Related Neurodegenerative Disorders Research Articles

Identification of novel susceptibility loci for Guam neurodegenerative disease: challenges of genome scans in genetic isolates.

Amyotrophic lateral sclerosis/parkinsonism-dementia complex (ALS/PDC) is a fatal neurodegenerative disease found in the Chamorro people of Guam and other Pacific Island populations. The etiology is unknown, although both genetic and environmental factors appear important. To identify loci for ALS/PDC, we conducted both genome-wide linkage and association analyses, using approximately 400 microsatellite markers, in the largest sample assembled to date, comprising a nearly complete sample of all living and previously sampled deceased cases. A single, large, complex pedigree was ascertained from a village on Guam, with smaller families and a case-control sample ascertained from the rest of Guam by population-based neurological screening and archival review. We found significant evidence for two regions with novel ALS/PDC loci on chromosome 12 and supportive evidence for the involvement of the MAPT region on chromosome 17. D12S1617 on 12p gave the strongest evidence of linkage (maximum LOD score, Z(max) = 4.03) in our initial scan, with additional support in the complete case-control sample in the form of evidence of allelic association at this marker and another nearby marker. D12S79 on 12q also provided significant evidence of linkage (Z(max) = 3.14) with support from flanking markers. Our results suggest that ALS/PDC may be influenced by as many as three loci, while illustrating challenges that are intrinsic in genetic analyses of isolated populations, as well as analytical strategies that are useful in this context. Elucidation of the genetic basis of ALS/PDC should improve our understanding of related neurodegenerative disorders including Alzheimer disease, Parkinson disease, frontotemporal dementia and ALS.

RNA oxidation in Alzheimer disease and related neurodegenerative disorders

RNA oxidation and its biological effects are less well studied compared to DNA oxidation. However, RNA may be more susceptible to oxidative insults than DNA, for RNA is largely single-stranded and its bases are not protected by hydrogen bonding and less protected by specific proteins. Also, cellular RNA locates in the vicinity of mitochondria, the primary source of reactive oxygen species. Oxidative modification can occur not only in protein-coding RNAs, but also in non-coding RNAs that have been recently revealed to contribute towards the complexity of the mammalian brain. Damage to coding and non-coding RNAs will cause errors in proteins and disturbances in the regulation of gene expression. While less lethal than mutations in the genome and not inheritable, such sublethal damage to cells might be associated with underlying mechanisms of degeneration, especially age-associated neurodegeneration that is commonly found in the elderly population. Indeed, oxidative RNA damage has been described recently in most of the common neurodegenerative disorders including Alzheimer disease, Parkinson disease, dementia with Lewy bodies and amyotrophic lateral sclerosis. Of particular interest, the accumulating evidence obtained from studies on either human samples or experimental models coincidentally suggests that oxidative RNA damage is a feature in vulnerable neurons at early-stage of these neurodegenerative disorders, indicating that RNA oxidation actively contributes to the onset or the development of the disorders. Further investigations aimed at understanding of the processing mechanisms related to oxidative RNA damage and its consequences may provide significant insights into the pathogenesis of neurodegenerative disorders and lead to better therapeutic strategies.

Identification of the amino acid sequence motif of α-synuclein responsible for macrophage activation

α-Synuclein (Syn) is implicated in the pathogenesis of PD and related neurodegenerative disorders. Recent studies have also shown that α-synuclein can activate microglia and enhance dopaminergic neurodegeneration. The mechanisms of microglia activation by α-synuclein, however, are not well understood. In this study, we found that not only α-synuclein but also β- and γ-synucleins activated macrophages (RAW 264.7) in vitro. Macrophages treated with synuclein proteins secreted TNF-α in a dose-dependent manner. Synuclein family proteins also increased mRNA transcription of COX-2 and iNOS. Two α-synuclein deletion mutants, SynΔNAC and Syn61–140, activated macrophages, while deletion mutants Syn1–60 and Syn96–140 did not significantly activate them. Finally, we demonstrated that macrophage activation by α-synuclein was accompanied by phosphorylation of ERK. These results suggest that synuclein family proteins can activate macrophages, and that macrophage activation needs both the N-terminal and C-terminal domains of α-synuclein, but not the central NAC region.

Modeling Tauopathy in the Fruit Fly Drosophila melanogaster

The fruit fly Drosophila melanogaster has emerged as a powerful system in which to model human disease. This review focuses on the utility of the fly to model tau-dependent neurodegeneration, a hallmark of Alzheimer's disease and related neurodegenerative disorders. I provide a detailed description of fly tauopathy models and summarize a number of studies that demonstrate their ability to recapitulate both primary features of tauopathy, including tau-induced neurodegeneration and phosphorylation, and secondary features, including oxidative stress, cell-cycle activation and changes in the actin cytoskeleton. Important genetic and biochemical insights are discussed, and future directions proposed.

Altered functional properties of a TRPM2 variant in Guamanian ALS and PD

Two related neurodegenerative disorders, Western Pacific amyotrophic lateral sclerosis (ALS) and parkinsonism-dementia (PD), originally occurred at a high incidence on Guam, in the Kii peninsula of Japan, and in southern West New Guinea more than 50 years ago. These three foci shared a unique mineral environment characterized by the presence of severely low levels of Ca(2+) and Mg(2+), coupled with high levels of bioavailable transition metals in the soil and drinking water. Epidemiological studies suggest that genetic factors also contribute to the etiology of these disorders. Here, we report that a variant of the transient receptor potential melastatin 2 (TRPM2) gene may confer susceptibility to these diseases. TRPM2 encodes a calcium-permeable cation channel highly expressed in the brain that has been implicated in mediating cell death induced by oxidants. We found a heterozygous variant of TRPM2 in a subset of Guamanian ALS (ALS-G) and PD (PD-G) cases. This variant, TRPM2(P1018L), produces a missense change in the channel protein whereby proline 1018 (Pro(1018)) is replaced by leucine (Leu(1018)). Functional studies revealed that, unlike WT TRPM2, P1018L channels inactivate. Our results suggest that the ability of TRPM2 to maintain sustained ion influx is a physiologically important function and that its disruption may, under certain conditions, contribute to disease states.

Stratification of Patients is the Way to Go to Develop Neuroprotective/Disease-Modifying Drugs for Alzheimer's Disease

Development of effective neuroprotective drugs for Alzheimer's disease (AD) is a formidable challenge because this disease is multifactorial and heterogeneous. Although AD is characterized histopathologically by the presence of numerous amyloid-beta plaques and neurofibrillary degeneration of abnormally hyperphosphorylated tau in the brain, these two hallmark lesions do not exist in any fixed proportion in this disease. Furthermore, in the brains of some normal aged individuals, there are as many amyloid-beta plaques seen as in typical cases of AD. On the other hand, extensive neurofibrillary degeneration of abnormally hyperphosphorylated tau and dementia but in the absence of amyloid-beta plaques occur in several related neurodegenerative disorders called tauopathies. More than one molecular mechanism has been described for the development of amyloid-beta as well as neurofibrillary degeneration of abnormally hyperphosphorylated tau. Thus, AD apparently results from several different etiopathogenic mechanisms and offers numerous rational therapeutic targets. We have discovered that there are at least five different subgroups of AD, and future studies are likely to identify additional subgroups. The employment of these subgroups of AD in clinical trials can markedly increase the success in developing specific and potent therapeutic drugs.

Increased Dosage of Dyrk1A Alters Alternative Splicing Factor (ASF)-regulated Alternative Splicing of Tau in Down Syndrome

Two groups of tau, 3R- and 4R-tau, are generated by alternative splicing of tau exon 10. Normal adult human brain expresses equal levels of them. Disruption of the physiological balance is a common feature of several tauopathies. Very early in their life, individuals with Down syndrome (DS) develop Alzheimer-type tau pathology, the molecular basis for which is not fully understood. Here, we demonstrate that Dyrk1A, a kinase encoded by a gene in the DS critical region, phosphorylates alternative splicing factor (ASF) at Ser-227, Ser-234, and Ser-238, driving it into nuclear speckles and preventing it from facilitating tau exon 10 inclusion. The increased dosage of Dyrk1A in DS brain due to trisomy of chromosome 21 correlates to an increase in 3R-tau level, which on abnormal hyperphosphorylation and aggregation of tau results in neurofibrillary degeneration. Imbalance of 3R- and 4R-tau in DS brain by Dyrk1A-induced dysregulation of alternative splicing factor-mediated alternative splicing of tau exon 10 represents a novel mechanism of neurofibrillary degeneration and may help explain early onset tauopathy in individuals with DS.

Scattering differentiates Alzheimer disease in vitro

The molecular bases of Alzheimer disease and related neurodegenerative disorders are becoming better understood, but the means for definitive diagnosis and monitoring in vivo remain lacking. Near-infrared optical spectroscopy offers a potential solution. We acquired transmission and reflectance spectra of thin brain tissue slabs, from which we calculated wavelength-dependent absorption and reduced scattering coefficients from 470-1000 nm. The reduced scattering coefficients in the near infrared clearly differentiated Alzheimer from control specimens. Diffuse reflectance spectra of gross brain tissue in vitro confirmed this observation. These results suggest a means for diagnosing and monitoring Alzheimer disease in vivo, using near-infrared optical spectroscopy.

Beyond Parkinson Disease: Amyotrophic Lateral Sclerosis and the Axon Guidance Pathway

BackgroundWe recently described a genomic pathway approach to study complex diseases. We demonstrated that models constructed using single nucleotide polymorphisms (SNPs) within axon guidance pathway genes were highly predictive of Parkinson disease (PD) susceptibility, survival free of PD, and age at onset of PD within two independent whole-genome association datasets. We also demonstrated that several axon guidance pathway genes represented by SNPs within our final models were differentially expressed in PD.Methodology/Principal FindingsHere we employed our genomic pathway approach to analyze data from a whole-genome association dataset of amyotrophic lateral sclerosis (ALS); and demonstrated that models constructed using SNPs within axon guidance pathway genes were highly predictive of ALS susceptibility (odds ratio = 1739.73, p = 2.92×10−60), survival free of ALS (hazards ratio = 149.80, p = 1.25×10−74), and age at onset of ALS (R2 = 0.86, p = 5.96×10−66). We also extended our analyses of a whole-genome association dataset of PD, which shared 320,202 genomic SNPs in common with the whole-genome association dataset of ALS. We compared for ALS and PD the genes represented by SNPs in the final models for susceptibility, survival free of disease, and age at onset of disease and noted that 52.2%, 37.8%, and 34.9% of the genes were shared respectively.Conclusions/SignificanceOur findings for the axon guidance pathway and ALS have prior biological plausibility, overlap partially with PD, and may provide important insight into the causes of these and related neurodegenerative disorders.

Proteomic identification of novel proteins associated with Lewy bodies

The manifestation of Lewy bodies (LB) in the brain is a hallmark of Parkinson's disease. Here, we present a comprehensive analysis of protein elements in Lewy bodies by comparative mass spectrometry. Cortical LB inclusions were enriched by sucrose gradient centrifugation from postmortem brains, and a negative control sample was prepared from specimen without LB pathology. Whereas approximately 550 proteins were identified in the LB-enriched sample by mass spectrometry, quantitative comparison with the control sample revealed that approximately 40 proteins were co-enriched with alpha-synuclein, the major component in Lewy bodies. As expected, the list of proteins included previously reported constituents, such as those involved in protein folding, membrane trafficking and oxidative stress. More interestingly, we discovered in the LB-enriched sample several kinases (MAPKK1/MEK1, protein kinase C, and doublecortin-like kinase), a novel deubiquitinating enzyme (otubain 1), and numerous ubiquitin ligases (KPC and SCF). The proteomic studies provide enzyme candidates to investigate the regulation of alpha-synuclein and/or other LB proteins, which may contribute to the formation of Lewy bodies and the toxicity of alpha-synuclein in the related neurodegenerative disorders.

A novel p38α MAPK inhibitor suppresses brain proinflammatory cytokine up-regulation and attenuates synaptic dysfunction and behavioral deficits in an Alzheimer's disease mouse model

BackgroundAn accumulating body of evidence is consistent with the hypothesis that excessive or prolonged increases in proinflammatory cytokine production by activated glia is a contributor to the progression of pathophysiology that is causally linked to synaptic dysfunction and hippocampal behavior deficits in neurodegenerative diseases such as Alzheimer's disease (AD). This raises the opportunity for the development of new classes of potentially disease-modifying therapeutics. A logical candidate CNS target is p38α MAPK, a well-established drug discovery molecular target for altering proinflammatory cytokine cascades in peripheral tissue disorders. Activated p38 MAPK is seen in human AD brain tissue and in AD-relevant animal models, and cell culture studies strongly implicate p38 MAPK in the increased production of proinflammatory cytokines by glia activated with human amyloid-beta (Aβ) and other disease-relevant stressors. However, the vast majority of small molecule drugs do not have sufficient penetrance of the blood-brain barrier to allow their use as in vivo research tools or as therapeutics for neurodegenerative disorders. The goal of this study was to test the hypothesis that brain p38α MAPK is a potential in vivo target for orally bioavailable, small molecules capable of suppressing excessive cytokine production by activated glia back towards homeostasis, allowing an improvement in neurologic outcomes.MethodsA novel synthetic small molecule based on a molecular scaffold used previously was designed, synthesized, and subjected to analyses to demonstrate its potential in vivo bioavailability, metabolic stability, safety and brain uptake. Testing for in vivo efficacy used an AD-relevant mouse model.ResultsA novel, CNS-penetrant, non-toxic, orally bioavailable, small molecule inhibitor of p38α MAPK (MW01-2-069A-SRM) was developed. Oral administration of the compound at a low dose (2.5 mg/kg) resulted in attenuation of excessive proinflammatory cytokine production in the hippocampus back towards normal in the animal model. Animals with attenuated cytokine production had reductions in synaptic dysfunction and hippocampus-dependent behavioral deficits.ConclusionThe p38α MAPK pathway is quantitatively important in the Aβ-induced production of proinflammatory cytokines in hippocampus, and brain p38α MAPK is a viable molecular target for future development of potential disease-modifying therapeutics in AD and related neurodegenerative disorders.

Role of Ubiquitin Protein Ligases in the Pathogenesis of Polyglutamine Diseases

The accumulation of intracellular protein deposits as inclusion bodies is the common pathological hallmark of most age related neurodegenerative disorders including polyglutamine diseases. Appearances of aggregates of the misfolded mutant disease proteins suggest that the cells are unable to efficiently degrade them, and failure of clearance leads to the severe disturbances of the cellular quality control system. The quality control ubiquitin ligases are now increasingly implicated in the biology of polyglutamine diseases, Parkinson's diseases, Amyotrophic lateral sclerosis and Alzheimer's disease. Here we review the recent studies that have revealed a critical role of E3 ubiquitin ligases in understanding the pathogenesis of polyglutamine diseases.

PS2 protein expression is upregulated by sex steroids in the cerebral cortex of aging mice

Mutations in presenilin (PS) genes cause majority of early onset Alzheimer's disease (AD), an age related neurodegenerative disorder. PS proteins undergo proteolytic cleavage to produce biologically active fragments, which constitute the catalytic core of the γ-secretase enzyme. This enzyme cleaves β-amyloid precursor protein (βAPP) to generate Aβ peptides, which are influenced by sex steroids. Recently we have reported the downregulation of PS1 expression by sex steroids in the brain of adult mice. Here we have examined the effect of gonadectomy and subsequent administration of gonadal hormones 17β-estradiol and testosterone on the level of PS2 C-terminal fragment (CTF) in the cerebral cortex of adult and old AKR strain mice of both sexes. PS2 expression was downregulated following gonadectomy, but upregulated by supplementation of gonadal steroids in both age groups and sexes. Thus these results demonstrate up-regulation of PS2 protein expression by sex steroids, which in turn may influence PS2 associated brain functions.

Regaining the Lost Past

People with Alzheimer’s disease or related neurodegenerative disorders may progress from absentmindedness and difficulty learning new things to a heartbreaking state in which they forget their own past and no longer recognize the faces of people they love. Expression of p25 (a truncated form of the p35 regulatory subunit of cyclin-dependent kinase 5) is associated with various neurodegenerative disorders, and the CK-p25 Tg mouse, in which p25 expression--and thereby neuronal loss--can be conditionally regulated, is a model for these disorders. Fischer et al . found that exposing CK-p25 Tg mice to an enriched environment (after neuronal loss had occurred and learning was impaired) improved their associative and spatial learning. Moreover, environmental enrichment led to the recovery of long-term memories that had apparently been lost. Experiments in wild-type mice revealed that exposure to an enriched environment stimulated histone acetylation in the hippocampus and cortex. The histone deacetylase (HDAC) inhibitor sodium butyrate (SB) improved associative and spatial learning in wild-type and CK-p25 Tg mice (after p25 induction) and, like environmental enrichment, enhanced dendritic and synaptic growth in CK-p25 Tg mice without affecting brain atrophy or neuronal loss. Furthermore, SB enabled the recovery of lost memories. The authors conclude that their data raise the possibility that HDAC inhibitors may provide a means to enable people with neurodegenerative disorders associated with memory loss to recover lost memories. Sweatt provides thoughtful commentary, noting (as do the authors) that HDAC inhibitors may affect the acetylation status of proteins other than histones. A. Fischer, F. Sananbenesi, X. Wang, M. Dobbin, L. H. Tsai, Recovery of learning and memory is associated with chromatin remodelling. Nature 447 , 178-182 (2007). [PubMed] J. D. Sweatt, Down memory lane. Nature 447 , 151-152 (2007). [PubMed]

S/P and T/P phosphorylation is critical for tau neurotoxicity in Drosophila

The microtubule-associated protein tau is hyperphosphorylated abnormally in AD and related neurodegenerative disorders. Many phospho epitopes created by proline directed kinases (SP/TP sites) show relative specificity for disease states. To test whether phosphorylation at the disease-associated SP/TP sites affects tau toxicity in vivo, we expressed a form of tau in Drosophila in which all SP/TP sites are mutated to alanine. We find that blocking phosphorylation at SP/TP motifs markedly reduces tau toxicity in vivo. Using phosphorylation-specific antibodies, we identify a positive correlation between increased phosphorylation at disease-associated sites and neurotoxicity. We use the phosphorylation-incompetent version of tau to show that kinase and phosphatase modifiers of tau neurotoxicity, including cdk5/p35, the JNK kinase hemipterous and PP2A act via SP/TP phosphorylation sites. We provide direct evidence in an animal model system to support the role of phosphorylation at SP/TP sites in playing a critical role in tau neurotoxicity.

Dibenzothiazoles as novel amyloid-imaging agents

Novel dibenzothiazole derivatives were synthesized and evaluated as amyloid-imaging agents. In vitro quantitative binding studies using AD brain tissue homogenates showed that the dibenzothiazole derivatives displayed high binding affinities with K i values in the nanomolar range (6.8–36 nM). These derivatives are relatively lipophilic with partition coefficients (logP oct) in the range of 1.25–3.05. Preliminary structure–activity relationship studies indicated dibenzothiazole derivatives bearing electron-donating groups exhibited higher binding affinities than those bearing electron-withdrawing groups. A lead compound was selected for its high binding affinity and radiolabeled with [ 125I] through direct radioiodination using sodium [ 125I] iodide in the presence of Chloramine T. The radioligand (4-[2,6′]dibenzothiazolyl-2′-yl-2-[ 125I]-phenylamine) displayed moderate lipophilicity (logP oct, 2.70), very good brain uptake (3.71 ± 0.63% ID/g at 2 min after iv injection in mice), and rapid washout from normal brains (0.78% and 0.43% ID/g at 30 and 60 min, respectively). These studies indicated that lipophilic dibenzothiazole derivatives represent a promising pharmacophore for the development of novel amyloid-imaging agents for potential application in Alzheimer’s disease and related neurodegenerative disorders.

LRRK2 Expression in Normal and Pathologic Human Brain and in Human Cell Lines

Mutations in the leucine-rich repeat kinase 2 gene (LRRK2) have been recently identified in families with autosomal-dominant late-onset Parkinson disease. We report that by reverse transcriptase-polymerase chain reaction, the mRNA of LRRK2 is expressed in soluble extracts of human brain, liver, and heart and in cultured human astrocytes, microglia, and oligodendroglia as well as in human neuroblastoma cell lines. We find by Western blotting using a polyclonal antibody of the leucine-rich repeat kinase 2 protein (Lrrk2) specific for C-terminal residues 2511-2527 that an apparent full-length protein and several of its fractions are expressed in soluble extracts of normal human brain. By immunocytochemistry, the antibody recognizes neurons, and more weakly astrocytes and microglia, in normal brain tissue. It intensely labels Lewy bodies in Parkinson disease and related neurodegenerative disorders. It also labels a subset of neurofibrillary tangles in Alzheimer disease and the Parkinsonism dementia complex of Guam (PDCG). It labels thorn-shaped astrocytes and oligodendroglial coiled bodies in PDCG; oligodendroglial inclusions in multiple system atrophy; Pick bodies in Pick disease; nuclear and cytoplasmic inclusions in Huntington disease; and intraneuronal and glial inclusions in amyotrophic lateral sclerosis. In summary, LRRK2 is constitutively expressed in neurons and also in glial cells of human brain. It strongly associates with pathological inclusions in several neurodegenerative disorders.

Targeting protein aggregation in neurodegeneration – lessons from polyglutamine disorders

Polyglutamine diseases, such as Huntington’s disease, are among the most common inherited neurodegenerative disorders. They share salient clinical and pathological features with major sporadic neurodegenerative diseases, such as Alzheimer’s disease, Parkinson’s disease and amyotropic lateral sclerosis. Over the last decade, protein aggregation has emerged as a common pathological hallmark in neurodegenerative diseases and has, therefore, attracted considerable attention as a likely shared therapeutic target. Because of their clearly defined molecular genetic basis, polyglutamine diseases have allowed researchers to dissect the relationship between neurodegeneration and protein aggregation. In this review, the authors discuss recent progress in understanding polyglutamine-mediated neurotoxicity, and discuss the most promising therapeutic strategies being developed in the polyglutamine diseases and related neurodegenerative disorders.

O2-05-07: Minocycline reduces tau phosphorylation and protects against Aβ-induced neurotoxicity

Minocycline is a semi–synthetic, second–generation tetracycline derivative. Recent research has indicated that in addition to its antimicrobial properties, minocycline is also a potent anti–inflammatory, anti–apoptotic and neuroprotective agent when tested in models of neurodegenerative disease. To determine if minocycline may have therapeutic value for the treatment of Alzheimer's disease by preventing tau hyperphosphorylation and amyloid–induced neurotoxicity. We have investigated the effect of minocycline, both in primary neuronal cultures, and in NFT–forming tau transgenic mice (htau line). Treatment of cultures with fibrillar and oligomeric Aβ42 over a period of 48 hours resulted in progressive neuronal death, which was prevented by pre–treatment of cultures with minocycline. Application of minocycline to control cultures also resulted in increased cell survival, demonstrating the neuroprotective properties of this agent. There is increasing evidence to suggest that kinase activation resulting in tau phosphorylation is an important factor in the demise of neurons that results from exposure to Aβ. To investigate this mechanism we have examined the effects of minocycline treatment on pathological tau phosphorylation in response to Aβ in culture, and also during tangle development in htau mice. We find that minocycline treatment results in significantly reduced tau phosphorylation at several epitopes of relevance to AD, including the CP13 and PHF1 epitopes at S202 and S396/404 respectively. The reduced tau phosphorylation we have observed correlates well with decreased cell death following treatment. Furthermore, after minocycline treatment we find reduced activity of specific tau protein kinases, which may account for the decreased tau phosphorylation we have observed. In addition to modulating kinase activities, we find reduced levels of GFAP after minocycline treatment indicating that minocycline can induce suppression of glial responses. These results suggest a dual neuroprotective effect of minocycline with inhibition of kinase activities preventing pathological tau phosphorylation and suppression of glial responses preventing neuroinflammation. At the doses used minocycline is known to be both safe and well tolerated. These results suggest that minocycline may be an effective therapeutic agent for the treatment of Alzheimer's disease and related neurodegenerative disorders. This work was supported by a fellowship grant from the Alzheimer's Society.

P1-361: Mapping genes affecting age-at-onset of Alzheimer’s disease in the chromosome 8

Identification of genes that affect age–at–onset (AAO) of AD has a profound impact in understanding the genetics of AD, and will potentially improve prevention and treatment in this aging–related neurodegenerative disorder. We previously conducted a whole genome screen for AAO of AD using 449 families and identified several linkage regions including chromosome 8q. The linkage region of chromosome 8q covers 46cM interval (between 119 to 165cM; LOD>1.0) with a peak multipoint LOD score of 2.09. To further map the gene(s) that is responsible for the LOD peak in this region of the chromosome 8, using variance–component procedure in SOLAR with increased sample size, and using Illumina Goldengate genotyping method. We genotyped four micro–satellite markers under this linkage region using additional 158 families with the same linkage analysis as in our previous study. We then followed up a 1–LOD score down region from the linkage peak using high density SNPs by genotyping 1536 Illumina Goldengate SNPs for association study to fine map the region. We obtained an updated two–point LOD score of 2.34 at the marker D8S1179. In addition, the new analysis narrowed the peak region to about 20cM (between 128 to149cM; LOD>1.0). We are currently genotyping 1536 Illumina Goldengate SNP OPA to follow up the ∼24mb area between q23.3 to q24.23 in Chr8 under the new linkage peak. These data strengthen the evidence for a potential gene(s) in this region contributing to the genetic etiology of AAO in AD.