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Abnormalities of the endosomal-lysosomal network (ELN) are a signature feature of Alzheimer's disease (AD). These include the earliest known cytopathology that is specific to AD and that affects endosomes and induces the progressive failure of lysosomes, each of which are directly linked by distinct mechanisms to neurodegeneration. The origins of ELN dysfunction and β-amyloidogenesis closely overlap, which reflects their common genetic basis, the established early involvement of endosomes and lysosomes in amyloid precursor protein (APP) processing and clearance, and the pathologic effect of certain APP metabolites on ELN functions. Genes that promote β-amyloidogenesis in AD (APP, PSEN1/2, and APOE4) have primary effects on ELN function. The importance of primary ELN dysfunction to pathogenesis is underscored by the mutations in more than 35 ELN-related genes that, thus far, are known to cause familial neurodegenerative diseases even though different pathogenic proteins may be involved. In this article, I discuss growing evidence that implicates AD gene-driven ELN disruptions as not only the antecedent pathobiology that underlies β-amyloidogenesis but also as the essential partner with APP and its metabolites that drive the development of AD, including tauopathy, synaptic dysfunction, and neurodegeneration. The striking amelioration of diverse deficits in animal AD models by remediating ELN dysfunction further supports a need to integrate APP and ELN relationships, including the role of amyloid-β, into a broader conceptual framework of how AD arises, progresses, and may be effectively therapeutically targeted.--Nixon, R. A. Amyloid precursor protein and endosomal-lysosomal dysfunction in Alzheimer's disease: inseparable partners in a multifactorial disease. [ABSTRACT FROM AUTHOR]

Amyotrophic lateral sclerosis (ALS) is a progressive motor neuron disease with a poorly understood cause and no effective treatment. Given that calpains mediate neurodegeneration in other pathological states and are abnormally activated in ALS, we investigated the possible ameliorative effects of inhibiting calpain over-activation in hSOD1G93A transgenic (Tg) mice in vivo by neuron-specific over-expression of calpastatin (CAST), the highly selective endogenous inhibitor of calpains. Our data indicate that over-expression of CAST in hSOD1G93A mice, which lowered calpain activation to levels comparable to wild-type mice, inhibited the abnormal breakdown of cytoskeletal proteins (spectrin, MAP2 and neurofilaments), and ameliorated motor axon loss. Disease onset in hSOD1G93A/CAST mice compared to littermate hSOD1G93A mice is delayed, which accounts for their longer time of survival. We also find that neuronal over-expression of CAST in hSOD1G93A transgenic mice inhibited production of putative neurotoxic caspase-cleaved tau and activation of Cdk5, which have been implicated in neurodegeneration in ALS models, and also reduced the formation of SOD1 oligomers. Our data indicate that inhibition of calpain with CAST is neuroprotective in an ALS mouse model. [ABSTRACT FROM AUTHOR]

In the United States, estimates indicate there are between 250,000 and 400,000 individuals with Down syndrome (DS), and nearly all will develop Alzheimer's disease (AD) pathology starting in their 30s. With the current lifespan being 55 to 60 years, approximately 70% will develop dementia, and if their life expectancy continues to increase, the number of individuals developing AD will concomitantly increase. Pathogenic and mechanistic links between DS and Alzheimer's prompted the Alzheimer's Association to partner with the Linda Crnic Institute for Down Syndrome and the Global Down Syndrome Foundation at a workshop of AD and DS experts to discuss similarities and differences, challenges, and future directions for this field. The workshop articulated a set of research priorities: (1) target identification and drug development, (2) clinical and pathological staging, (3) cognitive assessment and clinical trials, and (4) partnerships and collaborations with the ultimate goal to deliver effective disease-modifying treatments. [ABSTRACT FROM AUTHOR]

Autophagy is a lysosomal degradative process which recycles cellular waste and eliminates potentially toxic damaged organelles and protein aggregates. The important cytoprotective functions of autophagy are demonstrated by the diverse pathogenic consequences that may stem from autophagy dysregulation in a growing number of neurodegenerative disorders. In many of the diseases associated with autophagy anomalies, it is the final stage of autophagy-lysosomal degradation that is disrupted. In several disorders, including Alzheimer's disease ( AD), defective lysosomal acidification contributes to this proteolytic failure. The complex regulation of lysosomal p H makes this process vulnerable to disruption by many factors, and reliable lysosomal p H measurements have become increasingly important in investigations of disease mechanisms. Although various reagents for p H quantification have been developed over several decades, they are not all equally well suited for measuring the p H of lysosomes. Here, we evaluate the most commonly used p H probes for sensitivity and localisation, and identify Lyso Sensor yellow/blue-dextran, among currently used probes, as having the optimal profile of properties for measuring lysosomal p H. In addition, we review evidence that lysosomal acidification is defective in AD and extend our original findings, of elevated lysosomal p H in presenilin 1 ( PS1)-deficient blastocysts and neurons, to additional cell models of PS1 and PS1/2 deficiency, to fibroblasts from AD patients with PS1 mutations, and to neurons in the PS/ APP mouse model of AD. [ABSTRACT FROM AUTHOR]

The Β-site APP cleaving enzyme-1 (BACE1) mediates the first cleavage of the Β-amyloid precursor protein (APP) to yield the amyloid Β-peptide (AΒ), a key pathogenic agent in Alzheimer's disease (AD). Using a proteomic approach based on in-cell chemical cross-linking and tandem affinity purification (TAP), we herein identify sorting nexin 6 (SNX6) as a BACE1-associated protein. SNX6, a PX domain protein, is a putative component of retromer, a multiprotein cargo complex that mediates the retrograde trafficking of the cation-independent mannose-6-phosphate receptor (CI-MPR) and sortilin. RNA interference suppression of SNX6 increased BACE1-dependent secretion of soluble APP (sAPPΒ) and cell-associated fragments (C99), resulting in increased AΒ secretion. Furthermore, SNX6 reduction led to elevated steady-state BACE1 levels as well as increased retrograde transport of BACE1 in the endocytic pathway, suggesting that SNX6 modulates the retrograde trafficking and basal levels of BACE1, thereby regulating BACE1-mediated APP processing and AΒ biogenesis. Our study identifies a novel cellular pathway by which SNX6 negatively modulates BACE1-mediated cleavage of APP. [ABSTRACT FROM AUTHOR]

Individuals with Down syndrome develop β-amyloid deposition characteristic of early-onset Alzheimer’s disease (AD) in mid-life, presumably because of an extra copy of the chromosome 21-located amyloid precursor protein ( App) gene. App mRNA and APP metabolite levels were assessed in the brains of Ts65Dn mice, a mouse model of Down syndrome, using quantitative PCR, western blot analysis, immunoprecipitation, and ELISAs. In spite of the additional App gene copy, App mRNA, APP holoprotein, and all APP metabolite levels in the brains of 4-month-old trisomic mice were not increased compared with the levels seen in diploid littermate controls. However starting at 10 months of age, brain APP levels were increased proportional to the App gene dosage imbalance reflecting increased App message levels in Ts65Dn mice. Similar to APP levels, soluble amino-terminal fragments of APP (sAPPα and sAPPβ) were increased in Ts65Dn mice compared with diploid mice at 12 months but not at 4 months of age. Brain levels of both Aβ40 and Aβ42 were not increased in Ts65Dn mice compared with diploid mice at all ages examined. Therefore, multiple mechanisms contribute to the regulation towards diploid levels of APP metabolites in the Ts65Dn mouse brain. [ABSTRACT FROM AUTHOR]

As axons myelinate, establish a stable neurofilament network, and expand in caliber, neurofilament proteins are extensively phosphorylated along their C-terminal tails, which is recognized by the monoclonal antibody, RT-97. Here, we demonstrate in vivo that RT-97 immunoreactivity (IR) is generated by phosphorylation at KSPXK or KSPXXXK motifs and requires flanking lysines at specific positions. extracellular signal regulated kinase 1,2 (ERK1,2) and pERK1,2 levels increase in parallel with phosphorylation at the RT-97 epitope during early postnatal brain development. Purified ERK1,2 generated RT-97 on both KSP motifs on recombinant NF-H tail domain proteins , while cdk5 phosphorylated only KSPXK motifs. RT-97 epitope generation in primary hippocampal neurons was regulated by extensive cross-talk among ERK1,2, c-Jun N-terminal kinase 1,2 (JNK1,2) and cdk5. Inhibition of both ERK1,2 and JNK1,2 completely blocked RT-97 generation. Cdk5 influenced RT-97 generation indirectly by modulating JNK activation. In mice, cdk5 gene deletion did not significantly alter RT-97 IR or ERK1,2 and JNK activation. In mice lacking the cdk5 activator P35, the partial suppression of cdk5 activity increased RT-97 IR by activating ERK1,2. Thus, cdk5 influences RT-97 epitope generation partly by modulating ERKs and JNKs, which are the two principal kinases regulating neurofilament phosphorylation. The regulation of a single target by multiple protein kinases underscores the importance of monitoring other relevant kinases when the activity of a particular one is blocked. [ABSTRACT FROM AUTHOR]

Studies in continuously cultured cells have established that familial Alzheimer’s disease (FAD) mutant presenilin 1 (PS1) delays exit of the amyloid precursor protein (APP) from the trans-Golgi network (TGN). Here we report the first description of PS1-regulated APP trafficking in cerebral neurons in culture and in vivo. Using neurons from transgenic mice or a cell-free APP transport vesicle biogenesis system derived from the TGN of those neurons, we demonstrated that knocking-in an FAD-associated mutant PS1 transgene was associated with delayed kinetics of APP arrival at the cell surface. Apparently, this delay was at least partially attributable to impaired exit of APP from the TGN, which was documented in the cell-free APP transport vesicle biogenesis assay. To extend the study to APP and carboxyl terminal fragment (CTF) trafficking to cerebral neurons in vivo, we performed subcellular fractionation of brains from APP transgenic mice, some of which carried a second transgene encoding an FAD-associated mutant form of PS1. The presence of the FAD mutant PS1 was associated with a slight shift in the subcellular localization of both holoAPP and APP CTFs toward iodixanol density gradient fractions that were enriched in a marker for the TGN. In a parallel set of experiments, we used an APP : furin chimeric protein strategy to test the effect of artificially forcing TGN concentration of an APP : furin chimera that could be a substrate for β- and γ-cleavage. This chimeric substrate generated excess Aβ42 when compared with wildtype APP. These data indicate that the presence of an FAD-associated mutant human PS1 transgene is associated with redistribution of the APP and APP CTFs in brain neurons toward TGN-enriched fractions. The chimera experiment suggests that TGN-enrichment of a β-/γ-secretase substrate may play an integral role in the action of mutant PS1 to elevate brain levels of Aβ42. [ABSTRACT FROM AUTHOR]

The visualization of β-amyloid plaque deposition in brain, a key feature of Alzheimer's disease (AD), is important for the evaluation of disease progression and the efficacy of therapeutic interventions. In this study, β-amyloid plaques in the PS/APP transgenic mouse brain, a model of human AD pathology, were detected using MR microscopy without contrast reagents. β-Amyloid plaques were clearly visible in the cortex, thalamus, and hippocampus of fixed brains of PS/APP mice. The distribution of plaques identified by MRI was in excellent agreement with those found in the immunohistological analysis of the same brain sections. It was also demonstrated that image contrast for β-amyloid plaques was present in freshly excised nonfixed brains. Furthermore, the detection of β-amyloid plaques was achieved with a scan time as short as 2 hr, approaching the scan time considered reasonable for in vivo imaging. Magn Reson Med 52:538-544, 2004. © 2004 Wiley-Liss, Inc. [ABSTRACT FROM AUTHOR]

Aging and apolipoprotein E ( APOE) isoform are among the most consistent risks for the development of Alzheimer's disease (AD). Metabolic factors that modulate risk have been elusive, though oxidative reactions and their by-products have been implicated in human AD and in transgenic mice with overt histological amyloidosis. We investigated the relationship between the levels of endogenous murine amyloid β (Aβ) peptides and the levels of a marker of oxidation in mice that never develop histological amyloidosis [i.e. APOE knockout (KO) mice with or without transgenic human APOEℇ3 or human APOEℇ4 alleles]. Aging-, gender-, and APOE-genotype-dependent changes were observed for endogenous mouse brain Aβ40 and Aβ42 peptides. Levels of the oxidized lipid F2-isoprostane (F2- isoPs) in the brains of the same animals as those used for the Aβ analyses revealed aging- and gender-dependent changes in APOE KO and in human APOEℇ4 transgenic KO mice. Human APOEℇ3 transgenic KO mice did not exhibit aging- or gender-dependent increases in F2- isoPs. In general, the changes in the levels of brain F2- isoPs in mice according to age, gender, and APOE genotype mirrored the changes in brain Aβ levels, which, in turn, paralleled known trends in the risk for human AD. These data indicate that there exists an aging-dependent, APOE-genotype-sensitive rise in murine brain Aβ levels despite the apparent inability of the peptide to form histologically detectable amyloid. Human APOEℇ3, but not human APOEℇ4, can apparently prevent the aging-dependent rise in murine brain Aβ levels, consistent with the relative risk for AD associated with these genotypes. The fidelity of the brain Aβ/F2- isoP relationship across multiple relevant variables supports the hypothesis that oxidized lipids play a role in AD pathogenesis, as has been suggested by recent evidence that F2- isoPs can stimulate Aβ generation and aggregation. [ABSTRACT FROM AUTHOR]

The cerebral deposition of amyloid β-peptide, a central event in Alzheimer's disease (AD) pathogenesis, begins several years before the onset of clinical symptoms. Noninvasive detection of AD pathology at this initial stage would facilitate intervention and enhance treatment success. In this study, high-field MRI was used to detect changes in regional brain MR relaxation times in three types of mice: 1) transgenic mice (PS/APP) carrying both mutant genes for amyloid precursor protein (APP) and presenilin (PS), which have high levels and clear accumulation of β-amyloid in several brain regions, starting from 10 weeks of age; 2) transgenic mice (PS) carrying only a mutant gene for presenilin (PS), which show subtly elevated levels of Aβ-peptide without β-amyloid deposition; and 3) nontransgenic (NTg) littermates as controls. The transverse relaxation time T2, an intrinsic MR parameter thought to reflect impaired cell physiology, was significantly reduced in the hippocampus, cingulate, and retrosplenial cortex, but not the corpus callosum, of PS-APP mice compared to NTg. No differences in T1 values or proton density were detected between any groups of mice. These results indicate that T2 may be a sensitive marker of abnormalities in this transgenic mouse model of AD. Magn Reson Med 51:794-798, 2004. © 2004 Wiley-Liss, Inc. [ABSTRACT FROM AUTHOR]

The enzyme γ-secretase catalyzes the intramembrane proteolytic cleavage that generates the amyloid β-peptide from the β-amyloid precursor protein. The presenilin (PS) protein is one of the four integral membrane protein components of the mature γ-secretase complex. The PS protein is itself subjected to endoproteolytic processing, generating stable N- and C-terminal fragment (NTF and CTF, respectively) heterodimers. Here we demonstrate that coexpression of PS1 NTF and CTF functionally mimics expression of the full-length PS1 protein and restores γ-secretase activity in PS-deficient mammalian cells. The coexpressed fragments re-associate with each other inside the cell, where they also interact with nicastrin, another γ-secretase complex component. Analysis of γ-secretase activity following the expression of mutant forms of NTF and CTF, under conditions bypassing endoproteolysis, indicated that the putatively catalytic Asp257 and Asp385 residues have a direct effect on γ-secretase activity. Moreover, we demonstrate that expression of the wild-type CTF rescues endoproteolytic cleavage of C-terminally truncated PS1 molecules that are otherwise uncleaved and inactive. Recovery of cleavage is critically dependent on the integrity of Asp385. Taken together, our findings indicate that ectopically expressed NTF and CTF restore functional γ-secretase complexes and that the presence of full-length PS1 is not a requirement for proper complex assembly. [ABSTRACT FROM AUTHOR]

Abstract : We have shown previously that phosphate groups on the amino-terminal head domain region of the middle molecular mass subunit of neurofilament proteins (NF-M) are added by second messenger-dependent protein kinases. Here, we have identified Ser23 as a specific protein kinase A phosphorylation site on the native NF-M subunit and on two synthetic peptides, S1 (14 RRVPTETRSSF24 ) and S2 (21 RSSFSRVSGSPSSGFRSQSWS41 ), localized within the amino-terminal head domain region. Ser23 was identified as a phosphorylation site on the 32 P-labeled α-chymotryptic peptide that carried >80% of the 32 P-phosphates incorporated into the NF-M subunit by protein kinase A. The synthetic peptides S1 and S2 were phosphorylated 18 and two times more efficiently by protein kinase A than protein kinase C, respectively. Neither of the peptides was phosphorylated by casein kinase II. The sequence analyses of the chemically modified phosphorylated serine residues showed that Ser23 was the major site of phosphorylation for protein kinase A on both S1 and S2 peptides. Low levels of incorporation of 32 P-phosphates into Ser22 , Ser28 , and Ser32 by protein kinase A were also observed. Protein kinase C incorporated 32 P-phosphates into Ser22 , Ser23 , Ser25 , Ser28 , Ser32 , and a threonine residue, but none of these sites could be assigned as a major site of phosphorylation. Analyses of the phosphorylated synthetic peptides by liquid chromatography-tandem mass spectrometry also showed that protein kinase A phosphorylated only one site on peptide S1 and that ions with up to four phosphates were detected on peptide S2 . Analysis of the data from the tandem ion trap mass spectrometry by... [ABSTRACT FROM AUTHOR]

Traces the evolution of concepts on the transport and assembly of the axonal cytoskeleton, with emphasis on developmental biology. Synthesis of cytoskeletal proteins; Evolving models of slow axonal transport; Reconciling old and modern findings of the transport of axonal cytoskeleton.

In this article, the author offers information related to the cellular handling and clearance of proteins including the amyloid beta peptide (Abeta) and role of autophagy in Abeta secretion in patient suffering from Alzheimer's disease (AD). Topics discussed include beta-amyloid precursor protein (APP), autophagy dysfunction seen in AD and neurodegradation.