks not limited to brain amyloid. Moreover, Alzheimer's changes in neurochemistry of Ab, tau, neuronal cytoskeleton, and oxidative stress reactions likely represent physiological transitory mechanisms aiming to compensate impaired brain cholesterol dynamics and/or associated neurotransmission and synaptic plasticity failure.

Authors key words:
Alzheimer's disease, amyloid beta precursor, cytoskeleton, Down syndrome, learning, memory, lipoprotein receptor, LTP, neurodegeneration marker, oxidative stress, PHF tau phosphorylation, plaque, phospholipids, synaptic plasticity, secretase, SREBP

Choline in the aging brain.
Katz-Brull R, Koudinov AR, Degani H
[PubMed] [FullText]
Brain Research (2002)  4 October 951(2): 158

Abstract

Proton magnetic resonance spectroscopy has been increasingly utilized in brain research to monitor non-invasively metabolites such as N-acetyl aspartate (NAA), creatine (Cr) and choline (Cho). We present here studies of the effect of aging on the ratios of these metabolites measured in the rat brain in vivo and on choline transport and lipid synthesis in rat brain slices, in vitro. The in vivo studies indicated that the ratios of Cho/NAA and Cho/Cr increased in the aged hippocampus, whereas the ratio of Cr/NAA was similar in the aged and adult hippocampus. These three ratios remained similar in the cortex of adult and aged rats. The in vitro studies revealed that in the aged cortex and the aged hippocampus the activity of the low-affinity choline uptake increased, possibly compensating for a decrease in the high-affinity uptake activity and the rate of choline diffusion. The incorporation of choline into phospholipids exhibited high and low affinity kinetics which were not modified by aging.

Authors key words:
Proton magnetic resonance spectroscopy; High-affinity and low-affinity choline transport; Diffusion; Choline-phospholipid synthesis; Cortex; Hippocampus

Essential Role for Cholesterol in Synaptic Plasticity and Neuronal Degeneration.
Koudinov AR, Koudinova NV

FASEB J (2001) Published online June 27, 2001. 10.1096/fj.00-0815fje
FASEB J Exress Summary:  FASEB J. 2001 15 (10): 1858-1860.
 

Abstract

There is no understanding of the role of cholesterol and phospholipids in the mechanisms of synaptic function and neurodegeneration. Here we report that cholesterol disbalance is critical for synaptic transmission and plasticity as investigated by a study of paired pulse facilitation (PPF) and long-term potentiation (LTP). Extracellular recording of field evoked postsynaptic potentials showed enhanced PPF ratio and an impairment of LTP in CA1 subfield of adult rat ex-vivo hippocampal slices subjected to cyclodextrin- or normal human CSF-HDL3-mediated cholesterol efflux. Immunofluorescence with antibodies against neurofilament and tau revealed that cholesterol and phospholipids depletion causes alteration of normal hippocampal neurites and appearance of PHF-tau in the mossy fibers. We further find that LTP and amyloid beta protein increase [14C]acetate label incorporation into newly synthesized hippocampal membrane lipids. Our results indicate importance of neuronal cholesterol redistribution and synthesis for synaptic plasticity and neurodegeneration.

Authors key words:
Amyloid beta protein, Alzheimer’s disease, Down’s syndrome, cholesterol and phospholipid efflux and synthesis, hippocampal slices, Niemann-Pick type C disease, tau phosphorylation

Neuroscience Letters (2001) 16 November 314: 115-118

Abstract

Several previous studies reported the association of the soluble form of amyloid b (sAb) protein, a major constituent of amyloid deposits in Alzheimer's disease (AD), with normal blood, cerebrospinal  fluid (CSF) and central nervous system high density lipoproteins (HDLs). Present report aimed to elucidate the pattern of sAb and apolipoprotein (apo) distribution in AD CSF-HDL subfractions. We studied AD CSF-HDL subfractions by SDS/PAGE and immunoblot analysis after CSF fractionation via density flotation ultracentrifugation.  AD CSF was characterized by i) increased sAb and apolipoprotein content of the HDL1, and ii) sAb association with apoE and apoJ in HDL₂, HDL₃ and very high density lipoproteins. The finding supports our proposed hypothesis that upregulation of brain cholesterol dynamics is a fundamental event in the pathophysiology of Alzheimer's disease and that sAb binding to apolipoprotein and lipid may have important structure-functional consequences.

Authors key words:
Diagnostic; Lipoprotein receptor; Neuritic plaque; Phospholipids; Reverse cholesterol transport; Synaptic plasticity

Clin Med Health Res (2001) Published online December 17, 2001. clinmed/2001110002

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Abstract
There is no direct evidence that brain amyloid affects neuronal function. In this report we studied hippocampal slices from non-mutated human amyloid precursor protein (APP695) transgenic- and age-matched non-transgenic control mice. We aimed to differentiate separate actions of the aged (25.5 months) transgenic mice plaque-like amyloid and diffuse amyloid of the non-transgenic mice (verified by immunohistochemistry and Congo Red fluorescence) on synaptic plasticity. Extracellular recording of CA1 field excitatory postsynaptic potentials in vitro revealed impairment of input/output characteristics, long-term potentiation, and the delay of few milliseconds in initial post-tetanic traces in aged transgenic versus control mice hippocampal slices. Our results indicate that amyloid plaque (and not diffuse amyloid) may cause synaptic dysfunction, and suggest importance factors other then brain amyloid in pre-plaque stages of Alzheimer’s disease and in Down syndrome.

Alzheimer’s Abeta1-40 Peptide Modulates Lipid Synthesis In Neuronal Cultures And
Intact Rat Fetal Brain Under Normoxic And Oxidative Stress Conditions.

Koudinova NV, Koudinov AR, Yavin E
Neurochem Res 2000 May 25: 653-660

The effect of amyloid beta (Abeta), the major constituent of the Alzheimer's (AD) brain on lipid metabolism was investigated in cultured nerve cells and in a fetal rat brain model. Differentiated (NGF) and undifferentiated PC12 cells or primary cerebral cell cultures were incubated with [14C]acetate in the absence or presence of Abeta1-40. Incorporation of label into lipid species was determined after lipid extraction and TLC separation. Phosphatidylcholine (PC) and phosphatidylserine (PS) synthesis was increased by Abeta1-40, in a dose dependent manner, an effect which was more pronounced in differentiated PC12 cells. A significant proportion of radioactivity (5-6%) was released into the medium with a radioactivity distribution similar to that of the cellular lipids. Cholesterol and PC were the highest labeled medium lipids. Increasing Abeta1-40 concentration up to 0.1 microg/ml in cerebral cells but not in PC12 cells, caused a relative increase (1.5 fold) in release of PS, while that of PE decreased. Stimulation of PS release may possibly be associated with apoptotic cell death. Abeta1-40 peptide (5 microg) was administered intraperitonealy into rat fetuses (18 days gestation) along with [14C]acetate (2microCi/fetus). After 24 h, the maternal-fetal blood supply was occluded for 20 min (ischemia) followed by 15 min reperfusion. Fetuses were killed and liver and brain tissue subjected to lipid extraction and radioactivity determination after TLC. Abeta1-40 peptide increased synthesis of different classes of lipids up to 20-40% in brain tissue compared to controls. Labeling of liver lipids was decreased by Abeta1-40 by 20-30%. A general decrease in synthesis of lipids was observed after ischemia/reperfusion. Our data suggest that Abeta1-40 peptide regulates normal lipid biosynthesis but under ischemia it compromises it. The latter finding may confirm the oxidative stress etiology in AD and suggests that Abeta1-40 modulation of lipid metabolism may have Alzheimer's pathological relevance, particularly at high peptide concentrations.

Key Words:
Alzheimer disease; neuronal cells; oxidative stress; beta amyloid peptide; phospholipid metabolism; cholesterol

Unilateral GluR2(B) hippocampal knockdown: a novel partial seizure model in the developing rat.
Friedman LK, Koudinov AR
J Neurosci 1999 Nov 1 19:21 9412-25

Abstract

    Kainic acid (KA) induces status epilepticus in both adult and young rats but with different consequences on pathology and gene expression. In adults, GluR2(B) AMPA subunit expression is markedly reduced in CA3 neurons before neurodegeneration. In pups, the GluR2(B) subunit is sustained, possibly contributing to neuronal survival. Mechanisms underlying the reduced vulnerability of developing neurons to seizures was investigated by examining the effects of unilateral microinfusions of GluR2(B) antisense oligodeoxynucleotides (AS-ODNs) into the hippocampus of young rats in the presence or absence of a subconvulsive dose of KA. GluR2(B) AS-ODN infusions resulted in spontaneous seizure-like behavior, high stimulus intensity population spikes in the absence of long-term potentiation, and neurodegeneration of CA3 neurons lateral to the infusion site. Electroencephalography revealed paroxysmal activity and high-frequency high-amplitude discharges associated with vigorous and continuous scratching, wildrunning, or bilateral jerking movements. Pups lacking phenotypic behavior exhibited high-rhythmic oscillations and status epilepticus by the dose of KA used.  Radiolabeled AS-ODNs accumulated throughout the ipsilateral dorsal hippocampus. GluR2(B) but not GluR1(A) receptor protein was markedly reduced after GluR2(B) knockdown. In contrast, GluR1(A) knockdown reduced GluR1(A)  but not GluR2(B) protein without change in behavior or morphology. Therefore, unilateral downregulation of hippocampal GluR2(B) but not GluR1(A) protein reduces the seizure threshold and survival of CA3 neurons in the immature hippocampus, possibly providing a novel partial seizure model in the developing rat.

Preliminary account:
5th IBRO World Congress of Neuroscience, July 11-15, 1999, Jerusalem, Israel. Abstract book. p.192.

HDL phospholipid: a natural inhibitor of Alzheimer's amyloid beta-fibrillogenesis?
Koudinov AR, Koudinova NV, Berezov TT, Ivanov YD
Clin Chem Lab Med 1999 Oct 37:10 993-4

Abstract
Alzheimer's (AD) amyloid beta (Ab) is a major constituent of AD brain amyloid deposits and is a normal soluble protein (sAb) of plasma and cerebrospinal fluid high density lipoprotein (HDL). The secondary structure of Ab1-40  in dimyristoilphosphatidylcholine phospholipid (PL) environment  was studied by
Raman spectroscopy in the amide-I band. The ratio of  four secondary structure contents, alpha-helix : beta-sheet : beta-turn : random, were 13 : 53 : 21 : 13 and 2 : 58 : 26 : 14 for the PL bound and solubilized Ab, respectively. Additional spectral analysis in the 2800-2900 cm(-1) range revealed the disordering of PL bilayer by the incorporated Ab. Present data imply i) important role of PL, an HDL and membrane major lipid
structural constituent, in natural physiologic modulation of sAb solubility, and ii) change of membrane PL by the peptide. Both effects may be of special importance in AD and related disorders.

Supported in part by .

Beta-amyloid: Alzheimer's disease and brain beta-amyloidoses
Koudinov AR, Berezov TT, and Koudinova NV
Biochemistry (Moscow) 1999 July 64: 7 752-757

Abstract
This review considers some aspects of the biochemistry of beta-amyloid, a protein which produces insoluble deposits in the brain. These deposits are a specific morphological feature of Alzheimer's disease, Down's syndrome, and senile dementia. Our contribution. to the concept of a soluble form of beta-amyloid as of a normal human protein is presented.

Biochemical assay for amyloid beta deposits to distinguish Alzheimer's disease from other dementias.
Kaplan B, Haroutunian V, Koudinov A, Patael Y, Pras M, Gallo G
Clin Chim Acta 1999 Feb 280: 1-2 147-59

Abstract
Biochemical markers for Alzheimer's disease (AD) are of great value for precise diagnosis and in studies of the pathogenetic processes of this disease. A new biochemical assay allowing to differentiate AD from other forms of dementia is described. The assay is based on the extraction of amyloid beta (A beta) from milligram amounts of brain tissue by using 20% acetonitrile in 0.1% trifluoroacetic acid and its detection by Western blotting. The presence of the 4 kDa A beta was demonstrated in all cases of AD (n = 8) that were diagnosed by the independent histopathological examination of the postmortem tissues. No A beta was found in tissue extracts from seven out of eight cases of other forms of dementia. In contrast to other biochemical techniques of A beta detection in brain, the developed assay is simple; it does not require any special equipment and allows detection of A beta using milligram amounts of brain tissue.

Alzheimer's amyloid beta and lipid metabolism: a missing link ?
Koudinov AR, Berezov TT, and Koudinova NV
FASEB J 1998 September 12: 12 1097-1099

Supported in part by .

Alzheimer's Amyloid beta Interaction with Normal Human Plasma High Density Lipoprotein: Association with Apolipoprotein and Lipids.

Koudinov AR, Berezov TT, Kumar A, Koudinova NV
Clin Chim Acta 1998 February 23 270: 2 75-84

Abstract
Herein we report studies of Alzheimer's amyloid beta protein (Ab) interaction with normal human plasma HDL aiming to clarify to which, apolipoprotein or lipid, lipoprotein (LP) structural constituent soluble Ab is primarily bound. Purified HDLs were incubated with the biotinylated Ab1-40 followed by the LP repurification by Size Exclusion (SE) HPLC. SDS/PAGE, Immunoblot and N-terminal sequence analysis of the biotin-Ab positive protein bands revealed that Ab is bound to many apolipoproteins of the HDL, mainly apoA-I, apoA-II, apoE and apoJ. On the other hand, reconstituted protein free HDL lipid particles also bind Ab peptide and inhibit its aggregation, as intact HDL does. This was assessed by SE-HPLC, SDS/PAGE, immunoblot analysis, ultrastructural electron microscopy and congo red staining for b amyloid fibrils. Our data imply that Ab binding to lipids may play an important role in maintaining the peptide soluble and thus be particularly relevant to Ab normal and pathologic biochemistry and physiology.

Supported in part by .

Protein of memory loss
(Belok Zabivchivosti)

[ in Russian lay language ]

Koudinova NV, Koudinov AR
Chemistry and Life: XXI Century 1998, 1, 23-28

Abstract
Recently we reported that the soluble form of amyloid beta protein (sAbeta) in normal human plasma and cerebrospinal fluid is associated with lipoprotein (LP) particles. In this paper we tested the sAbeta secretion by cells in association with LP in the model of the human hepatoma HepG2 cell line. These cells secreted sAbeta to the culture media and expressed intracellular sAbeta immunoreactivity. Soluble Abeta in the cell supernatant was detected in 200-300 kDa LP complexes in association with apoA-I, apoJ, transthyrethin and phospholipids, triglycerides and free and esterified cholesterol. This was assessed by size exclusion HPLC, immunoprecipitation with corresponding antibodies and by analysis of sAbeta associated metabolically-labeled lipids, respectively. Our results suggest that sAbeta to LP association represents a unique mechanism, governing the normal biology of sAbeta.

Multiple inhibitory effects of Alzheimer's peptide Abeta1-40 on lipid biosynthesis
in cultured human HepG2 cells.
Koudinova NV, Berezov TT, Koudinov AR
FEBS Lett 1996 Oct 21 395: 2-3 204-6

Abstract
Herein we describe the inhibitory effect of the synthetic peptide Abeta1-40, homologous to the major high-density lipoprotein-associated species of Alzheimer's amyloid beta protein (Abeta), on lipid biosynthesis in human hepatic HepG2 cells. This culture synthesizes various lipids from [14C]acetate as a precursor. Treatment of cells with different concentrations of Abeta1-40 decreased the syntheses of various radiolabeled lipid species. The decrease reached saturation at peptide concentrations equal to 10-100 ng/ml. The lipids whose synthesis was decreased most were free and esterified cholesterol and phospholipids. This inhibitory effect suggests that Abeta protein may modulate physiological intracellular lipid syntheses. It may also be of special importance in the pathological condition, and contribute to the neurodegeneration, in Alzheimer's disease and related disorders.

Abstract
Cerebrovascular and parenchymal amyloid deposits found in brains of Alzheimer's disease, Down's syndrome and normal aging are mainly composed of aggregated amyloid beta protein (Abeta), a unique peptide 39 to 44 amino acids long. A similar but soluble Abeta (s Abeta) has been identified in plasma, cerebrospinal fluid (CSF) and cell supernatants, indicating that it is a normal protein. We report here that s Abeta in normal human plasma and CSF is complexed to high density lipoprotein (HDL) 3 and very high density lipoprotein (VHDL). Biotinylated synthetic peptide Abeta1-40 was traced in normal human plasma in in vitro experiments. Both tracer biotin-labeled Abeta1-40 and native sAbeta were specifically recovered in HDL3 and VHDL as it was assessed in immunoprecipitation experiments of purified plasma lipoproteins and lipoprotein depleted plasma. This fact prompted us to ascertain whether the interaction of sAbeta with HDL does occur in normal human CSF in vivo. For this purpose normals human CSF was fractionated by means of sequential flotation ultracentrifugation. The presence of sAbeta in the resulting lipoprotein fractions as well as in the lipoprotein depleted CSF was analysed by immunoblot analysis, electron and immune-electron microscopy and native size exclusion chromatography. Immunoblot analysis with 6E10 monoclonal anti-Abeta antibodies revealed s A beta association with all HDL subspecies of CSF, primarily HDL3 and VHDL and immunoelectron microscopy confirmed an association of sAbeta with CSF-HDL particles of 16.8 +/- 3.2 nm. Native size exclusion chromatography followed by immunoblot analysis with antibodies against Abeta and different apoliproproteins indicated an association of sAbeta with HDL complexes of approximately 200 kDa molecular weight. Soluble Abeta association with HDL3 and VHDL may be involved in maintaining the solubility of Abeta in biological fluids and points to a possible role of lipoproteins and lipoprotein lipid in the biology of aminoloidogenic peptides.

Alzheimer's peptides Abeta1-40 and Abeta1-28 inhibit the plasma cholesterol esterification rate.
Koudinov AR, Koudinova NV, Berezov TT
Biochem Mol Biol Int 1996 Apr 38: 4 747-52

Abstract
The amyloid fibrils of Alzheimer's disease and Down's syndrome amyloid deposits are composed mainly of aggregated amyloid beta protein (Abeta) which also exists in a soluble form. It has been shown that both Alzheimer's disease and Down's syndrome share another common feature: the decrease in plasma cholesterol esterification in affected individuals. In the present work the effect of synthetic peptides Abeta1-40 and Abeta1-28 on normal human plasma cholesterol esterification rate was studied. Both peptides at a concentration of 1 ng/ml inhibited plasma cholesterol esterification rate to 40-50 % of control value. Statistical analysis showed no differences in the effect of Abeta1-40 and Abeta1-28 on the inhibition, suggesting the importance of Abeta sequence 1-28 for this effect.

Biochemical characterization of Alzheimer's soluble amyloid beta protein in human cerebrospinal fluid: association with high density lipoproteins.
Koudinov AR, Koudinova NV, Kumar A, Beavis RC, Ghiso J
Biochem Biophys Res Commun 1996 Jun 25 223: 3 592-7

Abstract
The soluble form of Alzheimer's amyloid beta protein (sAbeta) is associated with high density lipoproteins (HDL) in normal human plasma (BBRC, 1994, 205, 1164-1171). Since sAbeta is also present in cerebrospinal fluid (CSF) and the lipoprotein pattern of CSF is different from that of plasma, it was of interest to ascertain whether the interaction of sAbeta with HDL also occurs in CSF. Normal human CSF lipoproteins were obtained by sequential flotation ultracentrifugation and analyzed for the presence of sAbeta via immunoblot, size-exclusion chromatography, immunoelectron microscopy, N-terminal sequence and mass-spectrometry analyses. Soluble Abeta was associated with CSF-HDL particles of 16.8 +/- 3.2 nm in diameter and approximately 200 kDa of relative molecular mass. A approximately 4.3 kDa component purified by HPLC was immunoreactive with anti-Abeta antibodies and exhibited an N-terminal sequence identical to the Abeta peptide with a mass of 4325.1 Da, indicating that the main sAbeta specie associated with CSF-HDL is Abeta1-40.

The soluble form of Alzheimer's amyloid beta protein is complexed to high density lipoprotein 3 and very high density lipoprotein in normal human plasma.
Koudinov A, Matsubara E, Frangione B, Ghiso J
Biochem Biophys Res Commun 1994 Dec 15 205:2 1164-71

Abstract
The amyloid fibrils of Alzheimer's neuritic plaques and cerebral blood vessels are mainly composed of aggregated forms of a 39 to 44 amino acids peptide, named amyloid beta (Abeta). A similar although soluble form of Abeta (sAbeta) has been identified in plasma, cerebrospinal fluid and cell culture supernatants, indicating that it is produced under physiologic conditions. We report here that sAbeta in normal human plasma is associated with lipoprotein particles, in particular to the HDL3 and VHDL fractions where it is complexed to ApoJ and, to a lesser extent, to ApoAI. This was assessed by immunoprecipitation experiments of purified plasma lipoproteins and lipoprotein-depleted plasma and confirmed by means of amino acid sequence analysis. Moreover, biotinylated synthetic peptide Abeta 1-40 was traced in normal human plasma in in vitro experiments. As in the case of sAbeta, biotinylated Abeta1-40 was specifically recovered in the HDL3 and VHDL fractions. This data together with the previous demonstration that Abeta1-40 is taken up into the brain via a specific mechanism and possibly as an Abeta 1-40-ApoJ complex indicate a role for HDL3- and VHDL-containing ApoJ in the transport of the peptide in circulation and suggest their involvement in the delivery of sAbeta across the blood-brain barrier.

The cerebrospinal-fluid soluble form of Alzheimer's amyloid beta is complexed to SP-40,40 (apolipoprotein J), an inhibitor of the complement membrane-attack complex.
Ghiso J, Matsubara E, Koudinov AR, Choi-Miura NH, Tomita M, Wisniewski T, Frangione B
Biochem J 1993 Jul 1 293 ( Pt 1) 27-30

Abstract
The amyloid fibrils deposited in Alzheimer's neuritic plaque cores and cerebral blood vessels are mainly composed of aggregated forms of a unique peptide, 39-42 amino acids long, named amyloid beta (A beta). A similar, although soluble, A beta ('sA beta') has been identified in cerebrospinal fluid, plasma and cell supernatants, indicating that it is normally produced by proteolytic processing of its precursor protein, amyloid precursor protein (APP). Using direct binding experiments we have isolated and characterized an 80 kDa circulating protein that specifically interacts with a synthetic peptide identical with A beta. The protein was unmistakably identified as SP-40,40 or ApoJ, a cytolytic inhibitor and lipid carrier, by means of amino acid sequence and immunoreactivity with specific antibodies. Immunoprecipitation with anti-SP-40,40 retrieved soluble A beta from cerebrospinal fluid, indicating that the interaction occurs in vivo.