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1.  Function, therapeutic potential and cell biology of BACE proteases: current status and future prospects 
Journal of neurochemistry  2014;130(1):4-28.
The β-site APP cleaving enzymes 1 and 2 (BACE1 and BACE2) were initially identified as transmembrane aspartyl proteases cleaving the amyloid precursor protein (APP). BACE1 is a major drug target for Alzheimer’s disease because BACE1-mediated cleavage of APP is the first step in the generation of the pathogenic amyloid-β peptides. BACE1, which is highly expressed in the nervous system, is also required for myelination by cleaving neuregulin 1. Several recent proteomic and in vivo studies usingBACE1-andBACE2-deficient mice demonstrate a much wider range of physiological substrates and functions for both proteases within and outside of the nervous system. For BACE1 this includes axon guidance, neurogenesis, muscle spindle formation, and neuronal network functions, whereas BACE2 was shown to be involved in pigmentation and pancreatic β-cell function. This review highlights the recent progress in understanding cell biology, substrates, and functions of BACE proteases and discusses the therapeutic options and potential mechanism-based liabilities, in particular for BACE inhibitors in Alzheimer’s disease.
PMCID: PMC4086641  PMID: 24646365
Alzheimer’s disease; BACE1; BACE2; protease; regulated intramembrane proteolysis; secretase
2.  Postsynaptic Target Specific Synaptic Dysfunctions in the CA3 Area of BACE1 Knockout Mice 
PLoS ONE  2014;9(3):e92279.
Beta-amyloid precursor protein cleaving enzyme 1 (BACE1), a major neuronal β-secretase critical for the formation of β-amyloid (Aβ) peptide, is considered one of the key therapeutic targets that can prevent the progression of Alzheimer’s disease (AD). Although a complete ablation of BACE1 gene prevents Aβ formation, we previously reported that BACE1 knockouts (KOs) display presynaptic deficits, especially at the mossy fiber (MF) to CA3 synapses. Whether the defect is specific to certain inputs or postsynaptic targets in CA3 is unknown. To determine this, we performed whole-cell recording from pyramidal cells (PYR) and the stratum lucidum (SL) interneurons in the CA3, both of which receive excitatory MF terminals with high levels of BACE1 expression. BACE1 KOs displayed an enhancement of paired-pulse facilitation at the MF inputs to CA3 PYRs without changes at the MF inputs to SL interneurons, which suggests postsynaptic target specific regulation. The synaptic dysfunction in CA3 PYRs was not restricted to excitatory synapses, as seen by an increase in the paired-pulse ratio of evoked inhibitory postsynaptic currents from SL to CA3 PYRs. In addition to the changes in evoked synaptic transmission, BACE1 KOs displayed a reduction in the frequency of miniature excitatory and inhibitory postsynaptic currents (mEPSCs and mIPSCs) in CA3 PYRs without alteration in mEPSCs recorded from SL interneurons. This suggests that the impairment may be more global across diverse inputs to CA3 PYRs. Our results indicate that the synaptic dysfunctions seen in BACE1 KOs are specific to the postsynaptic target, the CA3 PYRs, independent of the input type.
PMCID: PMC3956924  PMID: 24637500
3.  BACE1, a Major Determinant of Selective Vulnerability of the Brain to Amyloid-β Amyloidogenesis, is Essential for Cognitive, Emotional, and Synaptic Functions 
A transmembrane aspartyl protease termed β-site APP cleavage enzyme 1 (BACE1) that cleaves the amyloid-β precursor protein (APP), which is abundant in neurons, is required for the generation of amyloid-β (Aβ) peptides implicated in the pathogenesis of Alzheimer’s disease (AD). We now demonstrate that BACE1, enriched in neurons of the CNS, is a major determinant that predisposes the brain to Aβ amyloidogenesis. The physiologically high levels of BACE1 activity coupled with low levels of BACE2 and α-secretase anti-amyloidogenic activities in neurons is a major contributor to the accumulation of Aβ in the CNS, whereas other organs are spared. Significantly, deletion of BACE1 in APPswe;PS1ΔE9 mice prevents both Aβ deposition and age-associated cognitive abnormalities that occur in this model of Aβ amyloidosis. Moreover, Aβ deposits are sensitive to BACE1 dosage and can be efficiently cleared from the CNS when BACE1 is silenced. However, BACE1 null mice manifest alterations in hippocampal synaptic plasticity as well as in performance on tests of cognition and emotion. Importantly, memory deficits but not emotional alterations in BACE1-/- mice are prevented by coexpressing APPswe;PS1ΔE9 transgenes, indicating that other potential substrates of BACE1 may affect neural circuits related to emotion. Our results establish BACE1 and APP processing pathways as critical for cognitive, emotional, and synaptic functions, and future studies should be alert to potential mechanism-based side effects that may occur with BACE1 inhibitors designed to ameliorate Aβ amyloidosis in AD.
PMCID: PMC2564291  PMID: 16354928
BACE1 null mice; selective vulnerability; Aβ amyloidosis; Alzheimer’s; cognition; synaptic plasticity; RNAi
5.  Treatment with bexarotene, a compound that increases apolipoprotein-E, provides no cognitive benefit in mutant APP/PS1 mice 
Though the precise cause(s) of Alzheimer’s disease (AD) remain unknown, there is strong evidence that decreased clearance of β-amyloid (Aβ) from the brain can contribute to the disease. Therapeutic strategies to promote natural Aβ clearance mechanisms, such as the protein apolipoprotein-E (APOE), hold promise for the treatment of AD. The amount of APOE in the brain is regulated by nuclear receptors including retinoid X receptors (RXRs). Drugs that activate RXRs, including bexarotene, can increase APOE and ABCA1 production, and have been shown to decrease the Aβ burden and improve cognition in mouse models of Aβ amyloidosis. Although recent bexarotene studies failed to replicate the rapid clearance of Aβ from brains, behavioral and cognitive effects of this compound remain controversial.
In efforts to clarify these behavioral findings, mutant APP/PS1 mice were acutely dosed with bexarotene. While ABCA1 was upregulated in mutant APP/PS1 mice treated with bexarotene, this drug failed to attenuate Aβ plaques or cognitive deficits in these mice.
We recommend rigorous preclinical study to evaluate the mechanism and utility of such a compound for AD therapy.
PMCID: PMC3693923  PMID: 23764200
Bexarotene; Alzheimer’s disease; Mouse model; RXR agonist; APOE; Cognition
6.  Rodent models of TDP-43: Recent advances 
Brain research  2012;1462:26-39.
Recently, missense mutations in the gene TARDBP encoding TDP-43 have been linked to familial ALS. The discovery of genes encoding these RNA binding proteins, such as TDP-43 and FUS/TLS, raised the notion that altered RNA metabolism is a major factor underlying the pathogenesis of ALS. To begin to unravel how mutations in TDP-43 cause dysfunction and death of motor neurons, investigators have employed both gain- and loss-of-function studies in rodent model systems. Here, we will summarize major findings from the initial sets of TDP-43 transgenic and knockout rodent models, identify their limitations, and point to future directions toward clarification of disease mechanism(s) and testing of therapeutic strategies that ultimately may lead to novel therapy for this devastating disease.
PMCID: PMC3613131  PMID: 22608070
TDP-43; Transgenic; Conditional knockout
7.  Specific domains in anterior pharynx-defective 1 determine its intramembrane interactions with nicastrin and presenilin 
Neurobiology of aging  2010;33(2):277-285.
γ-Secretase, a multi-subunit transmembrane protease comprised of presenilin, nicastrin, presenilin enhancer 2, and anterior pharynx-defective 1, participates in the regulated intramembrane proteolysis of Type I membrane proteins including the amyloid precursor protein (APP). Although Aph-1 is thought to play a structural role in the assembly of γ-secretase complex and several transmembrane domains (TMDs) of Aph-1 have been shown to be critical for its function, the importance of the other domains of Aph-1 remains elusive. We screened a series of Aph-1 mutants and focused on 9 mutations distributed in 6 different TMDs of human APH-1aS, assessing their ability to complement mouse embryonic fibroblasts lacking Aph-1. We showed that mutations in TMD4 (G126) and TMD5 (H171) of Aph-1a prevented the formation of the Nct/Aph-1 subcomplex. Importantly, although mutations in TMD3 (Q83/E84/R85) and TMD6 (H197) of APH-1aS did not affect Nct/Aph-1 subcomplex formation, both mutations prevented further association/endoproteolysis of PS1. We propose a model that identifies critical TMDs of Aph-1 for associations with Nct and PS for the stepwise assembly of γ-secretase components.
PMCID: PMC2904414  PMID: 20382452
γ-Secretase; Aph-1; Nct; PS; mutagenesis; transmembrane domain
8.  Arc/Arg3.1 Regulates an Endosomal Pathway Essential for Activity-Dependent β-Amyloid Generation 
Cell  2011;147(3):615-628.
Assemblies of β-amyloid (Aβ) peptides are pathological mediators of Alzheimer's Disease (AD) and are produced by the sequential cleavages of amyloid precursor protein (APP) by β-secretase (BACE1) and γ-secretase. The generation of Aβ is coupled to neuronal activity, however the molecular basis is unknown. Here, we report that the immediate early gene Arc is required for activity-dependent generation of Aβ. Arc is a postsynaptic protein that recruits endophilin2/3 and dynamin to early/recycling endosomes that traffic AMPA receptors to reduce synaptic strength in both Hebbian and non-Hebbian forms of plasticity. The Arc-endosome also traffics APP and BACE1, and Arc physically associates with presenilin1 (PS1) to regulate γ-secretase trafficking and confer activity-dependence. Genetic deletion of Arc reduces Aβ load in a transgenic mouse model of AD. In concert with the finding that patients with AD can express anomalously high levels of Arc, we hypothesize that Arc participates in the pathogenesis of AD.
PMCID: PMC3207263  PMID: 22036569
9.  Reduced BACE1 activity enhances clearance of myelin debris and regeneration of axons in the injured peripheral nervous system 
The Journal of Neuroscience  2011;31(15):5744-5754.
β- site amyloid precursor protein (APP) cleaving enzyme 1 (BACE1) is an aspartyl protease best known for its role in generating the amyloid β peptides that are present in plaques of Alzheimer's Disease. BACE1 has been an attractive target for drug development. In cultured embryonic neurons BACE1-cleaved N-terminal APP is further processed to generate a fragment that can trigger axonal degeneration, suggesting a vital role for BACE1 in axonal health. In addition, BACE1 cleaves neuregulin 1 type III, a protein critical for myelination of peripheral axons by Schwann cells during development. Here, we asked if axonal degeneration or axonal regeneration in adult nerves might be affected by inhibition or elimination of BACE1. We report that BACE1 knockout and wild-type nerves degenerated at a similar rate after axotomy and to a similar extent in the experimental neuropathies produced by administration of paclitaxel and acrylamide. These data indicate N-APP is not the sole culprit in axonal degeneration in adult nerves. Unexpectedly, however, we observed that BACE1 knockout mice had markedly enhanced clearance of axonal and myelin debris from degenerated fibers, accelerated axonal regeneration, and earlier reinnervation of neuromuscular junctions, compared to littermate controls. These observations were reproduced in part by pharmacological inhibition of BACE1. These data suggest BACE1 inhibition as a therapeutic approach to accelerate regeneration and recovery after peripheral nerve damage.
PMCID: PMC3302726  PMID: 21490216
10.  Increased Expression of PS1 Is Sufficient to Elevate the Level and Activity of γ-Secretase In Vivo 
PLoS ONE  2011;6(11):e28179.
Increase in the generation and deposition of amyloid-β (Aβ) plays a central role in the development of Alzheimer's Disease (AD). Elevation of the activity of γ-secretase, a key enzyme required for the generation for Aβ, can thus be a potential risk factor in AD. However, it is not known whether γ-secretase can be upregulated in vivo. While in vitro studies showed that expression of all four components of γ-secretase (Nicastrin, Presenilin, Pen-2 and Aph-1) are required for upregulation of γ-secretase, it remains to be established as to whether this is true in vivo. To investigate whether overexpressing a single component of the γ-secretase complex is sufficient to elevate its level and activity in the brain, we analyzed transgenic mice expressing either wild type or familial AD (fAD) associated mutant PS1. In contrast to cell culture studies, overexpression of either wild type or mutant PS1 is sufficient to increase levels of Nicastrin and Pen-2, and elevate the level of active γ-secretase complex, enzymatic activity of γ-secretase and the deposition of Aβ in brains of mice. Importantly, γ-secretase comprised of mutant PS1 is less active than that of wild type PS1-containing γ-secretase; however, γ-secretase comprised of mutant PS1 cleaves at the Aβ42 site of APP-CTFs more efficiently than at the Aβ40 site, resulting in greater accumulation of Aβ deposits in the brain. Our data suggest that whereas fAD-linked PS1 mutants cause early onset disease, upregulation of PS1/γ-secretase activity may be a risk factor for late onset sporadic AD.
PMCID: PMC3226664  PMID: 22140537
11.  Amyloid Precursor Protein Processing and Alzheimer’s Disease 
Annual review of neuroscience  2011;34:185-204.
Alzheimer’s disease (AD), the leading cause of dementia worldwide, is characterized by the accumulation of the β-amyloid peptide (Aβ) within the brain along with hyperphosphorylated and cleaved forms of the microtubule-associated protein tau. Genetic, biochemical, and behavioral research suggest that physiologic generation of the neurotoxic Aβ peptide from sequential amyloid precursor protein (APP) proteolysis is the crucial step in the development of AD. APP is a single-pass transmembrane protein expressed at high levels in the brain and metabolized in a rapid and highly complex fashion by a series of sequential proteases, including the intramembranous γ-secretase complex, which also process other key regulatory molecules. Why Aβ accumulates in the brains of elderly individuals is unclear but could relate to changes in APP metabolism or Aβ elimination. Lessons learned from biochemical and genetic studies of APP processing will be crucial to the development of therapeutic targets to treat AD.
PMCID: PMC3174086  PMID: 21456963
Neurodegeneration; dementia; BACE1; α-secretase; γ-secretase; aging
12.  Selectivity to amyloid-β precursor protein cleavage provides hope against Alzheimer's 
Toward development of a safe and effective treatment for Alzheimer's disease, Elan Pharmaceuticals reported a novel γ-secretase inhibitor that specifically targets the cleavage of amyloid-β precursor protein, opening the way to design of substrate-specific γ-secretase inhibitors that would reduce the amyloid burden without significant adverse events.
PMCID: PMC3226269  PMID: 21418547
13.  Mossy fiber LTP deficits in BACE1 knockouts can be rescued by activation of α7 nicotinic acetylcholine receptors 
Beta-site amyloid precursor protein cleaving enzyme 1 (BACE1)–the neuronal β-secretase responsible for producing β-amyloid (Aβ) peptides–emerged as one of the key therapeutic targets of Alzheimer's disease (AD). Although complete ablation of the BACE1 gene prevents Aβ formation, we reported that BACE1 knockout mice display severe presynaptic deficits at mossy fiber (MF) to CA3 synapses in the hippocampus, a major locus of BACE1 expression. We also found that the deficits are likely due to abnormal presynaptic Ca2+ regulation. Cholinergic system has been implicated in AD, in some cases involving Ca2+-permeable α7-nicotinic acetylcholine receptors (nAChRs). Here we report that brief application of nicotine, via α7-nAChRs, can restore mossy fiber LTP (mfLTP) in BACE1 knockouts. Our data suggest that activating α7-nAChRs can recover the presynaptic deficits in BACE1 knockouts.
PMCID: PMC3137882  PMID: 20943921
long-term potentiation; presynaptic; paired-pulse facilitation; beta-secretase; Alzheimer's disease; α7-nAchR
14.  An Overview of APP Processing Enzymes and Products 
Neuromolecular medicine  2010;12(1):1-12.
The generation of amyloid β-peptide (Aβ) by enzymatic cleavages of the β-amyloid precursor protein (APP) has been at the center of Alzheimer’s disease (AD) research. While the basic process of β- and γ-secretase-mediated generation of Aβ is text book knowledge, new aspects of Aβ and other cleavage products have emerged in recent years. Also our understanding of the enzymes involved in APP proteolysis has increased dramatically. All of these discoveries contribute to a more complete understanding of APP processing and the physiological and pathological roles of its secreted and intracellular protein products. Understanding APP processing is important for any therapeutic strategy aimed at reducing Aβ levels in AD. In this review we provide a concise description of the current state of understanding the enzymes involved in APP processing, the cleavage products generated by different processing patterns, and the potential functions of those cleavage products.
PMCID: PMC2889200  PMID: 20232515
Amyloid beta; α-secretase; β-secretase; γ-secretase; APP; AICD
15.  The β-Secretase Enzyme BACE in Health and Alzheimer's Disease: Regulation, Cell Biology, Function, and Therapeutic Potential 
The β-amyloid (Aβ) peptide is the major constituent of amyloid plaques in Alzheimer's disease (AD) brain and is likely to play a central role in the pathogenesis of this devastating neurodegenerative disorder. The β-secretase, β-site amyloid precursor protein cleaving enzyme (BACE1; also called Asp2, memapsin 2), is the enzyme responsible for initiating Aβ generation. Thus, BACE is a prime drug target for the therapeutic inhibition of Aβ production in AD. Since its discovery 10 years ago, much has been learned about BACE. This review summarizes BACE properties, describes BACE translation dysregulation in AD, and discusses BACE physiological functions in sodium current, synaptic transmission, myelination, and schizophrenia. The therapeutic potential of BACE will also be considered. This is a summary of topics covered at a symposium held at the 39th annual meeting of the Society for Neuroscience and is not meant to be a comprehensive review of BACE.
PMCID: PMC2879048  PMID: 19828790
16.  Modeling an Anti-Amyloid Combination Therapy for Alzheimer's Disease 
Science translational medicine  2010;2(13):13ra1.
As only symptomatic treatments are now available for Alzheimer's disease (AD), safe and effective mechanism-based therapies remain a great unmet need for patients with this neurodegenerative disease. Although γ-secretase and BACE1 [β-site β-amyloid (Aβ) precursor protein (APP) cleaving enzyme 1] are well-recognized therapeutic targets for AD, untoward side effects associated with strong inhibition or reductions in amounts of these aspartyl proteases have raised concerns regarding their therapeutic potential. Although moderate decreases of either γ-secretase or BACE1 are not associated with mechanism-based toxicities, they provide only modest benefits in reducing Aβ in the brains of APPswe/PS1ΔE9 mice. Because the processing of APP to generate Aβ requires both γ-secretase and BACE1, it is possible that moderate reductions of both enzymes would provide additive and significant protection against Aβ amyloidosis. Here, we test this hypothesis and assess the value of this novel anti-amyloid combination therapy in mutant mice. We demonstrate that genetic reductions of both BACE1 and γ-secretase additively attenuate the amyloid burden and ameliorate cognitive deficits occurring in aged APPswe/PS1ΔE9 animals. No evidence of mechanism-based toxicities was associated with such decreases in amounts of both enzymes. Thus, we propose that targeting both γ-secretase and BACE1 may be an effective and safe treatment strategy for AD.
PMCID: PMC2852193  PMID: 20371462
17.  Beta-amyloid precursor protein cleavage enzyme 1 (BACE1) knockouts display deficits in activity-dependent potentiation of synaptic transmission at mossy fiber to CA3 synapses in the hippocampus 
Beta-amyloid precursor protein cleavage enzyme 1 (BACE1) has been identified as a major neuronal β-secretase critical for the formation of β-amyloid (Aβ) peptide, which is thought responsible for the pathology of Alzheimer’s disease (AD). Therefore, BACE1 is one of the key therapeutic targets that can prevent the progression of AD. Previous studies showed that knocking out the BACE1 gene prevents Aβ formation, but results in behavioral deficits and specific synaptic dysfunctions at Schaffer collateral to CA1 synapses. However, BACE1 protein is most highly expressed at the mossy fiber projections in CA3. Here we report that BACE1 knockout mice display reduced presynaptic function, as measured by an increase in paired-pulse facilitation ratio. More dramatically, mossy fiber LTP, which is normally expressed via an increase in presynaptic release, was eliminated in the knockouts. While LTD was slightly larger in the BACE1 knockouts, it could not be reversed. The specific deficit in mossy fiber LTP was upstream of cAMP signaling, and could be “rescued” by transiently elevating extracellular Ca2+ concentration. These results suggest that BACE1 may play a critical role in regulating presynaptic function, especially activity-dependent strengthening of presynaptic release, at mossy fiber synapses.
PMCID: PMC2728626  PMID: 18753368
long-term potentiation; long-term depression; presynaptic; paired-pulse facilitation; beta-secretase; Alzheimer’s disease
18.  APH1 Polar Transmembrane Residues Regulate the Assembly and Activity of Presenilin Complexes* 
The Journal of Biological Chemistry  2009;284(24):16298-16307.
Complexes involved in the γ/ϵ-secretase-regulated intramembranous proteolysis of substrates such as the amyloid-β precursor protein are composed primarily of presenilin (PS1 or PS2), nicastrin, anterior pharynx defective-1 (APH1), and PEN2. The presenilin aspartyl residues form the catalytic site, and similar potentially functional polar transmembrane residues in APH1 have been identified. Substitution of charged (E84A, R87A) or polar (Q83A) residues in TM3 had no effect on complex assembly or activity. In contrast, changes to either of two highly conserved histidines (H171A, H197A) located in TM5 and TM6 negatively affected PS1 cleavage and altered binding to other secretase components, resulting in decreased amyloid generating activity. Charge replacement with His-to-Lys substitutions rescued nicastrin maturation and PS1 endoproteolysis leading to assembly of the formation of structurally normal but proteolytically inactive γ-secretase complexes. Substitution with a negatively charged side chain (His-to-Asp) or altering the structural location of the histidines also disrupted γ-secretase binding and abolished functionality of APH1. These results suggest that the conserved transmembrane histidine residues contribute to APH1 function and can affect presenilin catalytic activity.
PMCID: PMC2713549  PMID: 19369254
19.  Progressive Behavioral Deficits in DJ-1 Deficient Mice are Associated with Normal Nigrostriatal Function 
Neurobiology of disease  2007;29(3):505-514.
Loss-of-function mutations in the DJ-1 gene account for an autosomal recessive form of Parkinson’s disease (PD). To investigate the physiological functions of DJ-1 in vivo, we generated DJ-1 knockout (DJ-1-/-) mice. Younger (< 1year) DJ-1 -/- mice were hypoactive and had mild gait abnormalities. Older DJ-1-/-, however, showed decreased bodyweight and grip strength, and more severe gait irregularities compared to wild-type littermates. The basal level of extracellular dopamine, evoked dopamine release and dopamine receptor D2 sensitivity appeared normal in the striatum of DJ-1-/- mice, which was consistent with similar results between DJ-1-/- and controls in behavioral paradigms specific for the dopaminergic system. An examination of spinal cord, nerve and muscle tissues failed to identify any pathological changes that were consistent with the noted motor deficits. Taken together, our findings suggest that loss of DJ-1 leads to progressive behavioral changes without significant alterations in nigrostriatal dopaminergic and spinal motor systems.
PMCID: PMC2271119  PMID: 18187333
DJ-1; knockout mouse; Parkinson’s disease; dopamine; striatum; spinal cord; muscle; motor behavior
20.  Amyotrophic Lateral Sclerosis 2-Deficiency Leads to Neuronal Degeneration in Amyotrophic Lateral Sclerosis through Altered AMPA Receptor Trafficking 
Amyotrophic lateral sclerosis (ALS), the most common adult-onset motor neuron disease is caused by a selective loss of motor neurons. One form of juvenile onset autosomal recessive ALS (ALS2) has been linked to the loss of function of the ALS2 gene. The pathogenic mechanism of ALS2-deficiency, however, remains unclear. To further understand the function of alsin that is encoded by the full-length ALS2 gene, we screened proteins interacting with alsin. Here, we report that alsin interacted with glutamate receptor interacting protein 1 (GRIP1) both in vitro and in vivo, and colocalized with GRIP1 in neurons. In support of the physiological interaction between alsin and GRIP1, the subcellular distribution of GRIP1 was altered in ALS2-/- spinal motor neurons, which correlates with a significant reduction of AMPA-type glutamate receptor subunit 2 (GluR2) at the synaptic/cell surface of ALS2-/- neurons. The decrease of calcium-impermeable GluR2-containing AMPA receptors at the cell/synaptic surface rendered ALS2-/- neurons more susceptible to glutamate receptor-mediated neurotoxicity. Our findings reveal a novel function of alsin in AMPA receptor trafficking and provide a novel pathogenic link between ALS2-deficiency and motor neuron degeneration, suggesting a protective role of alsin in maintaining the survival of motor neurons.
PMCID: PMC2556290  PMID: 17093100
ALS2; knock-out mouse; motor neuron; GRIP1; AMPA receptor; excitotoxicity
21.  Messenger RNA Oxidation Occurs Early in Disease Pathogenesis and Promotes Motor Neuron Degeneration in ALS 
PLoS ONE  2008;3(8):e2849.
Accumulating evidence indicates that RNA oxidation is involved in a wide variety of neurological diseases and may be associated with neuronal deterioration during the process of neurodegeneration. However, previous studies were done in postmortem tissues or cultured neurons. Here, we used transgenic mice to demonstrate the role of RNA oxidation in the process of neurodegeneration.
Methodology/Principal Findings
We demonstrated that messenger RNA (mRNA) oxidation is a common feature in amyotrophic lateral sclerosis (ALS) patients as well as in many different transgenic mice expressing familial ALS-linked mutant copper-zinc superoxide dismutase (SOD1). In mutant SOD1 mice, increased mRNA oxidation primarily occurs in the motor neurons and oligodendrocytes of the spinal cord at an early, pre-symptomatic stage. Identification of oxidized mRNA species revealed that some species are more vulnerable to oxidative damage, and importantly, many oxidized mRNA species have been implicated in the pathogenesis of ALS. Oxidative modification of mRNA causes reduced protein expression. Reduced mRNA oxidation by vitamin E restores protein expression and partially protects motor neurons.
These findings suggest that mRNA oxidation is an early event associated with motor neuron deterioration in ALS, and may be also a common early event preceding neuron degeneration in other neurological diseases.
PMCID: PMC2481395  PMID: 18682740
22.  Loss of ALS2 Function Is Insufficient to Trigger Motor Neuron Degeneration in Knock-Out Mice But Predisposes Neurons to Oxidative Stress 
Amyotrophic lateral sclerosis (ALS), the most common motor neuron disease, is caused by a selective loss of motor neurons in the CNS. Mutations in the ALS2 gene have been linked to one form of autosomal recessive juvenile onset ALS (ALS2). To investigate the pathogenic mechanisms of ALS2, we generated ALS2 knock-out (ALS2−/−) mice. Although ALS2−/− mice lacked obvious developmental abnormalities, they exhibited age-dependent deficits in motor coordination and motor learning. Moreover, ALS2−/− mice showed a higher anxiety response in the open-field and elevated plus-maze tasks. Although they failed to recapitulate clinical or neuropathological phenotypes consistent with motor neuron disease by 20 months of age, ALS2−/− mice or primary cultured neurons derived from these mice were more susceptible to oxidative stress compared with wild-type controls. These observations suggest that loss of ALS2 function is insufficient to cause major motor deficits or motor neuron degeneration in a mouse model but predisposes neurons to oxidative stress.
PMCID: PMC2364727  PMID: 16107644
ALS2; knock-out mouse; motor neuron; motor coordination; motor learning; oxidative stress
23.  Nicastrin Is Critical for Stability and Trafficking but Not Association of Other Presenilin/γ-Secretase Components* 
The Journal of biological chemistry  2005;280(17):17020-17026.
γ-Secretase, which is responsible for the intramembranous cleavage of Alzheimer β-amyloid precursor protein and the signaling receptor Notch, is a multiprotein complex consisting of at least four components: presenilin (PS); nicastrin (Nct); APH-1 (anterior pharynx-defective-1); and presenilin enhancer-2 (PEN-2). Presenilin 1 (PS1) is known to be essential for the stability, interaction, and trafficking of the other PS1/γ-secretase components. However, the precise functions of the other components remain elusive. Here, we investigated the functions of Nct within the PS1/γ-secretase complex. We demonstrated that the loss of Nct expression in the embryonic fibroblast cells (Nct KO cells) results in dramatically decreased levels of APH-1, PEN-2, and PS1 fragments accompanied by a significant accumulation of full-length PS1. In the absence of Nct, PEN-2 and full-length PS1 are subjected to proteasome-mediated degradation, whereas the degradation of APH-1 is mediated by both proteasomal and lysosomal pathways. Unlike the case of wild type cells in which the γ-secretase complex mainly locates in the trans-Golgi network, the majority of residual PEN-2, APH-1, and the uncleaved full-length PS1 in Nct KO cells reside in the endoplasmic reticulum, which remain associated with each other in the absence of Nct. Interestingly, significant amounts of full-length PS1 and PEN-2, but not APH-1, are detected on the plasma membrane in Nct KO cells, suggesting the Nct-independent cell surface delivery of the PEN-2·PS1. Finally, the diminished PEN-2 protein level in Nct-deficient cells can be partially restored by overexpression of exogenous PS1, APH-1, or PEN-2 individually or collectively, indicating a dispensable role for Nct in controlling PEN-2 level. Taken together, our study demonstrates a critical role of Nct in the stability and proper intracellular trafficking of other components of the PS1/γ-secretase complex but not in maintaining the association of PEN-2, APH-1, and full-length PS1.
PMCID: PMC1201533  PMID: 15711015
24.  Association of γ-Secretase with Lipid Rafts in Post-Golgi and Endosome Membranes* 
The Journal of biological chemistry  2004;279(43):44945-44954.
Alzheimer’s disease-associated β-amyloid peptides (Aβ) are generated by the sequential proteolytic processing of amyloid precursor protein (APP) by β- and γ-secretases. There is growing evidence that cholesterol- and sphingolipid-rich membrane microdomains are involved in regulating trafficking and processing of APP. BACE1, the major γ-secretase in neurons is a palmi-toylated transmembrane protein that resides in lipid rafts. A subset of APP is subject to amyloidogenic processing by BACE1 in lipid rafts, and this process depends on the integrity of lipid rafts. Here we describe the association of all four components of the γ-secretase complex, namely presenilin 1 (PS1)-derived fragments, mature nicastrin, APH-1, and PEN-2, with cholesterol-rich detergent insoluble membrane (DIM) domains of non-neuronal cells and neurons that fulfill the criteria of lipid rafts. In PS1−/−/PS2−/− and NCT−/− fibroblasts, γ-secretase components that still remain fail to become detergent-resistant, suggesting that raft association requires γ-secretase complex assembly. Biochemical evidence shows that subunits of the γ-secretase complex and three TGN/endosome-resident SNAREs cofractionate in sucrose density gradients, and show similar solubility or insolubility characteristics in distinct non-ionic and zwitterionic detergents, indicative of their co-residence in membrane microdomains with similar protein-lipid composition. This notion is confirmed using magnetic immunoisolation of PS1- or syntaxin 6-positive membrane patches from a mixture of membranes with similar buoyant densities following Lubrol WX extraction or sonication, and gradient centrifugation. These findings are consistent with the localization of γ-secretase in lipid raft microdomains of post-Golgi and endosomes, organelles previously implicated in amyloidogenic processing of APP.
PMCID: PMC1201506  PMID: 15322084

Results 1-24 (24)