A673T Mutation Research Articles

The Icelandic Mutation (APP-A673T) Is Protective against Amyloid Pathology In Vivo.

A previous epidemiological study in Northern Europe showed that the A673T mutation (Icelandic mutation) in the amyloid precursor protein gene (APP) can protect against Alzheimer's disease (AD). While the effect of the A673T mutation on APP processing has been investigated primarily in vitro, its in vivo impact has not been evaluated. This is mainly because most existing AD mouse models carry the Swedish mutation. The Swedish and Icelandic mutations are both located near the β-cleavage site, and each mutation is presumed to have the opposite effect on β-cleavage. Therefore, in the AD mouse models with the Swedish mutation, its effects could compete with the effects of the Icelandic mutation. Here, we introduced the A673T mutation into App knock-in mice devoid of the Swedish mutation (AppG-F mice) to avoid potential deleterious effects of the Swedish mutation and generated AppG-F-A673T mice. APP-A673T significantly downregulated β-cleavage and attenuated the production of Aβ and amyloid pathology in the brains of these animals. The Icelandic mutation also reduced neuroinflammation and neuritic alterations. Both sexes were studied. This is the first successful demonstration of the protective effect of the Icelandic mutation on amyloid pathology in vivo. Our findings indicate that specific inhibition of the APP-BACE1 interaction could be a promising therapeutic approach. Alternatively, the introduction of the disease-protective mutation such as APP-A673T using in vivo genome editing technology could be a novel treatment for individuals at high risk for AD, such as familial AD gene mutation carriers and APOE ε4 carriers.

Precise and efficient insertion of A673T mutation in APP gene using MSYM

Precise and efficient insertion of A673T mutation in APP gene using MSYM

Exploring the Involvement of the Amyloid Precursor Protein A673T Mutation against Amyloid Pathology and Alzheimer's Disease in Relation to Therapeutic Editing Tools.

Alzheimer’s disease (AD) is biologically defined as a complex neurodegenerative condition with a multilayered nature that leads to a progressive decline in cognitive function and irreversible neuronal loss. It is one of the primary diseases among elderly individuals. With an increasing incidence and a high failure rate for pharmaceutical options that are merely symptom-targeting and supportive with many side effects, there is an urgent need for alternative strategies. Despite extensive knowledge on the molecular basis of AD, progress concerning effective disease-modifying therapies has proven to be a challenge. The ability of the CRISPR–Cas9 gene editing system to help identify target molecules or to generate new preclinical disease models could shed light on the pathogenesis of AD and provide promising therapeutic possibilities. Here, we sought to highlight the current understanding of the involvement of the A673T mutation in amyloid pathology, focusing on its roles in protective mechanisms against AD, in relation to the recent status of available therapeutic editing tools.

Insertion of the Icelandic Mutation (A673T) by Prime Editing: A Potential Preventive Treatment for Familial and Sporadic Alzheimer's Disease.

Alzheimer's disease (AD) is the result of abnormal processing of the amyloid precursor protein (APP) by β-secretase and γ-secretase, which leads to the formation of toxic β-amyloid peptides. The toxic β-amyloid peptides induce neuron death, memory problems, and AD development. Several APP mutations increase the risk of developing early-onset AD. However, the A673T mutation identified in the Icelandic population prevents AD development by reducing the cleavage of APP by β-secretase. In this study, we inserted the A673T mutation in human cells using the CRISPR prime editing (PE) technique. Repeated PE treatments resulted in the insertion of the A673T mutation in up to 49.2% of APP genes when a second nick was induced in the other DNA strand. When the protospacer adjacent motif used for PE was also mutated, up to 68.9% of the APP genes contained the protective A673T mutation. PE is a promising approach to introduce the A673T mutation precisely without mutating nearby nucleotides.

Insertion of the protective APP A673T mutation by CRISPR/Cas9 base editing or PRIME editing.

There is currently no treatment for Alzheimer disease (AD). However, the Icelandic mutation in the APP gene (A673T) has been shown to confer a protection against the onset and development of AD (Jonsson et al. Nature 2012). This single nucleotide mutation in APP exon 16 reduces the cleavage of the APP protein by the beta-secretase by 40% thus preventing the development of AD even in persons more than 95 years old. Our research group has initially shown that the presence of the A673T mutation in an APP gene reduced the secretion of beta-amyloid peptides even if there is also a FAD mutation in the gene. This is the case for 14 different FAD mutations. We have used CRISPR/Cas9 base editing and PRIME editing technologies to insert the A673T mutation in the APP gene. We have compared several different cytidine base editor complexes to achieve the most effective and accurate genome modification possible in HEK293T cells and in SH-SY5Y neuroblastomas. The insertion of the A673T mutation in cells containing the London mutation reduced the secretion of beta-amyloid peptides. We are currently using lentiviral vectors to infect neurons from a mouse model and human neurons induced from fibroblasts of a patient with the London mutation. The insertion of the protective Icelandic mutation in the APP gene using these editing technologies opens a new potential therapeutic avenue not only for Familial Alzheimer’s diseases but also for sporadic Alzheimer’s disease.

Insertion of the Icelandic mutation (A673T) in the APP gene using the CRISPR/Cas9 base editing and Prime editing technologies, a preventive treatment for Alzheimer?

AbstractBackgroundThere is currently no treatment for Alzheimer disease (AD). However, the Icelandic mutation in the APP gene (A673T) has been shown to confer a protection against the onset and development of AD (Jonsson et al. Nature 2012). This single nucleotide mutation in APP exon 16 reduces the cleavage of the APP protein by the beta‐secretase by 40% thus preventing the development of AD even in persons more than 95 years old.MethodOur research group has initially shown that the presence of the A673T mutation in an APP gene reduced the secretion of beta‐amyloid peptides even if there is also a FAD mutation in the gene. This is the case for 14 different FAD mutations.We have also used the CRISPR/Cas9 base editing technology to insert the A673T mutation in the APP gene. We have compared several different cytidine base editor complexes to achieve the most effective and accurate genome modification possible in HEK293T cells and in SH‐SY5Y neuroblastomas. The insertion of the A673T mutation in cells containing the London mutation reduced the secretion of beta‐amyloid peptides.ResultWe have more recently used the Prime editing technology to insert only the A673T mutation without inducing the mutation of other nearby nucleotides. Repeated Prime editing treatment permitted to insert this mutation in up to 64% of the APP gene in HEK293T cells.ConclusionThe insertion of the protective Icelandic mutation in the APP gene using these editing technologies opens a new potential preventive treatment for not only for Familial Alzheimer’s diseases but also for sporadic Alzheimer’s disease.

Development of a therapeutic approach for hereditary diseases with prime editing: A study on Alzheimer's disease

The amyloid precursor protein (APP) plays a role in the pathophysiology of Alzheimer's disease. Excess cleavage by β-secretase enzyme leads to the accumulation of β-amyloid peptides. In contrast, a rare variant of the APP gene (A673T) decreases cleavage by β-secretase. However, inserting this mutation with gene editing technology is a challenge. Our hypothesis is that prime editing allows a significant editing rate while inducing few off-target mutations in the cell leading to a significant reduction in the amount of pathological peptides produced. The objective of this study is to achieve a permanent insertion of the A673T mutation by prime editing and obtain evidence of the reduced Aβ peptides accumulation in vitro. Prime editing guide RNAs (pegRNA) were constructed according to different protospacer adjacent motifs (PAM) around the A673T mutation by varying the length of the primer binding site (PBS) and the reverse transcriptase template (RTT) sequence. Initially, one guide was used on the target gene (PE2). Afterwards, additional guides were used to favor DNA editing (PE3, PE3b). Different techniques were used to optimize editing, such as adding valproic acid, repeated transfections, modifying the PAM and the structure of the prime editor. The mutation was introduced in HEK 293T cells and later in neurons derived from fibroblasts of patients with Alzheimer's disease. The results were quantified by Sanger and deep sequencing. Prime editing demonstrated precise editing in up to 64% in HEK 293T cells (Figure 1) with no notable off-target mutations within the target window. In future experiments, the level of Aβ40-42 peptides will be measured in the derived neurons. Our approach aims to demonstrate the potential of prime editing in the development of a treatment for Alzheimer's disease based on the protective effect of A673T. Our project also aims to evaluate optimization methods for prime editing.

The Protective A673T Mutation of Amyloid Precursor Protein (APP) in Alzheimer's Disease.

Alzheimer's disease is a progressive neurodegenerative disorder characterized by extracellular amyloid beta peptides and neurofibrillary tangles consisted of intracellular hyperphosphorylated Tau in the hippocampus and cerebral cortex. Most of the mutations in key genes that code for amyloid precursor protein can lead to significant accumulation of these peptides in the brain and cause Alzheimer's disease. Moreover, some point mutations in amyloid precursor protein can cause familial Alzheimer's disease, such as Swedish mutation (KM670/671NL) and A673V mutation. However, recent studies have found that the A673T mutation in amyloid precursor protein gene can protect against Alzheimer's disease, even if it is located next to the Swedish mutation (KM670/671NL) and at the same site as A673V mutation, which are pathogenic. It makes us curious about the protective A673T mutation. Here, we summarize the most recent insights of A673T mutation, focus on their roles in protective mechanisms against Alzheimer's disease, and discuss their involvement in future treatment.

Base editing strategy for insertion of the A673T mutation in the APP gene to prevent the development of AD in vitro

The amyloid precursor protein (APP) is a transmembrane protein mostly found in neurons. Cleavage of this protein by β-secretase can lead to the formation of amyloid-β (Aβ) peptide plaque, which leads to Alzheimer’s disease. Genomic analysis of an Icelandic population that did not show symptoms of Alzheimer’s at an advanced age led to the discovery of the A673T mutation. This mutation can reduce β-secretase cleavage by 40%. We hypothesized that the insertion of this mutation in patients’ neurons could be an effective and sustainable method of slowing down or even stopping the progression of Alzheimer’s disease. We modified the APP gene in HEK293T cells and in SH-SY5Y neuroblastoma using a Cas9 nickase (Cas9n)-deaminase enzyme to convert the alanine codon to a threonine. Several Cas9n-deaminase variants were tested to compare their efficiency of conversion. The results were characterized and quantified by deep sequencing. We successfully introduced the A673T mutation in 53% of HEK293T cells alongside a new mutation (E674K), which seemed to further reduce Aβ peptide accumulation. Our approach aimed to provide a new strategy for the treatment of Alzheimer’s and in so doing, demonstrate the capacity of base editing techniques for treating genetic diseases.

The protective mutation A673T in amyloid precursor protein gene decreases Aβ peptides production for 14 forms of Familial Alzheimer's Disease in SH-SY5Y cells.

The deposition of Aβ plaques in the brain leads to the onset and development of Alzheimer's disease. The Amyloid precursor protein (APP) is cleaved by α-secretase (non-amyloidogenic processing of APP), however increased cleavage by β-secretase (BACE1) leads to the accumulation of Aβ peptides, which forms plaques. APP mutations mapping to exons 16 and 17 favor plaque accumulation and cause Familial Alzheimer Disease (FAD). However, a variant of the APP gene (A673T) originally found in an Icelandic population reduces BACE1 cleavage by 40%. A series of plasmids containing the APP gene, each with one of 29 different FAD mutations mapping to exon 16 and exon 17 was created. These plasmids were then replicated with the addition of the A673T mutation. Combined these formed the library of plasmids that was used in this study. The plasmids were transfected in neuroblastomas to assess the effect of this mutation on Aβ peptide production. The production of Aβ peptides was decreased for some FAD mutations due to the presence of the co-dominant A673T mutation. The reduction of Aβ peptide concentrations for the London mutation (V717I) even reached the same level as for A673T control in SH-SY5Y cells. These preliminary results suggest that the insertion of A673T in APP genes containing FAD mutations might confer a clinical benefit in preventing or delaying the onset of some FADs.

Optimization of in vitro gene delivery to neurons containing an APP gene with a mutation responsible for familial Alzheimer’s disease for the development of a base editing therapy

AbstractBackgroundThe Amyloïd Precursor Protein (APP) is preferentially cut by the α‐secretase enzyme in healthy individuals. However, an abnormal cleavage by β‐secretase leads to the accumulation of β‐amyloid peptides. Numerous APP genetic mutations cause Familial Alzheimer Disease (FAD). However, a rare APP gene variant (A673T) found in Icelanders drastically reduces cleavage by the β‐secretase. We hypothesized that the insertion of this A673T mutation in a patient’s genome using the CRISPR/Cas9 Base‐Editing Technology (Komor et al. Nature 2016) could be an effective method to slow down the progression of familial and sporadic forms of Alzheimer’s disease. The objective of this study is to eventually compare the efficiency by which a lentiviral or a dual‐AAV‐based vector could deliver a base‐editing complex (SpCas9VQR‐Target‐AID) into neurons in vitro.MethodNeurons were obtained using two methods. The first method involved differentiating fibroblasts from a FAD patient or a mouse model with a London (V717I) mutation into neurons (Shrigley et al. JoVE 2018) (Figure 1), while the second method involved isolating hippocampal neurons from prenatal NL/F/G mice (Seibenhener et al. JoVE 2012). The dual AAV complex was designed to deliver two parts of the base editor gene separated by inteins (Villiger et al. Nat Med 2018). Hippocampal or induced neurons were transduced with lentivirus or AAV1. Cas9 detection was performed by immunofluorescence and Western Blot. The β‐amyloid peptides in supernatant were characterized and quantified by Meso Scale Discovery’s Aβ kit.ResultWe have been able to detect the Cas9 protein in neurons by western blot and immunofluorescence (Figure 2). In subsequent experiments, we will determine which delivery method will reduce more the β‐amyloid peptide.ConclusionOur approach aims to confirm the protective effect of the A673T mutation against the development of familial and sporadic forms of Alzheimer’s disease as well as develop an efficient delivery method for the base editing complex which will lead to an in vivo application. Long‐term perspective: We aim to develop a completely new therapeutic approach for AD and FAD using base editing or the new PRIME editing technique (Anzalone et al. Nature 2019). This may eventually lead to a Phase I clinical trial.

Alzheimer's protection effect of A673T mutation may be driven by lower Aβ oligomer binding affinity.

Several mutations conferring protection against Alzheimer's disease (AD) have been described, none as profound as the A673T mutation, where carriers are four times less likely to get AD compared to noncarriers. This mutation results in reduced amyloid beta (Aβ) protein production in vitro and lower lifetime Aβ concentration in carriers. Better understanding of the protective mechanisms of the mutation may provide important insights into AD pathophysiology and identify productive therapeutic intervention strategies for disease modification. Aβ(1‐42) protein forms oligomers that bind saturably to a single receptor site on neuronal synapses, initiating the downstream toxicities observed in AD. Decreased formation, toxicity, or stability of soluble Aβ oligomers, or reduction of synaptic binding of these oligomers, may combine with overall lower Aβ concentration to underlie A673T’s disease protecting mechanism. To investigate these possibilities, we compared the formation rate of soluble oligomers made from Icelandic A673T mutant and wild type (wt) Aβ(1‐42) synthetic protein, the amount and intensity of oligomer bound to mature primary rat hippocampal/cortical neuronal synapses, and the potency of bound oligomers to impact trafficking rate in neurons in vitro using a physiologically relevant oligomer preparation method. At equal protein concentrations, mutant protein forms approximately 50% or fewer oligomers of high molecular weight (>50 kDa) compared to wt protein. Mutant oligomers are twice as potent at altering the cellular vesicle trafficking rate as wt at equivalent concentrations, however, mutant oligomers have a >4‐fold lower binding affinity to synaptic receptors (K d = 1,950 vs. 442 nM). The net effect of these differences is a lower overall toxicity at a given concentration. This study demonstrates for the first time that mutant A673T Aβ oligomers prepared with this method have fundamentally different assembly characteristics and biological impact from wt protein and indicates that its disease protecting mechanism may result primarily from the mutant protein's much lower binding affinity to synaptic receptors. This suggests that therapeutics that effectively reduce oligomer binding to synapses in the brain may be beneficial in AD.

Generation of a human induced pluripotent stem cell line (UEFi003-A) carrying heterozygous A673T variant in amyloid precursor protein associated with a reduced risk of Alzheimer's disease.

A673T mutation in the amyloid precursor protein (APP) is a rare variant associated with a reduced risk of late-onset Alzheimer's disease (AD) and age-related cognitive decline. The A673T mutation decreases beta-amyloid (Aβ) production and aggregation in neuronal cultures in vitro. Here we have identified a Finnish non-diseased male individual carrying a heterozygous A673T mutation, obtained a skin biopsy sample from him, and generated an iPSC line using commercially available integration-free Sendai virus-based kit. The established iPSC line retained the mutation, expressed pluripotency markers, had a normal karyotype, and differentiated into all three germ layers in vitro.

Mutations of beta-amyloid precursor protein alter the consequence of Alzheimer's disease pathogenesis.

Alzheimer's disease is pathologically defined by accumulation of extracellular amyloid-β (Aβ). Approximately 25 mutations in β-amyloid precursor protein (APP) are pathogenic and cause autosomal dominant Alzheimer's disease. To date, the mechanism underlying the effect of APP mutation on Aβ generation is unclear. Therefore, investigating the mechanism of APP mutation on Alzheimer's disease may help understanding of disease pathogenesis. Thus, APP mutations (A673T, A673V, E682K, E693G, and E693Q) were transiently co-transfected into human embryonic kidney cells. Western blot assay was used to detect expression levels of APP, beta-secretase 1, and presenilin 1 in cells. Enzyme-linked immunosorbent assay was performed to determine Aβ1-40 and Aβ1-42 levels. Liquid chromatography-tandem mass chromatography was used to examine VVIAT, FLF, ITL, VIV, IAT, VIT, TVI, and VVIA peptide levels. Immunofluorescence staining was performed to measure APP and early endosome antigen 1 immunoreactivity. Our results show that the protective A673T mutation decreases Aβ42/Aβ40 rate by downregulating IAT and upregulating VVIA levels. Pathogenic A673V, E682K, and E693Q mutations promote Aβ42/Aβ40 rate by increasing levels of CTF99, Aβ42, Aβ40, and IAT, and decreasing VVIA levels. Pathogenic E693G mutation shows no significant change in Aβ42/Aβ40 ratio because of inhibition of γ-secretase activity. APP mutations can change location from the cell surface to early endosomes. Our findings confirm that certain APP mutations accelerate Aβ generation by affecting the long Aβ cleavage pathway and increasing Aβ42/40 rate, thereby resulting in Alzheimer's disease.

Generation of a human induced pluripotent stem cell line from a patient with a rare A673T variant in amyloid precursor protein gene that reduces the risk for Alzheimer's disease

An amyloid precursor protein (APP) A673T mutation was found to be protective against Alzheimer's disease (AD) and cognitive decline in the Icelandic population and to associate with decreased levels of plasma β-amyloid in a Finnish population-based cohort. Human fibroblasts from a Finnish male individual carrying the protective mutation were used to generate integration-free induced pluripotent stem cell (iPSCs) line by Sendai virus technology. The iPSC line retained the mutation and expressed pluripotency markers, had a normal karyotype and differentiated into all three germ layers.

BACE1 Cleavage Site Selection Critical for Amyloidogenesis and Alzheimer's Pathogenesis.

Mutations in amyloid β precursor protein (APP) gene alter APP processing, either causing familial Alzheimer's disease (AD) or protecting against dementia. Under normal conditions, β-site APP cleaving enzyme 1 (BACE1) cleaves APP at minor Asp1 site to generate C99 for amyloid β protein (Aβ) production, and predominantly at major Glu11 site to generate C89, resulting in truncated Aβ production. We discovered that A673V mutation, the only recessive AD-associated APP mutation, shifted the preferential β-cleavage site of BACE1 in APP from the Glu11 site to the Asp1 site both in male and female transgenic mice in vivo and in cell lines and primary neuronal culture derived from timed pregnant rats in vitro, resulting in a much higher C99 level and C99/C89 ratio. All other mutations at this site, including the protective Icelandic A673T mutation, reduced C99 generation, and decreased the C99/C89 ratio. Furthermore, A673V mutation caused stronger dimerization between mutant and wild-type APP, enhanced the lysosomal degradation of the mutant APP, and inhibited γ-secretase cleavage of the mutant C99 to generate Aβ, leading to recessively inherited AD. The results demonstrate that APP673 regulates APP processing and the BACE1 cleavage site selection is critical for amyloidogenesis in AD pathogenesis, and implicate a pharmaceutical potential for targeting the APP673 site for AD drug development.SIGNIFICANCE STATEMENT β-site APP cleaving enzyme 1 (BACE1) is essential for amyloid β protein production. We discovered that A673V mutation shifted the BACE1 cleavage site from the Glu11 to the Asp1 site, resulting in much higher C99 level and C99/C89 ratio. All other mutations at this site of amyloid β precursor protein (APP) reduced C99 generation and decreased the C99/C89 ratio. Furthermore, A673V mutation resulted in stronger dimerization between mutant and wild-type APP, enhanced the lysosomal degradation of the mutant APP, and inhibited γ-secretase cleavage of the mutant C99 to generate amyloid β protein, leading to recessively inherited Alzheimer's disease (AD). The results demonstrate that APP673 regulates APP processing, and the BACE1 cleavage site selection is critical for amyloidogenesis in AD pathogenesis, and implicate a pharmaceutical potential for targeting the APP673 site for AD drug development.

The A673T mutation in the amyloid precursor protein reduces the production of β-amyloid protein from its β-carboxyl terminal fragment in cells.

IntroductionThe A673T mutation in the amyloid precursor protein (APP) protects against Alzheimer’s disease by reducing β-amyloid protein (Aβ) production. This mutation reduced the release of the soluble APP fragment (sAPPβ), which is processed by β-secretase, suggesting a concomitant decrease in the β-carboxyl fragment of APP (C99), which is a direct substrate of γ-secretase for Aβ production. However, it remains controversial whether the level of C99 is significantly reduced in cells expressing APP that carry A673T as the cause of reduced Aβ production. Here, we investigated the effect of the A673T mutation in C99 on γ-cleavage in cells.ResultsWe found that the level of C99 in cells expressing APP A673T was indistinctive of that observed in cells expressing wild-type APP, although the release of sAPPβ was significantly reduced in the APP A673T cells. In addition, our reconstituted β-secretase assay demonstrated no significant difference in β-cleavage on an APP fragment carrying the A673T mutation compared with the wild-type fragment. Importantly, cells expressing C99 containing the A673T mutation (C99 A2T; in accordance with the Aβ numbering) produced roughly half the level of Aβ compared with the wild-type C99, suggesting that the C99 A2T is an insufficient substrate of γ-secretase in cells. A cell-free γ-secretase assay revealed that Aβ production from the microsomal fraction of cells expressing C99 A2T was diminished. A sucrose gradient centrifugation analysis indicated that the levels of the C99 A2T that was codistributed with γ-secretase components in the raft fractions were reduced significantly.ConclusionsOur data indicate that the A673T mutation in APP alters the release of sAPPβ, but not the C99 level, and that the C99 A2T is an inefficient substrate for γ-secretase in cell-based assay.Electronic supplementary materialThe online version of this article (doi:10.1186/s40478-015-0247-6) contains supplementary material, which is available to authorized users.

A mutation protective against Alzheimer's disease renders amyloid β precursor protein incapable of mediating neurotoxicity

Expression of a familial Alzheimer's disease (AD)-linked mutant of amyloid β precursor protein (APP) or the binding of transforming growth factor β2 to wild-type (wt)-APP causes neuronal death by activating an intracellular death signal (a APP-mediated intracellular death signal) in the absence of the involvement of amyloid β (Aβ) toxicity in vitro. These neuronal death models may therefore be regarded as Aβ-independent neuronal death models related to AD. A recent study has shown that the A673T mutation in the APP isoform APP770 , corresponding to the A598T mutation in the most prevalent neuronal APP isoform APP695 (an AD-protective mutant of APP), is linked to a reduction in the incidence rate of AD. Consistent with this, cells expressing the AD-protective mutant of APP produce less Aβ than cells expressing wt-APP. In this study, transforming growth factor β2 caused death in cultured neuronal cells expressing wt-APP, but not in those expressing the AD-protective mutant of APP. This result suggests that the AD-protective mutation of APP reduces the incidence rate of AD by attenuating the APP-mediated intracellular death signal. In addition, a mutation that causes hereditary cerebral hemorrhage with amyloidosis-Dutch type also attenuated the APP-mediated intracellular death signal. The A598T mutation of amyloid precursor protein APP is linked to a reduction in the incidence rate of Alzheimer's disease (AD). This study shows that TGFβ2 causes death in neuronal cells expressing wild-type APP, but not in those expressing the AD-protective mutant of APP, suggesting that the AD-protective mutation of APP reduces the incidence rate of AD by attenuating the APP-mediated intracellular death signal.

Normal healthy donors possess plasma IgG reactive to a neutralizing epitope for toxic and amyloid-seeding beta-amyloid oligomers

Computational chemistry methods have been applied to investigate the observed decrease in affinity.Results:As expected, the Swedishmutation peptide showed the highest maximum velocity, while the KM value was comparable to that for the wild-type substrate. The A673T mutation substrate yielded a Vmax value in the same range as the wild-type, but the KM value was increased by 50 fold. These results suggest the turnover rate of the enzyme-peptide complex is not affected by the mutation on the peptide. However, interpreting KM as a surrogate for affinity, the mutation reduces the affinity of the substrate for the enzyme. Preliminary modelingresults indicate that suboptimal interaction between BACE and the T673 hydroxyl group, which do not offset its increase desolvation, might be one of the reasons affecting the free energy of binding of the mutant. We also confirmed previous observations that in cells overexpressing APPA673T, soluble APP beta (sAPPb) secretion, primary product of BACE1 cleavage, is reduced by 50% compared to cells overexpressing the wild type construct. Conclusions: Taken together, this data suggest that a reduction of BACE affinity for this mutated APP explains how it may protect against Alzheimer’s disease. Thus providing strong evidence that reducing BACE activity by inhibitors may be beneficial for the treatment of AD.

Effect of A673T mutation on APP processing by BACE1 and its significance for Alzheimer's disease therapeutic research

Effect of A673T mutation on APP processing by BACE1 and its significance for Alzheimer's disease therapeutic research