PMCCPMCCPMCC

Search tips
Search criteria 

Advanced

 
Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
J Alzheimers Dis. Author manuscript; available in PMC 2011 February 1.
Published in final edited form as:
PMCID: PMC3031860
NIHMSID: NIHMS266068

Regional selectivity of rab5 and rab7 protein up regulation in mild cognitive impairment and Alzheimer's disease

Abstract

Endocytic alterations are one of the earliest changes to occur in Alzheimer's disease (AD), and are hypothesized to be involved in the selective vulnerability of specific neuronal populations during the progression of AD. Previous microarray and real-time quantitative PCR (qPCR) experiments revealed an up regulation of the early endosomal effector rab5 and the late endosome constituent rab7 in the hippocampus of peopole with mild cognitive impairment (MCI) and AD. To assess whether these select rab GTPase gene expression changes are reflected in protein levels within selectively vulnerable brain regions (basal forebrain, frontal cortex, and hippocampus) and relatively spared areas (cerebellum and striatum), we performed immunoblot analysis using antibodies directed against rab5 and rab7 on postmortem human brain tissue harvested from cases with a premortem clinical diagnosis of no cognitive impairment (NCI), MCI and AD. Results indicate selective up regulation of both rab5 and rab7 levels within basal forebrain, frontal cortex, and hippocampus in MCI and AD, which also correlated with Braak staging. In contrast, no differences in protein levels were found in the less vulnerable cerebellum and striatum. These regional immunoblot assays are consistent with single cell gene expression data, and provide protein-based evidence for endosomal markers contributing to the vulnerability of cell types within selective brain regions during the progression of AD.

Keywords: basal forebrain, cerebellum, endosome, hippocampus, frontal cortex, mild cognitive impairment, rab GTPase, selective vulnerability, striatum

Introduction

In neurons, the endosomal-lysosomal pathway performs a multiplicity of integral functions including internalizing nutrients and neurotrophic factors, degrading and recycling receptors, and integrating signaling information to relevant intracellular pathways [1-3]. Endocytosis enables neurons to modify or degrade molecules from the cell surface into intracellular compartments. A family of small ras-related GTPase (rab) proteins highly regulate trafficking of vesicles from early to late endosomes and other organelles along endosomal-lysosomal pathways [4-8]. Endosomes also play a critical role in neuronal development and homeostasis and normal synaptic transmission [9-12], as well as neuronal dysfunction during the progression of Alzheimer's disease (AD) [13-15]. In addition, signaling endosomes contain rab GTPases and neurotrophin receptor signaling complexes, which are responsible for growth factor signal transduction from synaptic sites to the nucleus [10, 16, 17]. Specific rab GTPases, including the early endosome effector rab5 and late endosome constituent rab7, have been implicated in the regulation of nerve growth factor (NGF) signaling in vitro [16-19], and we have demonstrated that up regulation of rab5 down regulates the brain-derived neurotrophic receptor (BDNF) receptor TrkB [20].

Endosomal-lysosomal system dysfunction is one of the earliest disturbances observed in AD [2, 15, 21], and may be one of the fundamental mechanisms underlying neurodegenerative changes during the progression of AD. Increases in rab5, an effector molecule that promotes early endosome fusion, a positive mediator of endocytosis, regulates early endosomal enlargement [6, 17]. Enlargement of rab5-positive endosomes is a pathological feature that precedes cerebral and vascular amyloid-beta peptide (Aβ) deposition, neurofibrillary tangle (NFT) formation, and is selective for AD [21-23]. Many vulnerable cell types within the forebrain demonstrate enlarged endosomes and increased rab5 immunoreactivity in human AD as well as in animal models of AD that display endosomal disturbances [21, 22, 24]. rab5 overexpression affects several vulnerable cellular phenotypes including cholinergic basal forebrain neurons, hippocampal pyramidal neurons, and neocortical pyramidal neurons [2, 21, 24-26]. Up regulation of rab5 also reproduces key aspects of the early endosomal phenotype found in AD, and may have downstream effects in other compartments including late endosomes [27, 28].

Microarray analysis has demonstrated significant up regulation of select rab GTPases within vulnerable CA1 hippocampal pyramidal neurons harvested from people who died with a clinical diagnosis of mild cognitive impairment (MCI) and AD, including rab5 and the late endosome constituent rab7 [20]. Notably, up regulation of rab5 and rab7 in CA1 neurons also correlates with cognitive decline in the same cohort used for microarray analysis [20]. Regional real-time quantitative PCR (qPCR) analysis and immunoblot analysis demonstrated up regulation of both rab5 and rab7 in the hippocampus [20], further indicating that early and late endosome dysfunction occurs in one of the most pathologically vulnerable forebrain regions affected in MCI and AD [29, 30].

Up regulation of rab5 and rab7 expression within selectively vulnerable hippocampal neurons occurs during the progression of AD [20], suggesting that dysregulation of a select rab GTPase phenotype is a molecular pathogenic marker for neuronal dysfunction in other highly vulnerable regions of the brain early in the disease process. Therefore, we hypothesize that regions with neuronal cell types vulnerable to AD neurodegeneration will display select rab GTPase up regulation, whereas relatively spared regions will show little or no rab5 and/or rab7 dysregulation. To this end, a survey of several vulnerable regions (including basal forebrain, frontal cortex, and hippocampus) and relatively spared regions (including cerebellum and striatum) was performed via immunoblot analysis for rab5 and rab7 to assess whether differential expression of these two rab GTPases is a selective event in vulnerable regions in the MCI and/or AD brain, or conversely, that up regulation of these discrete endosomal markers is a global event during AD progression.

Materials and Methods

Brain tissue collection

This study was performed under the auspices of IRB guidelines administrated by the Nathan Kline Institute/New York University Langone Medical Center. Immunoblot analysis using antibodies directed against rab5 and rab7 was performed using brain samples obtained from a total of 82 postmortem human subjects. Cases were clinically categorized premortem with no cognitive impairment (NCI; n = 27), MCI insufficient to meet criteria for dementia (n = 17), and AD (n = 27). The MCI population was defined as persons with impaired cognitive testing who were not found to have frank dementia by a neurologist [31, 32], commensurate with the current consensus criteria for the clinical classification of MCI [33, 34]. Only cases with age at death > 65 years and postmortem interval (PMI) ≤ 36 hours were included in the study. Frozen brain tissues were obtained from the Rush Religious Orders Study (n = 45; http://www.rush.edu/rumc/page-R12394.html), the University of Pennsylvania Brain Bank (n = 24; Center for Neurodegenerative Disease Research; http://www.pennadc.org/), the Harvard Brain Bank (n = 10; Harvard Brain Tissue Resource Center; http://www.brainbank.mclean.org/) and the Emory Brain Bank (n = 3; Center for Neurodegenerative Disease; http://neurology.emory.edu/ENNCF/neuropathology/resources.php). Samples from each case were collected from the substantia innominata of the basal forebrain, cerebellum, frontal cortex {Brodmann area (BA) BA9 and BA10}, hippocampus, and striatum. However, tissue was not always available for each case (see Table I). Demographic information of the 82 cases is presented in Table II. Antemortem cognitive assessments collected within one year prior to death using the Mini Mental State Exam (MMSE) were available for 44 participants of the Religious Orders Study [35, 36], 15 people from the University of Pennsylvania Brain Bank, and 1 case from the Emory Brain Bank. Exclusion criteria included argyrophilic grain disease, frontotemporal dementia, Lewy body disease, mixed dementias, Parkinson's disease, and stroke. A board certified neuropathologist blinded to the clinical diagnosis performed a neuropathological diagnosis. Neuropathological designations were based on established criteria [37-39]. Tissue handling practices across brains banks are similar, and immunoblotting findings using frozen brain specimens from these repositories have been reported previously [20, 40]. Brain samples were stored at −80 °C until processed for immunoblot analysis.

Table I
Summary of tissue samples obtained from each source
Table II
Clinical and neuropathologic demographics.

Immunoblot analysis

Frozen regional brain samples were homogenized in a 20 mM Tris-HCl (pH 7.4) buffer containing 10% (w/v) sucrose, 1 mM ethylenediaminetetraacetic acid (EDTA), 5 mM ethylene glycol-bis (ß-aminoethylether)-N,N,N′,N′-tetra-acetic acid (EGTA), 2 mg/ml of the following: (aprotinin, leupeptin, and chymostatin), 1 mg/ml of the following: {pepstatin A, antipain, benzamidine, and phenylmethylsulfonyl fluoride (PMSF)}, 100 μg/ml of the following: {soybean trypsin inhibitor, Nα-p-tosyl-L-lysine chloromethyl ketone (TLCK), and N-tosyl-L-phenylalanine chloromethyl ketone (TPCK)}, 1 mM of the following: (sodium fluoride and sodium orthovanadate) and centrifuged as described previously [40-42]. All protease inhibitors were purchased from Sigma (St. Louis, MO). Identical amounts of homogenates (10 μg) were loaded into a gel electrophoresis apparatus, subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE; 4-15% gradient acrylamide gels; Bio-Rad, Hercules, CA), and transferred to nitrocellulose by electroblotting (Mini Transblot, Bio-Rad). Nitrocellulose membranes were blocked in blocking buffer (LiCor, Lincoln, NE) for 1 hour at 4 °C prior to being incubated with antibodies directed against rab5 (rab5A; rabbit polyclonal sc-309; Santa Cruz Biotechnology, Santa Cruz, CA; 1:1,000 dilution), rab7 (rabbit polyclonal sc-10767; Santa Cruz Biotechnology 1:1,000 dilution), or ß-tubulin (TUBB; monoclonal antibody T5293; Sigma, 1:1,000 dilution) in blocking buffer overnight at 4 °C. Specificity for these well-characterized antibodies has been demonstrated previously by the manufacturer {rab5 (http://datasheets.scbt.com/sc-309.pdf), rab7 (http://datasheets.scbt.com/sc-10767.pdf), TUBB (http://www.sigmaaldrich.com/etc/medialib/docs/Sigma/Datasheet/t5293dat.Par.0001.File.tmp/t5293dat.pdf)} and within individual research reports [43-46]. Moreover, rab5 [21, 28, 43], rab7 [28, 47], and TUBB [20, 40, 48] antibodies have been used previously by our group and independent laboratories for human brain analyses. Membranes were developed using affinity–purified secondary antibodies conjugated to IRDye 800 (Rockland, Gilbertsville, PA) and visualized using an infrared detection system (Odyssey, LiCor) [41]. Individual samples were assayed 2-4 times per antibody.

Data analysis

Immunoblots were quantified by densitometric software supplied with the instrument. Signal intensity of immunoreactive bands was normalized to TUBB immunoreactivity for each assay as described previously [40-42]. TUBB expression has been well established as a suitable protein for normalization in AD by numerous independent research groups. However, this may not be absolute, as microtubules have been shown to decrease in AD and aging [49]. Alterations in immunoreactive band intensity as well as demographic and clinical/neuropathological variables (age, gender, educational level, MMSE, and Braak scores) were compared across clinical diagnostic groups using Kruskal-Wallis test or Fisher's exact test with Bonferroni correction for pairwise comparisons [48, 50, 51], as appropriate. The association between rab5 and rab7 levels and Braak scores were assessed by Spearman rank correlation, as were the correlation of rab5 or rab7 levels between and within brain regions [40, 52]. The level of statistical significance was set at (p < 0.05).

Results

rab5 immunoblot analysis

Quantitative immunoblotting was performed using homogenates prepared from brain tissue obtained from the basal forebrain, cerebellum, frontal cortex, hippocampus, and striatum from NCI, MCI, and AD cases using a well-characterized rab5 antibody. Immunoblotting with the rab5 antibody identified an ~27 kilodalton (kDa) band, consistent with our previous evaluation of this antibody [20]. Quantitative analysis of rab5 protein levels (normalized to TUBB) demonstrated regionally selective differential regulation. Specifically, rab5 was significantly up regulated in the frontal cortex (p < 0.0001) and hippocampus (p < 0.0001) in MCI and AD (Fig. 1), indicating a prodromal increase of this early endosome effector during the progression of dementia (Table III). rab5 protein levels were up regulated in MCI and further up regulated in the AD basal forebrain (p < 0.003); only the comparison between NCI and AD reached statistical significance, in part due to the relatively small number of basal forebrain MCI samples. In contrast, no differential regulation of rab5 levels was observed in either the cerebellum or striatum (Table III). In addition, increased levels of rab5 in the basal forebrain, frontal cortex, and hippocampus correlated with Braak staging (r = 0.56; p < 0.001), suggesting an association between rab5 levels and the development of NFT pathology as well as cognitive decline during the progression of AD.

Figure 1
Representative immunoblots illustrating rab5 (left panel) and rab7 (right panel) protein levels in tissue homogenates derived from the cerebellum, frontal cortex, and hippocampus during the progression of AD. Up regulation of rab5 and rab7 was observed ...
Table III
rab5 levels (normalized to TUBB; mean ± standard deviation) in the five studied regions, by clinical diagnostic group.

rab7 immunoblot analysis

Immunoblot analysis of regional tissue homogenates using a well-characterized rab7 antibody identified an ~25 kDa band that demonstrated regionally selective differential regulation similar to that seen for rab5, though slightly less in magnitude. Specifically, significant up regulation of rab7 was observed in the frontal cortex (p < 0.0001) and hippocampus (p < 0.003) in MCI and AD (Fig. 1), demonstrating increased expression of this late endosome constituent during the progression of dementia (Table IV). As with the rab5 immunoblot analysis, rab7 expression was also elevated in MCI and further up regulated in the AD basal forebrain, although this comparison did not reach statistical significance. rab7 protein levels were stable within the cerebellum and striatum across all three groups examined. Up regulation of rab7 protein levels also correlated with Braak stage in the frontal cortex (r = 0.55; p < 0.001). Unlike rab5 protein levels, only a weak correlation was found between rab7 protein levels and Braak stage in the basal forebrain and hippocampus.

Table IV
rab5 levels (normalized to TUBB; mean ± standard deviation) in the five studied regions, by clinical diagnostic group.

Coordinate protein level analysis within and between the five brain regions

Within each of the five brain regions, levels of rab5 and rab7 were most strongly correlated in the frontal cortex and hippocampus (r = 0.59, r = 0.68; p < 0.001). On the other hand, correlation analysis between regions showed that, despite the small sample sizes, levels of rab5 in hippocampus displayed a strong correlation with levels in the basal forebrain (n = 6 with measures of rab5 available in both regions, r = 0.94; p < 0.05) and frontal cortex (n = 11, r = 0.90; p < 0.0002). We also found a corelation between rab5 levels in basal forebrain and frontal cortex (n = 15, r = 0.62; p < 0.01) as well as in cerebellum and striatum (n = 13, r = 0.66: p < 0.01). These correlative findings are consistent with the observed select up regulation of rab5 in the highly vulnerable basal forebrain, frontal cortex, and hippocampus and lack of rab5 regulation in the spared cerebellum and striatum in the early stages of dementia. In contrast, no significant coordinate expression of rab7 levels were observed between regions.

Discussion

Molecular and cellular evidence exists for endosomal abnormalities in AD, relevant animal models of neurodegeneration, and in vitro [11, 15, 17, 19, 21, 24, 28, 53, 54]. Notably, expression levels of genes regulating early endosomes (including rab5) and late endosomes (including rab7) were selectively up regulated in homogeneous populations of CA1 neurons from individuals with a clinical diagnosis of MCI and AD [20]. Importantly, levels of these genes were selectively increased among individuals with cognitive decline [20]. Based on these expression findings, a regional assessment of protein levels for select rab GTPases was performed using postmortem brains samples from regions that are selectively vulnerable to AD pathology compared to areas relatively spared by the disease. The present results indicate regionally selective up regulation of both rab5 and rab7, which correlate with cognitive decline and Braak NFT staging. Notably, we found that distinct rab GTPase protein levels are increased in regions of the neocortex, limbic system and basal forebrain that show a predilection for the early development of AD pathology [37, 55-57]. These findings underscore the importance of evaluating changes in early and late endosomal pathways during the progression of AD in human postmortem brain tissues as well as within appropriate animal and cellular models.

Early endosomes receive their contents via endocytosis and designate specific cargo for vesicular transport to late endosomes en route to lysosomes, deliver certain cargoes to the Golgi apparatus via the retromer, or recycle elements back to the plasma membrane [10, 58]. From early endosomes, materials are transported to endosome carrier vesicles or multivesicular bodies, which are responsible for transferring cargo from early endosomes to late endosomes [1, 59]. Late endosomes obtain enzymes for degradation, including acid hydrolases such as the cathepsin family of proteases, from the trans Golgi network or through fusion with lysosomes [60, 61]. Intracellular vesicular trafficking between compartments is regulated via specific rab GTPases [6, 8]. Specifically, rab GTPases contribute to vesicle formation, motility, docking, and fusion, and are considered regulatory switches of protein trafficking, transport, and degradation [4]. Distinct rab GTPases are associated with discrete organelles and/or compartments where they are functionally active. For example, rab5 regulates early endosome uptake and fusion, whereas rab7 mediates the fusion of late endosomes. Both rab5 and rab7 were differentially regulated as assessed via microarray, qPCR, and immunoblotting approaches in CA1 neurons [20], and by regional immunoblot analysis herein. Other rab GTPases, including rab3 (synaptic localization), rab4 (early endosome localization), and rab24 (presumed trafficking compartment localization) also display differential regulation in AD [20], and await further regional assessment.

Endosomes are integral mediators of metabolism and cellular communication through trafficking and signaling functions, and are linked to a variety of pathways implicated in the pathogenesis of AD, including amyloidogenic amyloid-ß precursor protein (APP) processing and neurotrophin signaling [10, 11, 28, 54, 62, 63]. Early endosomes are the first major sorting station on the endosomal-lysosomal pathway and the site of internalization and initial processing of proteins relevant to AD pathogenesis including APP, apolipoprotein E (ApoE), low-density lipoprotein, and low-density lipoprotein receptor-related protein, among others [15, 64]. Moreover, early endosomes contain APP secretases and/or secretase activities, demonstrating a relationship between endosomal-lysosomal pathway activity, APP processing, Aβ generation, and β-carboxyl-terminal fragment (βCTF) production [27, 65-67]. The morphological appearance of abnormal endosomes coincides with Aβ accumulation within neuronal endocytic compartments [26, 68]. Overexpression of rab5 in vitro increases Aβ and βCTF production [27, 69]. Recent studies implicate APP and βCTF, and exclude Aβ and αCTF, as the cause of endocytic pathway dysfunction in AD and Down syndrome (DS) [54]. Specifically, endosome defects in DS fibroblasts depend on the overexpression of APP and βCTF production. Knockdown of either APP or the β-secretase BACE1 via RNA interference restored both normal endosome function and morphology. Conversely, overexpressing APP or βCTF in normal human fibroblasts induced endosome pathology [54]. APP binds to a protein complex which includes rab5 [70]. Moreover, rab5 is markedly up regulated in DS fibroblasts, and altering rab5 expression creates a similar endosomal phenotype as manipulating βCTFs [28]. Further characterization of the role(s) of rab5 (and rab7) on APP processing and Aβ regulation, as well as understanding the endosome/neurotrophic signaling axis, is crucial to provide mechanistic data to aid in understanding the pathobiology of selectively vulnerability of basal forebrain, cortical, and hippocampal neurons during the progression of AD.

Our findings of increased rab5 and rab7 expression in MCI and AD support the concept that endocytic pathway abnormalities early in AD onset and during the progression of AD reflect an over activation of the endocytic pathway. The present data describing up regulation of select rab GTPases in selectively vulnerable brain regions to the disease process illustrates the importance of the endosomal pathway in the pathogenesis of AD and lends support to emerging genetic evidence implicating a growing number of genes which influence endocytosis as putative risk factors for AD [71-74]. In summary, up regulation of rab5 and rab7 protein levels was found within the basal forebrain, frontal cortex, and hippocampus across the progression of dementia, whereas no differences in expression levels were found in the less vulnerable cerebellum and striatum, confirming our gene expression findings in CA1 neurons [20], and further arguing that these endosomal markers contribute to the vulnerability of specific regions and cell types during the onset of the AD. The role that rab GTPases play in the development of the major pathological hallmarks of AD (including NFTs and amyloid plaques) continues to be an exciting area of future basic and translational research.

Acknowledgements

Support for this project comes from NIH grants AG17617, AG14449, AG10161, AG09466, and the Alzheimer's Association. We thank Irina Elarova and Shaona Fang for expert technical assistance. We are indebted to the altruism and support of the participants in the Religious Orders Study and of tissue donors to the brain banks at the University of Pennsylvania Center for Neurodegenerative Disease Research, the Harvard Tissue Resource Center, and the Emory Center for Neurodegenerative Disease.

References

1. Bishop NE. Dynamics of endosomal sorting. Int Rev Cytol. 2003;232:1–57. [PubMed]
2. Cataldo AM, Hamilton DJ, Barnett JL, Paskevich PA, Nixon RA. Properties of the endosomal-lysosomal system in the human central nervous system: disturbances mark most neurons in populations at risk to degenerate in Alzheimer's disease. J. Neurosci. 1996;16:186–199. [PubMed]
3. Nixon RA, Cataldo AM. The endosomal-lysosomal system of neurons: new roles. Trends Neurosci. 1995;18:489–496. [PubMed]
4. Ng EL, Tang BL. Rab GTPases and their roles in brain neurons and glia. Brain Res Rev. 2008;58:236–246. [PubMed]
5. Novick P, Brennwald P. Friends and family: the role of the Rab GTPases in vesicular traffic. Cell. 1993;75:597–601. [PubMed]
6. Seachrist JL, Ferguson SS. Regulation of G protein-coupled receptor endocytosis and trafficking by Rab GTPases. Life Sci. 2003;74:225–235. [PubMed]
7. Spang A. Vesicle transport: a close collaboration of Rabs and effectors. Curr Biol. 2004;14:R33–34. [PubMed]
8. Zerial M, Stenmark H. Rab GTPases in vesicular transport. Curr Opin Cell Biol. 1993;5:613–620. [PubMed]
9. Ibanez CF. Message in a bottle: long-range retrograde signaling in the nervous system. Trends Cell Biol. 2007;17:519–528. [PubMed]
10. Bronfman FC, Escudero CA, Weis J, Kruttgen A. Endosomal transport of neurotrophins: roles in signaling and neurodegenerative diseases. Dev Neurobiol. 2007;67:1183–1203. [PubMed]
11. Salehi A, Delcroix JD, Belichenko PV, Zhan K, Wu C, Valletta JS, Takimoto-Kimura R, Kleschevnikov AM, Sambamurti K, Chung PP, Xia W, Villar A, Campbell WA, Kulnane LS, Nixon RA, Lamb BT, Epstein CJ, Stokin GB, Goldstein LS, Mobley WC. Increased App expression in a mouse model of Down's syndrome disrupts NGF transport and causes cholinergic neuron degeneration. Neuron. 2006;51:29–42. [PubMed]
12. Wang B, Yang L, Wang Z, Zheng H. Amyolid precursor protein mediates presynaptic localization and activity of the high-affinity choline transporter. Proc Natl Acad Sci USA. 2007;104:14140–14145. [PubMed]
13. LoGiudice L, Matthews G. Endocytosis at ribbon synapses. Traffic. 2007;8:1123–1128. [PubMed]
14. Nixon RA, Mathews PM, Cataldo AM. The neuronal endosomal-lysosomal system in Alzheimer's disease. J Alzheimers Dis. 2001;3:97–107. [PubMed]
15. Nixon RA. Endosome function and dysfunction in Alzheimer's disease and other neurodegenerative diseases. Neurobiol Aging. 2005;26:373–382. [PubMed]
16. Valdez G, Philippidou P, Rosenbaum J, Akmentin W, Shao Y, Halegoua S. Trk-signaling endosomes are generated by Rac-dependent macroendocytosis. Proc Natl Acad Sci USA. 2007;104:12270–12275. [PubMed]
17. Deinhardt K, Salinas S, Verastegui C, Watson R, Worth D, Hanrahan S, Bucci C, Schiavo G. Rab5 and rab7 control endocytic sorting along the axonal retrograde transport pathway. Neuron. 2006;52:293–305. [PubMed]
18. Liu J, Lamb D, Chou MM, Liu YJ, Li G. Nerve growth factor-mediated neurite outgrowth via regulation of Rab5. Mol Biol Cell. 2007;18:1375–1384. [PMC free article] [PubMed]
19. Saxena S, Bucci C, Weis J, Kruttgen A. The small GTPase Rab7 controls the endosomal trafficking and neuritogenic signaling of the nerve growth factor receptor TrkA. J Neurosci. 2005;25:10930–10940. [PubMed]
20. Ginsberg SD, Alldred MJ, Counts SE, Cataldo AM, Neve RL, Jiang Y, Wuu J, Chao MV, Mufson EJ, Nixon RA, Che S. Microarray analysis of hippocampal CA1 neurons implicates early endosomal dysfunction during Alzheimer's disease progression. Biol Psychiatry. 2010 in press. [PMC free article] [PubMed]
21. Cataldo AM, Peterhoff CM, Troncoso JC, Gomez-Isla T, Hyman BT, Nixon RA. Endocytic pathway abnormalities precede amyloid beta deposition in sporadic Alzheimer's disease and Down syndrome: differential effects of APOE genotype and presenilin mutations. Am J Pathol. 2000;157:277–286. [PubMed]
22. Cataldo A, Rebeck GW, Ghetri B, Hulette C, Lippa C, Van Broeckhoven C, van Duijn C, Cras P, Bogdanovic N, Bird T, Peterhoff C, Nixon R. Endocytic disturbances distinguish among subtypes of Alzheimer's disease and related disorders. Ann Neurol. 2001;50:661–665. [PubMed]
23. Nixon RA, Cataldo AM. Lysosomal system pathways: genes to neurodegeneration in Alzheimer's disease. J Alzheimers Dis. 2006;9:277–289. [PubMed]
24. Cataldo AM, Petanceska S, Peterhoff CM, Terio NB, Epstein CJ, Villar A, Carlson EJ, Staufenbiel M, Nixon RA. App gene dosage modulates endosomal abnormalities of Alzheimer's disease in a segmental trisomy 16 mouse model of down syndrome. J Neurosci. 2003;23:6788–6792. [PubMed]
25. Cataldo AM, Barnett JL, Picroni C, Nixon RA. Increased neuronal endocytosis and protease delivery to early endosomes in sporadic Alzheimer's disease: neuropathologic evidence for a mechanism of increased ß-amyloidogenesis. J. Neurosci. 1997;17:6142–6151. [PubMed]
26. Cataldo AM, Petanceska S, Terio NB, Peterhoff CM, Durham R, Mercken M, Mehta PD, Buxbaum J, Haroutunian V, Nixon RA. Abeta localization in abnormal endosomes: association with earliest Abeta elevations in AD and Down syndrome. Neurobiol Aging. 2004;25:1263–1272. [PubMed]
27. Grbovic OM, Mathews PM, Jiang Y, Schmidt SD, Dinakar R, Summers-Terio NB, Ceresa BP, Nixon RA, Cataldo AM. Rab5-stimulated up-regulation of the endocytic pathway increases intracellular beta-cleaved amyloid precursor protein carboxyl-terminal fragment levels and Abeta production. J Biol Chem. 2003;278:31261–31268. [PubMed]
28. Cataldo AM, Mathews PM, Boiteau AB, Hassinger LC, Peterhoff CM, Jiang Y, Mullaney K, Neve RL, Gruenberg J, Nixon RA. Down syndrome fibroblast model of Alzheimer-related endosome pathology: accelerated endocytosis promotes late endocytic defects. Am J Pathol. 2008;173:370–384. [PubMed]
29. Hyman BT, Van Hoesen GW, Damasio AR. Memory-related neural systems in Alzheimer's disease: an anatomic study. Neurology. 1990;40:1721–1730. [PubMed]
30. Mufson EJ, Counts SE, Che S, Ginsberg SD. Neuronal gene expression profiling: uncovering the molecular biology of neurodegenerative disease. Prog Brain Res. 2006;158:197–222. [PubMed]
31. Bennett DA, Wilson RS, Schneider JA, Evans DA, Beckett LA, Aggarwal NT, Barnes LL, Fox JH, Bach J. Natural history of mild cognitive impairment in older persons. Neurology. 2002;59:198–205. [PubMed]
32. DeKosky ST, Ikonomovic MD, Styren SD, Beckett L, Wisniewski S, Bennett DA, Cochran EJ, Kordower JH, Mufson EJ. Upregulation of choline acetyltransferase activity in hippocampus and frontal cortex of elderly subjects with mild cognitive impairment. Ann Neurol. 2002;51:145–155. [PubMed]
33. Winblad B, Palmer K, Kivipelto M, Jelic V, Fratiglioni L, Wahlund LO, Nordberg A, Backman L, Albert M, Almkvist O, Arai H, Basun H, Blennow K, de Leon M, DeCarli C, Erkinjuntti T, Giacobini E, Graff C, Hardy J, Jack C, Jorm A, Ritchie K, van Duijn C, Visser P, Petersen RC. Mild cognitive impairment--beyond controversies, towards a consensus: report of the International Working Group on Mild Cognitive Impairment. J Intern Med. 2004;256:240–246. [PubMed]
34. Reisberg B, Ferris SH, Kluger A, Franssen E, Wegiel J, de Leon MJ. Mild cognitive impairment (MCI): a historical perspective. Int Psychogeriatr. 2008;20:18–31. [PubMed]
35. Bennett DA, Schneider JA, Bienias JL, Evans DA, Wilson RS. Mild cognitive impairment is related to Alzheimer disease pathology and cerebral infarctions. Neurology. 2005;64:834–841. [PubMed]
36. Mufson EJ, Ma SY, Dills J, Cochran EJ, Leurgans S, Wuu J, Bennett DA, Jaffar S, Gilmor ML, Levey AI, Kordower JH. Loss of basal forebrain P75(NTR) immunoreactivity in subjects with mild cognitive impairment and Alzheimer's disease. J Comp Neurol. 2002;443:136–153. [PubMed]
37. Braak H, Braak E. Neuropathological stageing of Alzheimer-related changes. Acta Neuropathol. 1991;82:239–259. [PubMed]
38. Hyman BT, Trojanowski JQ. Consensus recommendations for the postmortem diagnosis of Alzheimer disease from the National Institute on Aging and the Reagan Institute Working Group on diagnostic criteria for the neuropathological assessment of Alzheimer disease. J. Neuropathol. Exp. Neurol. 1997;56:1095–1097. [PubMed]
39. Mirra SS, Heyman A, McKeel D, Sumi SM, Crain BJ, Brownlee LM, Vogel FS, Hughes JP, van Belle G, Berg L. The Consortium to Establish a Registry for Alzheimer's Disease (CERAD). Part II. Standardization of the neuropathologic assessment of Alzheimer's disease. Neurology. 1991;41:479–486. [PubMed]
40. Counts SE, Nadeem M, Wuu J, Ginsberg SD, Saragovi HU, Mufson EJ. Reduction of cortical TrkA but not p75(NTR) protein in early-stage Alzheimer's disease. Ann Neurol. 2004;56:520–531. [PubMed]
41. Ginsberg SD. Glutamatergic neurotransmission expression profiling in the mouse hippocampus after perforant-path transection. Am J Geriatr Psychiatry. 2005;13:1052–1061. [PubMed]
42. Ginsberg SD, Hemby SE, Lee VM-Y, Eberwine JH, Trojanowski JQ. Expression profile of transcripts in Alzheimer's disease tangle-bearing CA1 neurons. Ann Neurol. 2000;48:77–87. [PubMed]
43. Lanzetti L, Palamidessi A, Areces L, Scita G, Di Fiore PP. Rab5 is a signalling GTPase involved in actin remodelling by receptor tyrosine kinases. Nature. 2004;429:309–314. [PubMed]
44. MacMicking JD, Taylor GA, McKinney JD. Immune control of tuberculosis by IFN-gamma-inducible LRG-47. Science. 2003;302:654–659. [PubMed]
45. He W, Ladinsky MS, Huey-Tubman KE, Jensen GJ, McIntosh JR, Bjorkman PJ. FcRn-mediated antibody transport across epithelial cells revealed by electron tomography. Nature. 2008;455:542–546. [PMC free article] [PubMed]
46. Siddiqui SS, Aamodt E, Rastinejad F, Culotti J. Anti-tubulin monoclonal antibodies that bind to specific neurons in Caenorhabditis elegans. J Neurosci. 1989;9:2963–2972. [PubMed]
47. Raymond AA, Gonzalez de Peredo A, Stella A, Ishida-Yamamoto A, Bouyssie D, Serre G, Monsarrat B, Simon M. Lamellar bodies of human epidermis: proteomics characterization by high throughput mass spectrometry and possible involvement of CLIP-170 in their trafficking/secretion. Mol Cell Proteomics. 2008;7:2151–2175. [PubMed]
48. Ginsberg SD, Che S, Wuu J, Counts SE, Mufson EJ. Down regulation of trk but not p75 gene expression in single cholinergic basal forebrain neurons mark the progression of Alzheimer's disease. J Neurochem. 2006;97:475–487. [PubMed]
49. Cash AD, Aliev G, Siedlak SL, Nunomura A, Fujioka H, Zhu X, Raina AK, Vinters HV, Tabaton M, Johnson AB, Paula-Barbosa M, Avila J, Jones PK, Castellani RJ, Smith MA, Perry G. Microtubule reduction in Alzheimer's disease and aging is independent of tau filament formation. Am J Pathol. 2003;162:1623–1627. [PubMed]
50. Counts SE, He B, Che S, Ikonomovic MD, Dekosky ST, Ginsberg SD, Mufson EJ. {alpha}7 Nicotinic receptor up-regulation in cholinergic basal forebrain neurons in Alzheimer disease. Arch Neurol. 2007;64:1771–1776. [PubMed]
51. Ginsberg SD, Che S, Counts SE, Mufson EJ. Shift in the ratio of three-repeat tau and four-repeat tau mRNAs in individual cholinergic basal forebrain neurons in mild cognitive impairment and Alzheimer's disease. J Neurochem. 2006;96:1401–1408. [PubMed]
52. SAS Institute Inc . SAS/STAT 9.1 User's Guide. SAS Publishing; Cary, NC: 2004.
53. Adamec E, Mohan PS, Cataldo AM, Vonsattel JP, Nixon RA. Up-regulation of the lysosomal system in experimental models of neuronal injury: implications for Alzheimer's disease. Neuroscience. 2000;100:663–675. [PubMed]
54. Jiang Y, Mullaney KA, Peterhoff CM, Che S, Schmidt SD, Boyer-Boiteau A, Ginsberg SD, Cataldo AM, Mathews PM, Nixon RA. Alzheimer's-related endosome dysfunction in Down syndrome is A{beta}-independent but requires APP and is reversed by BACE-1 inhibition. Proc Natl Acad Sci USA. 2010;107:1630–1635. [PubMed]
55. Hof PR, Morrison JH. The aging brain: morphomolecular senescence of cortical circuits. Trends Neurosci. 2004;27:607–613. [PubMed]
56. Mesulam MM, Mufson EJ, Levey AI, Wainer BH. Cholinergic innervation of cortex by the basal forebrain: cytochemistry and cortical connections of the septal area, diagonal band nuclei, nucleus basalis (substantia innominata), and hypothalamus in the rhesus monkey. J Comp Neurol. 1983;214:170–197. [PubMed]
57. Morrison JH, Hof PR. The organization of the cerebral cortex: from molecules to circuits. In: Magistretti PJ, editor. Discussions in Neuroscience, vol. 9. FESN-Elsevier; Geneva: 1992.
58. Bonanomi D, Benfenati F, Valtorta F. Protein sorting in the synaptic vesicle life cycle. Prog Neurobiol. 2006;80:177–217. [PubMed]
59. Gruenberg J. The endocytic pathway: a mosaic of domains. Nat Rev Mol Cell Biol. 2001;2:721–730. [PubMed]
60. Bright NA, Gratian MJ, Luzio JP. Endocytic delivery to lysosomes mediated by concurrent fusion and kissing events in living cells. Curr Biol. 2005;15:360–365. [PubMed]
61. Cowles CR, Odorizzi G, Payne GS, Emr SD. The AP-3 adaptor complex is essential for cargo-selective transport to the yeast vacuole. Cell. 1997;91:109–118. [PubMed]
62. Delcroix JD, Valletta J, Wu C, Howe CL, Lai CF, Cooper JD, Belichenko PV, Salehi A, Mobley WC. Trafficking the NGF signal: implications for normal and degenerating neurons. Prog Brain Res. 2004;146:3–23. [PubMed]
63. Howe CL, Mobley WC. Signaling endosome hypothesis: A cellular mechanism for long distance communication. J Neurobiol. 2004;58:207–216. [PubMed]
64. Nixon RA, Cataldo AM, Mathews PM. The endosomal-lysosomal system of neurons in Alzheimer's disease pathogenesis: a review. Neurochem Res. 2000;25:1161–1172. [PubMed]
65. Vassar R, Bennett BD, Babu-Khan S, Kahn S, Mendiaz EA, Denis P, Teplow DB, Ross S, Amarante P, Loeloff R, Luo Y, Fisher S, Fuller J, Edenson S, Lile J, Jarosinski MA, Biere AL, Curran E, Burgess T, Louis JC, Collins F, Treanor J, Rogers G, Citron M. Beta-secretase cleavage of Alzheimer's amyloid precursor protein by the transmembrane aspartic protease BACE. Science. 1999;286:735–741. [PubMed]
66. Huse JT, Pijak DS, Leslie GJ, Lee VM, Doms RW. Maturation and endosomal targeting of beta-site amyloid precursor protein-cleaving enzyme. The Alzheimer's disease beta-secretase. J Biol Chem. 2000;275:33729–33737. [PubMed]
67. Choi JH, Berger JD, Mazzella MJ, Morales-Corraliza J, Cataldo AM, Nixon RA, Ginsberg SD, Levy E, Mathews PM. Age-dependent dysregulation of brain amyloid precursor protein in the Ts65Dn Down syndrome mouse model. J Neurochem. 2009;110:1818–1827. [PMC free article] [PubMed]
68. Gouras GK, Almeida CG, Takahashi RH. Intraneuronal Abeta accumulation and origin of plaques in Alzheimer's disease. Neurobiol Aging. 2005;26:1235–1244. [PubMed]
69. Mathews PM, Guerra CB, Jiang Y, Grbovic OM, Kao BH, Schmidt SD, Dinakar R, Mercken M, Hille-Rehfeld A, Rohrer J, Mehta P, Cataldo AM, Nixon RA. Alzheimer's disease-related overexpression of the cation-dependent mannose 6-phosphate receptor increases Abeta secretion: role for altered lysosomal hydrolase distribution in beta-amyloidogenesis. J Biol Chem. 2002;277:5299–5307. [PubMed]
70. Laifenfeld D, Patzek LJ, McPhie DL, Chen Y, Levites Y, Cataldo AM, Neve RL. Rab5 mediates an amyloid precursor protein signaling pathway that leads to apoptosis. J Neurosci. 2007;27:7141–7153. [PubMed]
71. Nixon RA. Niemann-Pick Type C disease and Alzheimer's disease: the APP-endosome connection fattens up. Am J Pathol. 2004;164:757–761. [PubMed]
72. Nixon RA, Yang DS, Lee JH. Neurodegenerative lysosomal disorders: a continuum from development to late age. Autophagy. 2008;4:590–599. [PubMed]
73. Lambert JC, Heath S, Even G, Campion D, Sleegers K, Hiltunen M, Combarros O, Zelenika D, Bullido MJ, Tavernier B, Letenneur L, Bettens K, Berr C, Pasquier F, Fievet N, Barberger-Gateau P, Engelborghs S, De Deyn P, Mateo I, Franck A, Helisalmi S, Porcellini E, Hanon O, de Pancorbo MM, Lendon C, Dufouil C, Jaillard C, Leveillard T, Alvarez V, Bosco P, Mancuso M, Panza F, Nacmias B, Bossu P, Piccardi P, Annoni G, Seripa D, Galimberti D, Hannequin D, Licastro F, Soininen H, Ritchie K, Blanche H, Dartigues JF, Tzourio C, Gut I, Van Broeckhoven C, Alperovitch A, Lathrop M, Amouyel P. Genome-wide association study identifies variants at CLU and CR1 associated with Alzheimer's disease. Nat Genet. 2009;41:1094–1099. [PubMed]
74. Harold D, Abraham R, Hollingworth P, Sims R, Gerrish A, Hamshere ML, Pahwa JS, Moskvina V, Dowzell K, Williams A, Jones N, Thomas C, Stretton A, Morgan AR, Lovestone S, Powell J, Proitsi P, Lupton MK, Brayne C, Rubinsztein DC, Gill M, Lawlor B, Lynch A, Morgan K, Brown KS, Passmore PA, Craig D, McGuinness B, Todd S, Holmes C, Mann D, Smith AD, Love S, Kehoe PG, Hardy J, Mead S, Fox N, Rossor M, Collinge J, Maier W, Jessen F, Schurmann B, van den Bussche H, Heuser I, Kornhuber J, Wiltfang J, Dichgans M, Frolich L, Hampel H, Hull M, Rujescu D, Goate AM, Kauwe JS, Cruchaga C, Nowotny P, Morris JC, Mayo K, Sleegers K, Bettens K, Engelborghs S, De Deyn PP, Van Broeckhoven C, Livingston G, Bass NJ, Gurling H, McQuillin A, Gwilliam R, Deloukas P, Al-Chalabi A, Shaw CE, Tsolaki M, Singleton AB, Guerreiro R, Muhleisen TW, Nothen MM, Moebus S, Jockel KH, Klopp N, Wichmann HE, Carrasquillo MM, Pankratz VS, Younkin SG, Holmans PA, O'Donovan M, Owen MJ, Williams J. Genome-wide association study identifies variants at CLU and PICALM associated with Alzheimer's disease. Nat Genet. 2009;41:1088–1093. [PMC free article] [PubMed]