Amyloid Beta

Mutations in the amyloid precursor protein (APP) gene cause early-onset familial Alzheimer disease (AD) by affecting the formation of the amyloid ␤ (A␤) peptide, the major constituent of AD plaques. We expressed human APP 751 containing these mutations in the brains of transgenic mice. Two transgenic mouse lines develop pathological features reminiscent of AD. The degree of pathology depends on expression levels and specific mutations. A 2-fold overexpression of human APP with the Swedish double mutation at positions 670͞671 combined with the V717I mutation causes A␤ deposition in neocortex and hippocampus of 18-month-old transgenic mice. The deposits are mostly of the diffuse type; however, some congophilic plaques can be detected. In mice with 7-fold overexpression of human APP harboring the Swedish mutation alone, typical plaques appear at 6 months, which increase with age and are Congo Red-positive at first detection. These congophilic plaques are accompanied by neuritic changes and dystrophic cholinergic fibers. Furthermore, inf lammatory processes indicated by a massive glial reaction are apparent. Most notably, plaques are immunoreactive for hyperphosphorylated tau, reminiscent of early tau pathology. The immunoreactivity is exclusively found in congophilic senile plaques of both lines. In the higher expressing line, elevated tau phosphorylation can be demonstrated biochemically in 6-month-old animals and increases with age. These mice resemble major features of AD pathology and suggest a central role of A␤ in the pathogenesis of the disease.

Amyloid ␤ peptide (A␤), the principal proteinaceous component of amyloid plaques in brains of Alzheimer's disease patients, is derived by proteolytic cleavage of the amyloid precursor protein (APP). Proteolytic cleavage of APP by a putative ␣-secretase within the A␤ sequence precludes the formation of the amyloidogenic peptides and leads to the release of soluble APPs␣ into the medium. By overexpression of a disintegrin and metalloprotease (ADAM), classified as ADAM 10, in HEK 293 cells, basal and protein kinase C-stimulated ␣-secretase activity was increased severalfold. The proteolytically activated form of ADAM 10 was localized by cell surface biotinylation in the plasma membrane, but the majority of the proenzyme was found in the Golgi. These results support the view that APP is cleaved both at the cell surface and along the secretory pathway. Endogenous ␣-secretase activity was inhibited by a dominant negative form of ADAM 10 with a point mutation in the zinc binding site. Studies with purified ADAM 10 and A␤ fragments confirm the correct ␣-secretase cleavage site and demonstrate a dependence on the substrate's conformation. Our results provide evidence that ADAM 10 has ␣-secretase activity and many properties expected for the proteolytic processing of APP.

Fibrillogenesis of the amyloid ␤-protein (A␤) is a seminal pathogenetic event in Alzheimer's disease. Inhibiting fibrillogenesis is thus one approach toward disease therapy. Rational design of fibrillogenesis inhibitors requires elucidation of the stages and kinetics of A␤ fibrillogenesis. We report results of studies designed to examine the initial stages of A␤ oligomerization. Size exclusion chromatography, quasielastic light scattering spectroscopy, and electron microscopy were used to characterize fibrillogenesis intermediates. After dissolution in 0.1 M Tris-HCl, pH 7.4, and removal of preexistent seeds, A␤ chromatographed almost exclusively as a single peak. The molecules composing the peak had average hydrodynamic radii of 1.8 ؎ 0.2 nm, consistent with the predicted size of dimeric A␤. Over time, an additional peak, with a molecular weight >100,000, appeared. This peak contained predominantly curved fibrils, 6 -8 nm in diameter and <200 nm in length, which we have termed "protofibrils." The kinetics of protofibril formation and disappearance are consistent with protofibrils being intermediates in the evolution of amyloid fibers. Protofibrils appeared during the polymerization of A␤-(1-40), A␤-(1-42), and A␤-(1-40)-Gln 22 , peptides associated with both sporadic and inherited forms of Alzheimer's disease, suggesting that protofibril formation may be a general phenomenon in A␤ fibrillogenesis. If so, protofibrils could be attractive targets for fibrillogenesis inhibitors.

Nicastrin, a transmembrane glycoprotein, forms high molecular weight complexes with presenilin 1 and presenilin 2. Suppression of nicastrin expression in Caenorhabditis elegans embryos induces a subset of notch/glp-1 phenotypes similar to those induced by simultaneous null ...

Protein conformational diseases, including Alzheimer's, Huntington's, and Parkinson's diseases, result from protein misfolding, giving a distinct fibrillar feature termed amyloid. Recent studies show that only the globular (not fibrillar) conformation of amyloid proteins is sufficient to induce cellular pathophysiology. However, the 3D structural conformations of these globular structures, a key missing link in designing effective prevention and treatment, remain undefined as of yet. By using atomic force microscopy, circular dichroism, gel electrophoresis, and electrophysiological recordings, we show here that an array of amyloid molecules, including amyloid-␤(1-40), ␣-synuclein, ABri, ADan, serum amyloid A, and amylin undergo supramolecular conformational change. In reconstituted membranes, they form morphologically compatible ion-channel-like structures and elicit single ion-channel currents. These ion channels would destabilize cellular ionic homeostasis and hence induce cell pathophysiology and degeneration in amyloid diseases.

Neuroinflammation is a characteristic of pathologically affected tissue in several neurodegenerative disorders. These changes are particularly observed in affected brain areas of Alzheimer&#x27;s disease (AD) cases. They include an accumulation of large numbers of activated microglia and ...

Amyloid (8 protein (A(P) is the 40to 42residue polypeptide implicated in the pathogenesis of Alzheimer disease. We have incorporated this peptide into phosphatidylserine liposomes and then fused the liposomes with a planar bilayer. When incorporated into bilayers the A(3P forms channels, which generate linear current-voltage relationships in symmetrical solutions. A permeability ratio, PK/Pa, of 11 for the open A(3P channel was estimated from the reversal

Excessive cerebral accumulation of the 42-residue amyloid beta-protein (Abeta) is an early and invariant step in the pathogenesis of Alzheimer&#39;s disease. Many studies have examined the cellular production of Abeta from its membrane-bound precursor, including the role of the presenilin proteins therein, but almost nothing is known about how Abeta is degraded and cleared following its secretion. We previously screened neuronal and nonneuronal cell lines for the production of proteases capable of degrading naturally secreted Abeta under biologically relevant conditions and concentrations. The major such protease identified was a metalloprotease released particularly by a microglial cell line, BV-2. We have now purified and characterized the protease and find that it is indistinguishable from insulin-degrading enzyme (IDE), a thiol metalloendopeptidase that degrades small peptides such as insulin, glucagon, and atrial natriuretic peptide. Degradation of both endogenous and synthetic...

Age has a powerful effect on enhanced susceptibility to neurodegenerative diseases, including susceptibility to stroke and cognitive impairment (CI) even in optimally healthy individuals. We critically evaluated the notion that oxidative stress increases in aging brain. Rigorous studies show logarithmic age-dependent increases in oxidized proteins and oxidized DNA lesions. Decreased activity of antioxidant protective enzymes does not account for the observed increases. The reactivity of the lipid oxidation product 4-hydroxy-2-nonenal (HNE) with key mitochondria enzymes may be important in the age-dependent loss in energy generation and enhanced susceptibility of neurons to apoptosis. Age-dependent enhanced neuroinflammatory processes may play an important role in toxin generation that causes death or dysfunction of neurons in neurodegenerative diseases. Non-steroidal anti-inflammatory drugs (NSAIDs) show significant promise. Vitamin E supplementation did not show major beneficial effect on cognitive functions. Major clinical trials for Alzheimer&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;s disease (AD) involving cycloxygenase-II (COX II) inhibitors and amyloid-beta vaccination have been discontinued. Novel therapeutics based on blocking neuron damaging neuroinflammatory processes show great promise for abating dementia progression although they have yet to make it to clinical practice.

We have studied the fibrillogenesis of synthetic amyloid 13-protein-(1-40) fragment (Aj3) in 0.1 M HCI. At low pH, Af3 formed fibrils at a rate amenable to detailed monitoring by quasi-elastic light-scattering spectroscopy. Examination of the fibrils with circular dichroism spectroscopy and electron microscopy showed them to be highly similar to those found in amyloid plaques. We determined the hydrodynamic radii ofA,8 aggregates during the entire process of fibril nucleation and growth. Above an Aj3 concentration of :0.1 mM, the initial rate of elongation and the final size of fibrils were independent of Aj3 concentration. Below an A,j concen

Mitochondrial dysfunction has been proposed to play a pivotal role in neurodegenerative diseases, including Alzheimer's disease (AD). To address whether mitochondrial dysfunction precedes the development of AD pathology, we conducted mitochondrial functional analyses in female triple transgenic Alzheimer's mice (3xTg-AD) and age-matched nontransgenic (nonTg). Mitochondrial dysfunction in the 3xTg-AD brain was evidenced by decreased mitochondrial respiration and decreased pyruvate dehydrogenase (PDH) protein level and activity as early as 3 months of age. 3xTg-AD mice also exhibited increased oxidative stress as manifested by increased hydrogen peroxide production and lipid peroxidation. Mitochondrial amyloid beta (A␤) level in the 3xTg-AD mice was significantly increased at 9 months and temporally correlated with increased level of A␤ binding to alcohol dehydrogenase (ABAD). Embryonic neurons derived from 3xTg-AD mouse hippocampus exhibited significantly decreased mitochondrial respiration and increased glycolysis. Results of these analyses indicate that compromised mitochondrial function is evident in embryonic hippocampal neurons, continues unabated in females throughout the reproductive period, and is exacerbated during reproductive senescence. In nontransgenic control mice, oxidative stress was coincident with reproductive senescence and accompanied by a significant decline in mitochondrial function. Reproductive senescence in the 3xTg-AD mouse brain markedly exacerbated mitochondrial dysfunction. Collectively, the data indicate significant mitochondrial dysfunction occurs early in AD pathogenesis in a female AD mouse model. Mitochondrial dysfunction provides a plausible mechanistic rationale for the hypometabolism in brain that precedes AD diagnosis and suggests therapeutic targets for prevention of AD.

Amyloid ␤-peptide [A␤(1-42)] is central to the pathogenesis of Alzheimer's disease (AD), and the AD brain is under intense oxidative stress, including membrane lipid peroxidation. A␤(1-42) causes oxidative stress in and neurotoxicity to neurons in mechanisms that are inhibited by Vitamin E and involve the single methionine residue of this peptide. In particular, A␤ induces lipid peroxidation in ways that are inhibited by free radical antioxidants. Two reactive products of lipid peroxidation are the alkenals, 4-hydroxynonenal (HNE) and 2-propenal (acrolein). These alkenals covalently bind to synaptosomal protein cysteine, histidine, and lysine residues by Michael addition to change protein conformation and function. HNE or acrolein binding to proteins introduces a carbonyl to the protein, making the protein oxidatively modified as a consequence of lipid peroxidation. Immunoprecipitation of proteins from AD and control brain, obtained no longer than 4 h PMI, showed selective proteins are oxidatively modified in the AD brain. Creatine kinase (CK) and ␤-actin have increased carbonyl groups, and Glt-1, a glutamate transporter, has increased binding of HNE in AD. A␤(1-42) addition to synaptosomes also results in HNE binding to Glt-1, thereby coupling increased A␤(1-42) in AD brain to increased lipid peroxidation and its sequelae and possibly explaining the mechanism of glutatmate transport inhibition known in AD brain. A␤ also inhibits CK. Implications of these findings relate to decreased energy utilization, altered assembly of cytoskeletal proteins, and increased excitotoxicity to neurons by glutamate, all reported for AD. The epsilon-4 allele of the lipid carrier protein apolipoprotein E (APOE) allele is a risk factor for AD. Synaptosomes from APOE knockout mice are more vulnerable to A␤-induced oxidative stress (protein oxidation, lipid peroxidation, and ROS generation) than are those from wild-type mice. Further, synaptosomes from allele-specific APOE knock-in mice have tiered vulnerability to A␤(1-42)-induced oxidative stress, with APOE4 more vulnerable to A␤(1-42) than are those from APOE2 or APOE3 mice. These results are consistent with the notion of a coupling of the oxidative environment in AD brain and increased risk of developing this disorder. Taken together, the findings from in-vitro studies of lipid peroxidation induced by A␤(1-42) and postmortem studies of lipid peroxidation (and its sequelae) in AD brain may help explain the APOE allele-related risk for AD, some of the functional and structural alterations in AD brain, and strongly support a causative role of A␤(1-42)-induced oxidative stress in AD neurodegeneration.

A valid quantitative imaging method for the measurement of amyloid deposition in humans could improve Alzheimer's disease (AD) diagnosis and antiamyloid therapy assessment. Our group developed Pittsburgh Compound-B (PIB), an amyloid-binding radiotracer, for positron emission tomography (PET). The current study was aimed to further validate PIB PET through quantitative imaging (arterial input) and inclusion of subjects with mild cognitive impairment (MCI). Pittsburgh Compound-B studies were performed in five AD, five MCI, and five control subjects and five subjects were retested within 20 days. Magnetic resonance images were acquired for partial volume correction and region-of-interest definition (e.g., posterior cingulate: PCG; cerebellum: CER). Data were analyzed using compartmental and graphical approaches. Regional distribution volume (DV) values were normalized to the reference region (CER) to yield DV ratios (DVRs). Good agreement was observed between compartmental and Logan DVR values (e.g., PCG: r ¼ 0.89, slope ¼ 0.91); the Logan results were less variable. Nonspecific PIB retention was similar across subjects (n ¼ 15, Logan CER DV: 3.6370.48). Greater retention was observed in AD cortical areas, relative to controls (Po0.05). The PIB retention in MCI subjects appeared either 'AD-like' or 'control-like'. The mean test/ retest variation was B6% in primary areas-of-interest. The Logan analysis was the method-of-choice for the PIB PET data as it proved stable, valid, and promising for future larger studies and voxelbased statistical analyses. This study also showed that it is feasible to perform quantitative PIB PET imaging studies that are needed to validate simpler methods for routine use across the AD disease spectrum.

Synapse deterioration underlying severe memory loss in early Alzheimer's disease (AD) is thought to be caused by soluble amyloid beta (A␤) oligomers. Mechanistically, soluble A␤ oligomers, also referred to as A␤-derived diffusible ligands (ADDLs), act as highly specific pathogenic ligands, binding to sites localized at particular synapses. This binding triggers oxidative stress, loss of synaptic spines, and ectopic redistribution of receptors critical to plasticity and memory. We report here the existence of a protective mechanism that naturally shields synapses against ADDL-induced deterioration. Synapse pathology was investigated in mature cultures of hippocampal neurons. Before spine loss, ADDLs caused major downregulation of plasma membrane insulin receptors (IRs), via a mechanism sensitive to calcium calmodulin-dependent kinase II (CaMKII) and casein kinase II (CK2) inhibition. Most significantly, this loss of surface IRs, and ADDL-induced oxidative stress and synaptic spine deterioration, could be completely prevented by insulin. At submaximal insulin doses, protection was potentiated by rosiglitazone, an insulin-sensitizing drug used to treat type 2 diabetes. The mechanism of insulin protection entailed a marked reduction in pathogenic ADDL binding. Surprisingly, insulin failed to block ADDL binding when IR tyrosine kinase activity was inhibited; in fact, a significant increase in binding was caused by IR inhibition. The protective role of insulin thus derives from IR signaling-dependent downregulation of ADDL binding sites rather than ligand competition. The finding that synapse vulnerability to ADDLs can be mitigated by insulin suggests that bolstering brain insulin signaling, which can decline with aging and diabetes, could have significant potential to slow or deter AD pathogenesis.

The amyloid ␤-peptide (A␤) has been suggested to exert its toxicity intracellularly. Mitochondrial functions can be negatively affected by A␤ and accumulation of A␤ has been detected in mitochondria. Because A␤ is not likely to be produced locally in mitochondria, we decided to investigate the mechanisms for mitochondrial A␤ uptake. Our results from rat mitochondria show that A␤ is transported into mitochondria via the translocase of the outer membrane (TOM) machinery. The import was insensitive to valinomycin, indicating that it is independent of the mitochondrial membrane potential. Subfractionation studies following the import experiments revealed A␤ association with the inner membrane fraction, and immunoelectron microscopy after import showed localization of A␤ to mitochondrial cristae. A similar distribution pattern of A␤ in mitochondria was shown by immunoelectron microscopy in human cortical brain biopsies obtained from living subjects with normal pressure hydrocephalus. Thus, we present a unique import mechanism for A␤ in mitochondria and demonstrate both in vitro and in vivo that A␤ is located to the mitochondrial cristae. Importantly, we also show that extracellulary applied A␤ can be internalized by human neuroblastoma cells and can colocalize with mitochondrial markers. Together, these results provide further insight into the mitochondrial uptake of A␤, a peptide considered to be of major significance in Alzheimer's disease.

Recent studies have indicated an association between Alzheimer's disease (AD) and central nervous system (CNS) insulin resistance. However, the cellular mechanisms underlying the link between these two pathologies have not been elucidated. Here we show that signal transduction by neuronal insulin receptors (IR) is strikingly sensitive to disruption by soluble A␤ oligomers (also known as ADDLs). ADDLs are known to accumulate in AD brain and have recently been implicated as primary candidates for initiating deterioration of synapse function, composition, and structure. Using mature cultures of hippocampal neurons, a preferred model for studies of synaptic cell biology, we found that ADDLs caused a rapid and substantial loss of neuronal surface IRs specifically on dendrites bound by ADDLs. Removal of dendritic IRs was associated with increased receptor immunoreactivity in the cell body, indicating redistribution of the receptors. The neuronal response to insulin, measured by evoked IR tyrosine autophosphorylation, was greatly inhibited by ADDLs. Inhibition also was seen with added glutamate or potassium-induced depolarization. The effects on IR function were completely blocked by NMDA receptor antagonists, tetrodotoxin, and calcium chelator BAPTA-AM. Downstream from the IR, ADDLs induced a phosphorylation of Akt at serine 473 , a modification associated with neurodegenerative and insulin resistance diseases. These results identify novel factors that affect neuronal IR signaling and suggest that insulin resistance in AD brain is a response to ADDLs, which disrupt insulin signaling and may cause a brainspecific form of diabetes as part of an overall pathogenic impact on CNS synapses.-Zhao, W. Q., De Felice, F. G., Fernandez, S., Chen, H., Lambert, M. P., Quon, M. J., Krafft, G. A., Klein, W. L. Amyloid beta oligomers induce impairment of neuronal insulin receptors. FASEB J. 22, 246 -260 (2008)

There is a growing body of evidence to support a role for oxidative stress in Alzheimer's disease (AD), with increased levels of lipid peroxidation, DNA and protein oxidation products (HNE, 8-HO-guanidine and protein carbonyls respectively) in AD brains. The brain is a highly oxidative organ consuming 20% of the body's oxygen despite accounting for only 2% of the total body weight. With normal ageing the brain accumulates metals ions such iron (Fe), zinc (Zn) and copper (Cu). Consequently the brain is abundant in antioxidants to control and prevent the detrimental formation of reactive oxygen species (ROS) generated via Fenton chemistry involving redox active metal ion reduction and activation of molecular oxygen. In AD there is an over accumulation of the Amyloid β peptide (Aβ), this is the result of either an elevated generation from amyloid precursor protein (APP) or inefficient clearance of Aβ from the brain. Aβ can efficiently generate reactive oxygen species in the presence of the transition metals copper and iron in vitro. Under oxidative conditions Aβ will form stable dityrosine cross-linked dimers which are generated from free radical attack on the tyrosine residue at position 10. There are elevated levels of urea and SDS resistant stable linked Aβ oligomers as well as dityrosine cross-linked peptides and proteins in AD brain. Since soluble Aβ levels correlate best with the degree of degeneration [C.A. McLean, R.A. Cherny, F.W. Fraser, S.J. Fuller, M.J. Smith, K. Beyreuther, A.I. Bush, C.L. Masters, Soluble pool of Abeta amyloid as a determinant of severity of neurodegeneration in Alzheimer's disease, Ann. Neurol. 46 (1999) 860–866] we suggest that the toxic Aβ species corresponds to a soluble dityrosine cross-linked oligomer. Current therapeutic strategies using metal chelators such as clioquinol and desferrioxamine have had some success in altering the progression of AD symptoms. Similarly, natural antioxidants curcumin and ginkgo extract have modest but positive effects in slowing AD development. Therefore, drugs that target the oxidative pathways in AD could have genuine therapeutic efficacy.

Amyloid b-peptide (Ab) 1)42 oligomers have recently been discussed as intermediate toxic species in Alzheimer's disease (AD) pathology. Here we describe a new and highly stable Ab 1)42 oligomer species which can easily be prepared in vitro and is present in the brains of patients with AD and Ab 1)42-overproducing transgenic mice. Physicochemical characterization reveals a pure, highly water-soluble globular 60-kDa oligomer which we named 'Ab 1)42 globulomer'. Our data indicate that Ab 1)42 globulomer is a persistent structural entity formed independently of the fibrillar aggregation pathway. It is a potent antigen in mice and rabbits eliciting generation of Ab 1)42 globulomer-specific antibodies that do not cross-react with amyloid precursor protein, Ab 1)40 and Ab 1)42 monomers and Ab fibrils. Ab 1)42 globulomer binds specifically to dendritic processes of neurons but not glia in hippocampal cell cultures and completely blocks long-term potentiation in rat hippocampal slices. Our data suggest that Ab 1)42 globulomer represents a basic pathogenic structural principle also present to a minor extent in previously described oligomer preparations and that its formation is an early pathological event in AD. Selective neutralization of the Ab globulomer structure epitope is expected to have a high potential for treatment of AD.

We quantified the amount of amyloid ␤-peptide (A␤) immunoreactivity as well as amyloid deposits in a large cohort of transgenic mice overexpressing the V717F human amyloid precursor protein (APP V717F؉/؊ TG mice) with no, one, or two mouse apolipoprotein E (Apoe) alleles at various ages. Remarkably, no amyloid deposits were found in any brain region of APP V717F؉/؊ Apoe ؊/؊ TG mice as old as 22 mo of age, whereas age-matched APP V717F ؉/؊ Apoe ؉/؊ and Apoe ؉/؉ TG mice display abundant amyloid deposition. The amount of A␤ immunoreactivity in the hippocampus was also markedly reduced in an Apoe gene dose-dependent manner (Apoe ؉/؉ > Apoe ؉/؊ Ͼ Ͼ Apoe ؊/؊), and no A␤ immunoreactivity was detected in the cerebral cortex of APP V717F؉/؊ Apoe ؊/؊ TG mice at any of the time points examined. The absence of apolipoprotein E protein (apoE) dramatically reduced the amount of both A␤1-40 and A␤1-42 immunoreactive deposits as well as the resulting astrogliosis and microgliosis normally observed in APP V717F TG mice. ApoE immunoreactivity was detected in a subset of A␤ immunoreactive deposits and in virtually all thioflavine-Sfluorescent amyloid deposits. Because the absence of apoE alters neither the transcription or translation of the APP V717F transgene nor its processing to A␤ peptide(s), we postulate that apoE promotes both the deposition and fibrillization of A␤, ultimately affecting clearance of protease-resistant A␤/apoE aggregates. ApoE appears to play an essential role in amyloid deposition in brain, one of the neuropathological hallmarks of Alzheimer's disease.

In light of the rising prevalence of Alzheimer&#39;s disease (AD), new strategies to prevent, halt, and reverse this condition are needed urgently. Perturbations of brain network activity are observed in AD patients and in conditions that increase the risk of developing AD, suggesting that aberrant network activity might contribute to AD-related cognitive decline. Human amyloid precursor protein (hAPP) transgenic mice simulate key aspects of AD, including pathologically elevated levels of amyloid-β peptides in brain, aberrant neural network activity, remodeling of hippocampal circuits, synaptic deficits, and behavioral abnormalities. Whether these alterations are linked in a causal chain remains unknown. To explore whether hAPP/amyloid-β-induced aberrant network activity contributes to synaptic and cognitive deficits, we treated hAPP mice with different antiepileptic drugs. Among the drugs tested, only levetiracetam (LEV) effectively reduced abnormal spike activity detected by elect...

Progressive amyloid deposition in senile plaques and cortical blood vessels may play a central role in the pathogenesis of Alzheimer disease. We have used x-ray diffraction and electron microscopy to study the molecular organization and morphology of macromolecular assemblies formed by three synthetic peptides homologous to fi protein of brain amyloid: fl-(1-28), residues 1-28 of the fi protein; [Ala"Jfl-(1-28), P-(1-28) with alanine substituted for lysine at position 16; and fl-(18-28), residues 18-28 of the fi protein.

Amyloid-b (Ab) peptides, derived from the amyloid precursor protein, and the microtubule-associated protein tau are key pathogenic factors in Alzheimer's disease (AD). How exactly they impair cognitive functions is unknown. We assessed the effects of Ab and tau on axonal transport of mitochondria and the neurotrophin receptor TrkA, cargoes that are critical for neuronal function and survival and whose distributions are altered in AD. Ab oligomers rapidly inhibited axonal transport of these cargoes in wild-type neurons. Lowering tau levels prevented these defects without affecting baseline axonal transport. Thus, Ab requires tau to impair axonal transport, and tau reduction protects against Ab-induced axonal transport defects.

In Alzheimer disease (AD), neurons are thought to be subjected to the deleterious cytotoxic effects of activated microglia. We demonstrate that binding of amyloidbeta peptide (Aβ ) to neuronal Receptor for Advanced Glycation Endproduct (RAGE), a cell surface receptor for Aβ , induces macrophage-colony stimulating factor (M-CSF) by an oxidant sensitive, nuclear factor kappa B-dependent pathway. AD brain shows increased neuronal expression of M-CSF in proximity to Aβ deposits, and in cerebrospinal fluid from AD patients there was ≈ 5-fold increased M-CSF antigen (P < 0.01), compared with age-matched controls. M-CSF released by Aβ -stimulated neurons interacts with its cognate receptor, c-fms, on microglia, thereby triggering chemotaxis, cell proliferation, increased expression of the macrophage scavenger receptor and apolipoprotein E, and enhanced survival of microglia exposed to Aβ , consistent with pathologic findings in AD. These data delineate an inflammatory pathway triggered by engagement of Aβ on neuronal RAGE. We suggest that M-CSF, thus generated, contributes to the pathogenesis of AD, and that M-CSF in cerebrospinal fluid might provide a means for monitoring neuronal perturbation at an early stage in AD.

The receptor for advanced glycation end products (RAGE) is thought to be a primary transporter of -amyloid across the blood-brain barrier (BBB) into the brain from the systemic circulation, while the low-density lipoprotein receptor-related protein (LRP)-1 mediates transport of -amyloid out of the brain. To determine whether there are Alzhei-mer's disease (AD)-related changes in these BBBassociated -amyloid receptors, we studied RAGE, LRP-1, and -amyloid in human elderly control and AD hippocampi. In control hippocampi, there was robust RAGE immunoreactivity in neurons, whereas microvascular staining was barely detectable. LRP-1 staining, in contrast, was clearly evident within microvessels but only weakly stained neurons. In AD cases, neuronal RAGE immunoreactivity was signiWcantly decreased. An unexpected Wnding was the strongly positive microvascular RAGE immunoreactivity. No evidence for colocalization of RAGE and -amyloid was seen within either microvessels or senile plaques. A reversed pattern was evident for LRP-1 in AD. There was very strong staining for LRP-1 in neurons, with minimal microvascular staining. Unlike RAGE, colocalization of LRP-1 and -amyloid was clearly present within senile plaques but not microvessels. Western blot analysis revealed a much higher concentration of RAGE protein in AD hippocampi as compared with controls. Concentration of LRP-1 was increased in AD hippocampi, likely secondary to its colocalization with senile plaques. These data conWrm that AD is associated with changes in the relative distribution of RAGE and LRP-1 receptors in human hippocampus. They also suggest that the proportion of amyloid within the brains of AD patients that is derived from the systemic circulation may be signiWcant.

Extracellular deposition of amyloid fibrils is responsible for the pathology in the systemic amyloidoses and probably also in Alzheimer disease [Haass, C. & Selkoe, D. J. (1993) Cell 75, 1039-1042] and type II diabetes mellitus [Lorenzo, A., Razzaboni, B., Weir, G. C. & Yankner, B. A. (1994) Nature (London) 368, 756-760]. The fibrils themselves are relatively resistant to proteolysis in vitro but amyloid deposits do regress in vivo, usually with clinical benefit, if new amyloid fibril formation can be halted. Serum amyloid P component (SAP) binds to all types of amyloid fibrils and is a universal constituent of amyloid deposits, including the plaques, amorphous amyloid P protein deposits and neurofibrillary tangles ofAlzheimer disease [Coria, F., Castano, E.,

Microglia are associated with neuritic plaques in Alzheimer disease (AD) and serve as a primary component of the innate immune response in the brain. Neuritic plaques are fibrous deposits composed of the amyloid beta-peptide fragments (Abeta) of the amyloid precursor protein (APP). Numerous studies have shown that the immune cells in the vicinity of amyloid deposits in AD express mRNA and proteins for pro-inflammatory cytokines, leading to the hypothesis that microglia demonstrate classical (Th-1) immune activation in AD. Nonetheless, the complex role of microglial activation has yet to be fully explored since recent studies show that peripheral macrophages enter an &quot;alternative&quot; activation state. To study alternative activation of microglia, we used quantitative RT-PCR to identify genes associated with alternative activation in microglia, including arginase I (AGI), mannose receptor (MRC1), found in inflammatory zone 1 (FIZZ1), and chitinase 3-like 3 (YM1). Our findings c...

Alzheimer&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;s disease (AD) brains are characterized by extensive oxidative stress. Additionally, large depositions of amyloid beta-peptide (Abeta) are observed, and many researchers opine that Abeta is central to the pathogenesis of AD. Our laboratory combined these two observations in a comprehensive model for neurodegeneration in AD brains centered around Abeta-induced oxidative stress. Given the oxidative stress in AD and its potentially important role in neurodegeneration, considerable research has been conducted on the use of antioxidants to slow or reverse the pathology and course of AD. One source of antioxidants is the diet. This review examines the literature of the effects of endogenous and exogenous, nutritionally-derived antioxidants in relation to AD. In particular, studies of glutathione and other SH-containing antioxidants, vitamins, and polyphenolic compounds and their use in AD and modulation of Abeta-induced oxidative stress and neurotoxicity are reviewed.

Mutations in APP at the βand γ-secretase sites have been shown to cause familial forms of early-onset AD. These mutations increase the production of either total amyloid-β peptides (Aβ) or the more amyloidogenic Aβ1-42 species. In contrast, most mutations within the Aβ domain do not result in a full range of AD pathology but characteristically result in cerebrovascular pathology 1-3 . For example, the E693Q point mutation in APP (affecting residue 22 of Aβ) results in HCHWA-D, an autosomal-dominant form of CAA 4,5 . Those afflicted with HCHWA-D suffer from recurrent lobar cerebral hemorrhages, with an onset in the fifth decade of life 6 . At autopsy, extensive CAA is typically found in leptomeningeal arteries and cortical arterioles, and to a lesser extent in meningocortical veins. Unlike in AD, parenchymal amyloid plaques are not prominent in HCHWA-D, although diffuse parenchymal Aβ is found 7 . Because of these features of the disease, HCHWA-D has become the human genetic archetype of the Aβ congophilic angiopathy seen sporadically in many of the elderly and in the majority of those with AD 8,9 .

Alzheimer's disease (AD) is the most common type of dementia in the elderly. Products of oxidative and nitrosative stress (OS and NS, respectively) accumulate with aging, which is the main risk factor for AD. This provides the basis for the involvement of OS and NS in AD pathogenesis. OS and NS occur in biological systems due to the dysregulation of the redox balance, caused by a deficiency of antioxidants and/or the overproduction of free radicals. Free radical attack against lipids, proteins, sugars and nucleic acids leads to the formation of bioproducts whose detection in fluids and tissues represents the currently available method for assessing oxidative/nitrosative damage. Post-mortem and in-vivo studies have demonstrated an accumulation of products of free radical damage in the central nervous system and in the peripheral tissues of subjects with AD or mild cognitive impairment (MCI). In addition to their individual role, biomarkers for OS and NS in AD are associated with altered bioenergetics and amyloidbeta (Ab) metabolism. In this review we discuss the main results obtained in the field of biomarkers of oxidative/nitrosative stress in AD and MCI in humans, in addition to their potential role as a tool for diagnosis, prognosis and treatment efficacy in AD. ß

Standardized extract from the leaves of the Ginkgo biloba tree, labeled EGb761, has been used in clinical trials for its beneficial effects on brain functions, particularly in connection with agerelated dementias and Alzheimer's disease (AD). Substantial experimental evidence indicates that EGb761 protects against neuronal damage from a variety of insults, but its cellular and molecular mechanisms remain unknown. Using a neuroblastoma cell line stably expressing an AD-associated double mutation, we report that EGb761 inhibits formation of amyloid-␤ (A␤) fibrils, which are the diagnostic, and possibly causative, feature of AD.

Production of amyloid-␤ protein (A␤) is initiated by a ␤-secretase that cleaves the A␤ precursor protein (APP) at the N terminus of A␤ (the ␤ site). A recently identified aspartyl protease, BACE, cleaves the ␤ site and at residue 11 within the A␤ region of APP. Here we show that BACE2, a BACE homolog, cleaves at the ␤ site and more efficiently at a different site within A␤. The Flemish missense mutation of APP, implicated in a form of familial Alzheimer's disease, is adjacent to this latter site and markedly increases A␤ production by BACE2 but not by BACE. BACE and BACE2 respond identically to conservative ␤-site mutations, and alteration of a common active site Arg inhibits ␤-site cleavage but not cleavage within A␤ by both enzymes. These data suggest that BACE2 contributes to A␤ production in individuals bearing the Flemish mutation, and that selective inhibition of these highly similar proteases may be feasible and therapeutically advantageous.

Epidemiological evidence suggests that nonsteroidal anti-inflammatory drugs (NSAIDs) decrease the risk for Alzheimer's disease (AD). Certain NSAIDs can activate the peroxisome proliferatoractivated receptor-␥ (PPAR␥), which is a nuclear transcriptional regulator. Here we show that PPAR␥ depletion potentiates ␤secretase [␤-site amyloid precursor protein cleaving enzyme (BACE1)] mRNA levels by increasing BACE1 gene promoter activity. Conversely, overexpression of PPAR␥, as well as NSAIDs and PPAR␥ activators, reduced BACE1 gene promoter activity. These results suggested that PPAR␥ could be a repressor of BACE1. We then identified a PPAR␥ responsive element (PPRE) in the BACE1 gene promoter. Mutagenesis of the PPRE abolished the binding of PPAR␥ to the PPRE and increased BACE1 gene promoter activity. Furthermore, proinflammatory cytokines decreased PPAR␥ gene transcription, and this effect was supressed by NSAIDs. We also demonstrate that in vivo treatment with PPAR␥ agonists increased PPAR␥ and reduced BACE1 mRNA and intracellular ␤-amyloid levels. Interestingly, brain extracts from AD patients showed decreased PPAR␥ expression and binding to PPRE in the BACE1 gene promoter. Our data strongly support a major role of PPAR␥ in the modulation of amyloid-␤ generation by inflammation and suggest that the protective mechanism of NSAIDs in AD involves activation of PPAR␥ and decreased BACE1 gene transcription. amyloid ͉ inflammation ͉ Alzheimer's disease ͉ transgenic mice

Polymerization of the amyloid beta (Abeta) peptide into protease-resistant fibrils is a significant step in the pathogenesis of Alzheimer&amp;amp;amp;amp;amp;#39;s disease. It has not been possible to obtain detailed structural information about this process with conventional techniques because the peptide has limited solubility and does not form crystals. In this work, we present experimental results leading to a molecular level model for fibril formation. Systematically selected Abeta-fragments containing the Abeta16-20 sequence, previously shown essential for Abeta-Abeta binding, were incubated in a physiological buffer. Electron microscopy revealed that the shortest fibril-forming sequence was Abeta14-23. Substitutions in this decapeptide impaired fibril formation and deletion of the decapeptide from Abeta1-42 inhibited fibril formation completely. All studied peptides that formed fibrils also formed stable dimers and/or tetramers. Molecular modeling of Abeta14-23 oligomers in an antiparallel beta-sheet conformation displayed favorable hydrophobic interactions stabilized by salt bridges between all charged residues. We propose that this decapeptide sequence forms the core of Abeta-fibrils, with the hydrophobic C terminus folding over this core. The identification of this fundamental sequence and the implied molecular model could facilitate the design of potential inhibitors of amyloidogenesis.