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.
MR microscopy; transgenic mouse; Alzheimer’s disease; β-amyloid plaques
This study confirms the presence of iron, co-localized with Aβ plaques, in PS/APP mouse brain, using Perls’ stain for Fe3+ supplemented by 3,3′-diaminobenzidine (DAB) and Aβ immunohistochemistry in histological brains sections fixed with formalin or methacarn. In this study, the fixation process and the slice thickness did not interfere with the Perls’ technique. The presence of iron in β-amyloid plaques in PS/APP transgenic mice, a model of Alzheimer’s disease (AD) pathology, may explain previous reports of reductions of transverse relaxation time (T2) in MRI studies and represent the source of the intrinsic Aβ plaque MR contrast in this model.
β-amyloid; Alzheimer’s disease; Brain; Iron; MRI; Transgenic mice
While anti-human-Aβ immunotherapy clears brain β-amyloid plaques in Alzheimer's disease (AD), targeting additional brain plaque constituents to promote clearance has not been attempted. Endogenous murine Aβ is a minor β-amyloid plaque component in amyloid precursor protein transgenic AD models, which we show is ~2–8% of the total accumulated Aβ in various human APP transgenic mice. Murine Aβ co-deposits and co-localizes with human Aβ in amyloid plaques and the two Aβ species co-immunoprecipitate together from brain extracts. In the human APP transgenic mice Tg2576, passive immunization for eight weeks with a murine-Aβ-specific antibody reduced β-plaque pathology, robustly decreasing both murine and human Aβ levels. The immunized mice additionally showed improvements in two behavioral assays, odor habituation and nesting behavior. We conclude that passive anti-murine-Aβ immunization clears β-amyloid plaque pathology – including the major human Aβ component – and decreases behavioral deficits, arguing that targeting minor, endogenous brain plaque constituents can be beneficial, broadening the range of plaque-associated targets for AD therapeutics.
Alzheimer's disease; Aβ; co-deposition; immunization; immunotherapy
Microtubule-based axonal transport is believed to become globally
disrupted in Alzheimer’s disease in part due to alterations of tau
expression or phosphorylation. We previously showed that axonal transport rates
along retinal ganglion axons are unaffected by deletion of normal mouse tau or
by overexpression of wild-type human tau. Here, we report that htau mice
expressing 3-fold higher levels of human tau in the absence of mouse tau also
display normal fast and slow transport kinetics despite the presence of
abnormally hyperphosphorylated tau in some neurons. In addition, markers of slow
transport (neurofilament light subunit) and fast transport (snap25) exhibit
normal distributions along optic axons of these mice. These studies demonstrate
that human tau overexpression, even when associated with a limited degree of tau
pathology, does not necessarily impair general axonal transport function
in vivo. This investigation is contributed for the issue of
Journal of Alzheimer’s Disease dedicated to the memory of Inge
Grunke-Iqbal and to the celebration of her contributions to Alzheimer’s
Tau; tauopathy; Alzheimer’s disease; neurofilament; slow axonal transport; fast axonal transport
We report that neuronal overexpression of the endogenous inhibitor of calpains, calpastatin (CAST), in a mouse model of human Alzheimer’s disease (AD) β-amyloidosis, the APP23 mouse, reduces β-amyloid pathology and Aβ levels when comparing aged, double transgenic (tg) APP23/CAST with APP23 mice. Concurrent with Aβ plaque deposition, aged APP23/CAST mice show a decrease in the steady-state brain levels of the amyloid precursor protein (APP) and APP C-terminal fragments when compared to APP23 mice. This CAST-dependent decrease in APP metabolite levels was not observed in single tg CAST mice expressing endogenous APP or in younger, Aβ plaque predepositing APP23/CAST mice. We also determined that the CAST-mediated inhibition of calpain activity in the brain is greater in the CAST mice with β-amyloid pathology than in non-APP tg mice, as demonstrated by a decrease in calpain-mediated cytoskeleton protein cleavage. Moreover, aged APP23/CAST mice have reduced ERK1/2 activity and tau phosphorylation when compared to APP23 mice. In summary, in vivo calpain inhibition mediated by CAST transgene expression reduces Aβ pathology in APP23 mice, with our findings further suggesting that APP metabolism is modified by CAST overexpression as the mice develop β-amyloid pathology. Our results indicate that the calpain system in neurons is more responsive to CAST inhibition under conditions of β-amyloid pathology, suggesting that in the disease state neurons may be more sensitive to the therapeutic use of calpain inhibitors.
calpain; calpastatin; APP; Aβ; Alzheimer’s disease
Peripherin, a neuronal intermediate filament protein implicated in neurodegenerative disease, coexists with the neurofilament triplet proteins (NFL, NFM, and NFH) but has an unknown function. The earlier peak expression of peripherin than the triplet during brain development and its ability to form homopolymers, unlike the triplet, which are obligate heteropolymers, have supported a widely held view that peripherin and neurofilament triplet form separate filament systems. Here, we demonstrate, however, that despite a postnatal decline in expression, peripherin is as abundant as the triplet in the adult PNS and exists in a relatively fixed stoichiometry with these subunits. Peripherin exhibits a distribution pattern identical to those of triplet proteins in sciatic axons and co-localizes with NFL on single neurofilament by immunogold electron microscopy. Peripherin also co-assembles into a single network of filaments containing NFL, NFM, NFH with and without α-internexin in quadruple- or quintuple-transfected SW13 vim (−) cells. Genetically deleting NFL in mice dramatically reduces peripherin content in sciatic axons. Moreover, peripherin mutations has been shown to disrupt the neurofilament network in transfected SW13 vim(−) cells. These data show that peripherin and the neurofilament proteins are functionally interdependent. The results strongly support the view that rather than forming an independent structure, peripherin is a subunit of neurofilaments in the adult PNS. Our findings provide a basis for its close relationship with neurofilaments in PNS diseases associated with neurofilament accumulation.
peripherin; neurofilament; intermediate filament; cytoskeleton; peripheral nerve; ALS; PNS
Abnormally swollen regions of axons and dendrites (neurites) filled mainly with autophagy-related organelles represent the highly characteristic and widespread form of “neuritic dystrophy” in Alzheimer disease (AD), which implies dysfunction of autophagy and axonal transport. In this punctum, we discuss our recent findings that autophagic/lysosomal degradation is critical to proper axonal transport of autophagic vacuoles (AVs) and lysosomes. We showed that lysosomal protease inhibition induces defective axonal transport of specific cargoes, causing these cargoes to accumulate in axonal swellings that biochemically and morphologically resemble the dystrophic neurites in AD. Our findings suggest that a cargo-specific failure of axonal transport promotes neuritic dystrophy in AD, which involves a mechanism distinct from the global axonal transport deficits seen in some other neurodegenerative diseases.
Alzheimer disease; autophagy; axonal transport; dystrophic neurites; lysosomes; proteolysis
Cytoskeletal protein phosphorylation is frequently altered in neuropathologic states but little is known about changes during normal aging. Here we report that declining protein phosphatase activity, rather than activation of kinases, underlies aging-related neurofilament hyperphosphorylation. Purified PP2A or PP2B dephosphorylated the heavy neurofilament (NFH) subunit or its extensively phorphorylated carboxyl-terminal domain in vitro. In cultured primary hippocampal neurons, inhibiting either phosphatase induced NFH phosphorylation without activating known neurofilament kinases. Neurofilament phosphorylation in the mouse CNS, as reflected by levels of the RT-97 phosphoepitope associated with late axon maturation, more than doubled during the 12 month period after NFH expression plateaued at p21. This was accompanied by declines in levels and activity of PP2A but not PP2B, and no rise in activities of neurofilament kinases (Erk1,2, cdk5 and JNK1,2). Inhibiting PP2A in mice in vivo restored brain RT-97 to levels seen in young mice. Declining PP2A activity, therefore, can account for rising neurofilament phosphorylation in maturing brain, potentially compounding similar changes associated with adult-onset neurodegenerative diseases.
neurofilament; phosphorylation; dephosphorylation; kinases; phosphatases; maturation; aging; RT-97 epitope; immunoreactivity
Endocytic system dysfunction is one of the earliest disturbances that occur in Alzheimer’s disease (AD), and may underlie the selective vulnerability of cholinergic basal forebrain (CBF) neurons during the progression of dementia. Herein we report that genes regulating early and late endosomes are selectively upregulated within CBF neurons in mild cognitive impairment (MCI) and AD. Specifically, upregulation of rab4, rab5, rab7, and rab27 was observed in CBF neurons microdissected from postmortem brains of individuals with MCI and AD compared to age-matched control subjects with no cognitive impairment (NCI). Upregulated expression of rab4, rab5, rab7, and rab27 correlated with antemortem measures of cognitive decline in individuals with MCI and AD. qPCR validated upregulation of these select rab GTPases within microdissected samples of the basal forebrain. Moreover, quantitative immunoblot analysis demonstrated upregulation of rab5 protein expression in the basal forebrain of subjects with MCI and AD. The elevation of rab4, rab5, and rab7 expression is consistent with our recent observations in CA1 pyramidal neurons in MCI and AD. These findings provide further support that endosomal pathology accelerates endocytosis and endosome recycling, which may promote aberrant endosomal signaling and neurodegeneration throughout the progression of AD.
cognitive decline; endosome; microarray; mild cognitive impairment; rab5; and qPCR
Newly synthesized neurofilaments or protofilaments are incorporated into a highly stable stationary cytoskeleton network as they are transported along axons. Although the heavily phosphorylated carboxyl-terminal tail domains of the heavy and medium neurofilament (NF) subunits have been proposed to contribute to this process and particularly to stability of this structure, their function is still obscure. Here we show in NF-H/M tail deletion [NF-(H/M)tailΔ] mice that the deletion of both of these domains selectively lowers NF levels 3–6 fold along optic axons without altering either rates of subunit synthesis or the rate of slow axonal transport of NF. Pulse labeling studies carried out over 90 days revealed a significantly faster rate of disappearance of NF from the stationary NF network of optic axons in NF-(H/M)tailΔ mice. Faster NF disappearance was accompanied by elevated levels of NF-L proteolytic fragments in NF-(H/M)tailΔ axons. We conclude that NF-H and NF-M C-terminal domains do not normally regulate NF transport rates as previously proposed, but instead increase the proteolytic resistance of NF, thereby stabilizing the stationary neurofilament cytoskeleton along axons.
Despite the progress of the past two decades, the cause of Alzheimer's disease (AD) and effective treatments against it remains elusive. The hypothesis that amyloid-β (Aβ) peptides are the primary causative agents of AD retains significant support amongst researchers. Nonetheless, a growing body of evidence shows that Aβ peptides are unlikely to be the sole factor in AD etiology. Evidence that Aβ/amyloid-independent factors, including the actions of AD-related genes, also contribute significantly to AD pathogenesis was presented in a symposium at the 2010 annual meeting of the Society for Neuroscience. Here we summarize the studies showing how amyloid-independent mechanisms cause defective endo-lysosomal trafficking, altered intracellular signaling cascades or impaired neurotransmitter release and contribute to synaptic dysfunction and/or neurodegeneration, leading to dementia in AD. A view of AD pathogenesis that encompasses both the amyloid-dependent and -independent mechanisms will help fill the gaps in our knowledge and reconcile the findings that cannot be explained solely by the amyloid hypothesis.
The unique vulnerability of the olfactory system to Alzheimer’s disease (AD) provides a quintessential translational tool for understanding mechanisms of synaptic dysfunction and pathological progression in the disease. Using the Tg2576 mouse model of β-amyloidosis, we show aberrant, hyperactive olfactory network activity begins early in life, prior to detectable behavioral impairments or comparable hippocampal dysfunction and at a time when Aβ deposition is restricted to the olfactory bulb (OB). Hyperactive odor-evoked activity in the piriform cortex (PCX) and increased OB-PCX functional connectivity emerged at a time coinciding with olfactory behavior impairments. This hyperactive activity persisted until later-life when the network converted to a hyporesponsive state. This conversion was Aβ-dependent, as liver-x-receptor agonist treatment to promote Aβ degradation, rescued the hyporesponsive state and olfactory behavior. These data lend evidence to a novel working model of olfactory dysfunction in AD and, complimentary to other recent works, suggest that disease-relevant network dysfunction is highly dynamic and region specific, yet with lasting effects on cognition and behavior.
Neural network; olfactory bulb; olfactory cortex; Amyloid-β; APP
Autophagy, the major degradative pathway for organelles and long-lived proteins, is essential for the survival of neurons. Mounting evidence has implicated defective autophagy in the pathogenesis of several major neurodegenerative diseases, particularly Alzheimer's disease (AD). A continuum of abnormalities of the lysosomal system has been identified in neurons of the AD brain, including pathological endocytic pathway responses at the very earliest disease stage and a progressive disruption of autophagy leading to the massive buildup of incompletely digested substrates within dystrophic axons and dendrites. In this review, we examine research on autophagy in AD and evaluate evidence addressing the specific step or steps along the autophagy pathway that may be defective. Current evidence strongly points to disruption of substrate proteolysis within autolysosomes for the principal mechanism underlying autophagy failure in AD. In the most common form of familial early onset AD, mutant presenilin 1 disrupts autophagy directly by impeding lysosomal proteolysis while, in other forms of AD, autophagy impairments may involve different genetic or environmental factors. Attempts to restore more normal lysosomal proteolysis and autophagy efficiency in mouse models of AD pathology have yielded promising therapeutic effects on neuronal function and cognitive performance, demonstrating the relevance of autophagy failure to the pathogenesis of AD and the potential of autophagy modulation as a therapeutic strategy.
The extensive autophagic-lysosomal pathology in Alzheimer disease (AD) brain has revealed a major defect in the proteolytic clearance of autophagy substrates. Autophagy failure contributes on several levels to AD pathogenesis and has become an important therapeutic target for AD and other neurodegenerative diseases. We recently observed broad therapeutic effects of stimulating autophagic-lysosomal proteolysis in the TgCRND8 mouse model of AD that exhibits defective proteolytic clearance of autophagic substrates, robust intralysosomal amyloid-β peptide (Aβ) accumulation, extracellular β-amyloid deposition and cognitive deficits. By genetically deleting the lysosomal cysteine protease inhibitor, cystatin B (CstB), to selectively restore depressed cathepsin activities, we substantially cleared Aβ, ubiquitinated proteins and other autophagic substrates from autolysosomes/lysosomes and rescued autophagic-lysosomal pathology, as well as reduced total Aβ40/42 levels and extracellular amyloid deposition, highlighting the underappreciated importance of the lysosomal system for Aβ clearance. Most importantly, lysosomal remediation prevented the marked learning and memory deficits in TgCRND8 mice. Our findings underscore the pathogenic significance of autophagic-lysosomal dysfunction in AD and demonstrate the value of reversing this dysfunction as an innovative therapeutic strategy for AD.
autophagy; lysosome; cathepsin; cystatin B; proteolysis; Alzheimer disease; transgenic
This review critically examines progress in understanding the link between Alzheimer’s disease (AD) molecular pathogenesis and behavior, with an emphasis on the impact of amyloid-β. We present the argument that the AD research field requires more multi-faceted analyses into the impacts of Alzheimer’s pathogenesis which combine simultaneous molecular-, circuit-, and behavior-level approaches. Supporting this argument is a review of particular research utilizing similar, ‘systems-level’ methods in mouse models of AD. Related to this, a critique of common physiological and behavioral models is made – highlighting the likely usefulness of more refined and specific tools in understanding the relationship between candidate molecular pathologies and behavioral dysfunction. Finally, we propose challenges for future research which, if met, may greatly extend our current understanding of how AD molecular pathology impacts neural network function and behavior and possibly may lead to refinements in disease therapeutics.
Amyloid-β; APP; cognition; dementia; endocytosis; LTD; LTP; neural connectivity; presenilin; tau; rab5; synapse
In the hallmark neuritic dystrophy of Alzheimer’s disease (AD), autophagic vacuoles containing incompletely digested proteins selectively accumulate in focal axonal swellings, reflecting defects in both axonal transport and autophagy. Here, we investigated the possibility that impaired lysosomal proteolysis could be a basis for both defects leading to neuritic dystrophy. In living primary mouse cortical neurons expressing fluorescence-tagged markers, LC3-positive autophagosomes forming in axons rapidly acquired the endo-lysosomal markers, Rab7 and LAMP1, and underwent exclusive retrograde movement. Proteolytic clearance of these transported autophagic vacuoles was initiated upon fusion with bi-directionally moving lysosomes that increase in number at more proximal axon levels and in the perikaryon. Disrupting lysosomal proteolysis by either inhibiting cathepsins directly or by suppressing lysosomal acidification slowed the axonal transport of autolysosomes, late endosomes and lysosomes and caused their selective accumulation within dystrophic axonal swellings. Mitochondria and other organelles lacking cathepsins moved normally under these conditions, indicating that the general functioning of the axonal transport system was preserved. Dystrophic swellings induced by lysosomal proteolysis inhibition resembled in composition those in several mouse models of AD and also acquired other AD-like features, including immunopositivity for ubiquitin, APP, and neurofilament protein hyperphosphorylation. Restoration of lysosomal proteolysis reversed the affected movements of proteolytic Rab7 vesicles, which in turn, largely cleared autophagic substrates and reversed the axonal dystrophy. These studies identify the AD-associated defects in neuronal lysosomal proteolysis as a possible basis for the selective transport abnormalities and highly characteristic pattern of neuritic dystrophy associated with AD.
Autophagy, a major degradative pathway for proteins and organelles, is essential for survival of mature neurons. Extensive autophagic-lysosomal pathology in Alzheimer’s disease brain contributes to Alzheimer’s disease pathogenesis, although the underlying mechanisms are not well understood. Here, we identified and characterized marked intraneuronal amyloid-β peptide/amyloid and lysosomal system pathology in the Alzheimer’s disease mouse model TgCRND8 similar to that previously described in Alzheimer’s disease brains. We further establish that the basis for these pathologies involves defective proteolytic clearance of neuronal autophagic substrates including amyloid-β peptide. To establish the pathogenic significance of these abnormalities, we enhanced lysosomal cathepsin activities and rates of autophagic protein turnover in TgCRND8 mice by genetically deleting cystatin B, an endogenous inhibitor of lysosomal cysteine proteases. Cystatin B deletion rescued autophagic-lysosomal pathology, reduced abnormal accumulations of amyloid-β peptide, ubiquitinated proteins and other autophagic substrates within autolysosomes/lysosomes and reduced intraneuronal amyloid-β peptide. The amelioration of lysosomal function in TgCRND8 markedly decreased extracellular amyloid deposition and total brain amyloid-β peptide 40 and 42 levels, and prevented the development of deficits of learning and memory in fear conditioning and olfactory habituation tests. Our findings support the pathogenic significance of autophagic-lysosomal dysfunction in Alzheimer’s disease and indicate the potential value of restoring normal autophagy as an innovative therapeutic strategy for Alzheimer’s disease.
autophagy; lysosome; cystatin B; cathepsin; Alzheimer’s disease
Endocytic dysfunction and neurotrophin signaling deficits may underlie the selective vulnerability of hippocampal neurons during the progression of Alzheimer’s disease (AD), although there is little direct in vivo and biochemical evidence to support this hypothesis.
Microarray analysis of hippocampal CA1 pyramidal neurons acquired via laser capture microdissection (LCM) was performed using postmortem brain tissue. Validation was achieved using real-time quantitative PCR (qPCR) and immunoblot analysis. Mechanistic studies were performed using human fibroblasts subjected to overexpression with viral vectors or knockdown via siRNA.
Expression levels of genes regulating early endosomes (rab5) and late endosomes (rab7) are selectively up regulated in homogeneous populations of CA1 neurons from individuals with mild cognitive impairment (MCI) and AD. The levels of these genes are selectively increased as antemortem measures of cognition decline during AD progression. Hippocampal qPCR and immunoblot analyses confirmed increased levels of these transcripts and their respective protein products. Elevation of select rab GTPases regulating endocytosis paralleled the down regulation of genes encoding the neurotrophin receptors TrkB and TrkC. Overexpression of rab5 in cells suppressed TrkB expression, whereas knockdown of TrkB expression did not alter rab5 levels, suggesting that TrkB down regulation is a consequence of endosomal dysfunction associated with elevated rab5 levels in early AD.
These data support the hypothesis that neuronal endosomal dysfunction is associated with preclinical AD. Increased endocytic pathway activity, driven by elevated rab GTPase expression, may result in long term deficits in hippocampal neurotrophic signaling and represent a key pathogenic mechanism underlying AD progression.
laser capture microdissection; mild cognitive impairment; RNA amplification; qPCR; rab5; rab7; siRNA; TrkB
Autophagy is the process by which organelles and portions of the cytoplasm are degraded in lysosomes. Several different forms of autophagy are known that are distinguishable chiefly by the mode in which cargo is delivered to the lysosome for degradation. Ubiquilin was recently reported to regulate macroautophagy, the form of autophagy in which cytosolic cargo is packaged in a double-membrane structure or autophagosome that fuses with lysosomes for degradation. We confirm here using different morphological and biochemical procedures that ubiquilin is present in autophagosomes in HeLa cells and in brain and liver tissue of mouse. Coimmunoprecipitation studies indicated that ubiquilin binds the autophagosome marker LC3 in a complex and that reduction of ubiquilin expression reduces autophagosome formation, which correlates with a reduction in maturation of LC3-I to the LC3-II form of the protein. We found that ubiquilin is degraded during both macroautophagy and during chaperone-mediated autophagy (CMA), the latter of which involves the active transport of proteins into lysosomes. We discuss the implication of this degradation in mediating cross-talk between macroautophagy and CMA. Finally, we demonstrate that ubiquilin protects cells against starvation-induced cell death propagated by overexpression of mutant Alzheimer's disease PS2N141I protein and green fluorescent protein (GFP)-huntingtin exon-1 fusion protein containing 74 polyglutamines.
White matter disorders can involve injury to myelin or axons but the respective contribution of each to clinical course is difficult to evaluate non-invasively. Here, to develop a paradigm for further investigations of axonal pathology by MRI, we compared two genetic mouse models exhibiting relatively selective axonal or myelin deficits using quantitative MRI relaxography of the transverse relaxation times (T2) in vivo and ultrastructural morphometry. In HM-DKO mice, which lack genes encoding the heavy (NF-H) and medium (NF-M) subunits of neurofilaments, neurofilament content of large myelinated axons of the central nervous system (CNS) is markedly reduced in the absence of changes in myelin thickness and volume. In shiverer mutant mice, which lack functional myelin basic protein, CNS myelin sheath formation is markedly reduced but neurofilament content is normal. We observed increases in T2 in nearly all white matter in Shiverer mice compared to their wild type, while more subtle increases in T2 were observed in HM-DKO in the corpus callosum. White matter T2 was generally greater in Shiverer mice than HM-DKO mice. Ultrastructural morphometry of the corpus callosum, which exhibited the greatest T2 differences, confirmed that total cross sectional area occupied by axons was similar in the two mouse models and that the major ultrastructural differences, determined by morphometry, were an absence of myelin and larger unmyelinated axons in shiverer mice and absence of neurofilaments in HM-DKO mice. Our findings indicate that T2 is strongly influenced by myelination state and axonal volume, while neurofilament structure within the intra-axonal compartment has a lesser effect upon single compartment T2 estimates.
T2 relaxation; iron; white matter; neurofilament; microtubules; myelin; shiverer mice
The neurofilament light subunit (NF-L) binds to myosin Va (Myo Va) in neurons but the sites of interaction and functional significance are not clear. We show by deletion analysis that motor domain of Myo Va binds to the NF-L rod domain that forms the NF backbone. Loss of NF-L and Myo Va binding from axons significantly reduces the axonal content of ER, and redistributes ER to the periphery of axon. Our data are consistent with a novel function for NFs as a scaffold in axons for maintaining the content and proper distribution of vesicular organelles, mediated in part by Myo Va. Based on observations that the Myo Va motor domain binds to intermediate filament (IF) proteins of several classes, Myo Va interactions with IFs may serve similar roles in organizing organelle topography in different cell types.
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.
basal forebrain; cerebellum; endosome; hippocampus; frontal cortex; mild cognitive impairment; rab GTPase; selective vulnerability; striatum
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 immunureactivity is generated by phosphorylation at KSPXK or KSPXXXK motifs and requires flanking lysines at specific positions. ERK1,2 and pERK1,2 levels increase in parallel with phosphorylation at the RT-97 epitope during early post-natal 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 crosstalk among ERK1,2, 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.
phosphorylation; neurofilament; kinases; RT-97 epitope; crosstalk
Individuals with Down syndrome develop β-amyloid deposition characteristic of early-onset Alzheimer's disease (AD) in mid-life, presumably due to 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 qPCR, 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 to 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, sAPPα and sAPPβ levels were increased in Ts65Dn mice compared to 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.
amyloid precursor protein (APP); Down syndrome; animal model; trisomy; Alzheimer's disease