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Methods in Enzymology 2017The study of membrane protein function and structure requires their successful detection, expression, solubilization, and/or reconstitution, which poses a challenging... (Review)
Review
The study of membrane protein function and structure requires their successful detection, expression, solubilization, and/or reconstitution, which poses a challenging task and relies on the availability of suitable tools. Several research groups have successfully applied Nanobodies in the purification, as well as the functional and structural characterization of membrane proteins. Nanobodies are small, single-chain antibody fragments originating from camelids presenting on average a longer CDR3 which enables them to bind in cavities and clefts (such as active and allosteric sites). Notably, Nanobodies generally bind conformational epitopes making them very interesting tools to stabilize, dissect, and characterize specific protein conformations. In the clinic, several Nanobodies are under evaluation either as potential drug candidates or as diagnostic tools. In recent years, we have successfully generated high-affinity, conformation-sensitive anti-γ-secretase Nanobodies. γ-Secretase is a multimeric membrane protease involved in processing of the amyloid precursor protein with high clinical relevance as mutations in its catalytic subunit (Presenilin) cause early-onset Alzheimer's disease. Advancing our knowledge on the mechanisms governing γ-secretase intramembrane proteolysis through various strategies may lead to novel therapeutic avenues for Alzheimer's disease. In this chapter, we present the strategies we have developed and applied for the screening and characterization of anti-γ-secretase Nanobodies. These protocols could be of help in the generation of Nanobodies targeting other membrane proteins.
Topics: Alzheimer Disease; Amyloid Precursor Protein Secretases; Catalytic Domain; Epitopes; Humans; Membrane Proteins; Molecular Biology; Mutation; Presenilins; Protein Conformation; Single-Chain Antibodies; Single-Domain Antibodies; Structure-Activity Relationship
PubMed: 28065273
DOI: 10.1016/bs.mie.2016.10.029 -
Current Gene Therapy 2021Alzheimer's disease (AD) is the most common form of dementia in the elderly and this complex disorder is associated with environmental as well as genetic factors.... (Review)
Review
Alzheimer's disease (AD) is the most common form of dementia in the elderly and this complex disorder is associated with environmental as well as genetic factors. Early-onset AD (EOAD) and late-onset AD (LOAD, more common) are major identified types of AD. The genetics of EOAD is extensively understood, with three gene variants such as APP, PSEN1, and PSEN2 leading to the disease. Some common alleles, including APOE, are effectively associated with LOAD identified, but the genetics of LOAD is not clear to date. It has been accounted that about 5-10% of EOAD patients can be explained through mutations in the three familiar genes of EOAD. The APOE ε4 allele augmented the severity of EOAD risk in carriers, and the APOE ε4 allele was considered as a hallmark of EOAD. A great number of EOAD patients, who are not genetically explained, indicate that it is not possible to identify disease-triggering genes yet. Although several genes have been identified by using the technology of next-generation sequencing in EOAD families, including SORL1, TYROBP, and NOTCH3. A number of TYROBP variants are identified through exome sequencing in EOAD patients and these TYROBP variants may increase the pathogenesis of EOAD. The existence of the ε4 allele is responsible for increasing the severity of EOAD. However, several ε4 allele carriers propose the presence of other LOAD genetic as well as environmental risk factors that are not identified yet. It is urgent to find out missing genetics of EOAD and LOAD etiology to discover new potential genetic facets which will assist in understanding the pathological mechanism of AD. These investigations should contribute to developing a new therapeutic candidate for alleviating, reversing and preventing AD. This article, based on current knowledge, represents the overview of the susceptible genes of EOAD, and LOAD. Next, we represent the probable molecular mechanism that might elucidate the genetic etiology of AD and highlight the role of massively parallel sequencing technologies for novel gene discoveries.
Topics: Adaptor Proteins, Signal Transducing; Aged; Alleles; Alzheimer Disease; Amyloid beta-Protein Precursor; Apolipoproteins E; Disease Progression; Genetic Variation; Humans; LDL-Receptor Related Proteins; Membrane Proteins; Membrane Transport Proteins; Mutation; Presenilin-1; Presenilin-2
PubMed: 33231156
DOI: 10.2174/1566523220666201123112822 -
ELife Jan 2017The high levels of serine (S) and threonine (T) residues within the Presenilin 1 (PS1) N-terminus and in the large hydrophilic loop region suggest that the enzymatic...
The high levels of serine (S) and threonine (T) residues within the Presenilin 1 (PS1) N-terminus and in the large hydrophilic loop region suggest that the enzymatic function of PS1/γ-secretase can be modulated by its 'phosphorylated' and 'dephosphorylated' states. However, the functional outcome of PS1 phosphorylation and its significance for Alzheimer's disease (AD) pathogenesis is poorly understood. Here, comprehensive analysis using FRET-based imaging reveals that activity-driven and Protein Kinase A-mediated PS1 phosphorylation at three domains (domain 1: T74, domain 2: S310 and S313, domain 3: S365, S366, and S367), with S367 being critical, is responsible for the PS1 pathogenic 'closed' conformation, and resulting increase in the Aβ42/40 ratio. Moreover, we have established novel imaging assays for monitoring PS1 conformation in vivo, and report that PS1 phosphorylation induces the pathogenic conformational shift in the living mouse brain. These phosphorylation sites represent potential new targets for AD treatment.
Topics: Alzheimer Disease; Animals; Disease Models, Animal; Fluorescence Resonance Energy Transfer; Mice; Optical Imaging; Phosphorylation; Presenilin-1; Protein Conformation; Protein Domains; Protein Processing, Post-Translational
PubMed: 28132667
DOI: 10.7554/eLife.19720 -
Journal of Molecular Neuroscience : MN Nov 2016An imbalance in metal homeostasis is a prominent feature of Alzheimer's disease (AD). A wealth of evidence from independent studies over the past two and half decades... (Review)
Review
An imbalance in metal homeostasis is a prominent feature of Alzheimer's disease (AD). A wealth of evidence from independent studies over the past two and half decades has found changes to the distribution of brain iron, zinc, and copper in AD patients and animal models of the disease. Early research focused on the association of these metals with amyloid beta (Aβ), particularly extraneuronal Aβ plaque pathology. In contrast, there are numerous studies that have demonstrated a loss of iron-, zinc-, or copper-dependent cellular functions in AD animal and cell models, highlighting the importance of metal homeostasis in maintaining healthy brain function. Characterizing the molecular pathways that are impacted by iron, zinc, or copper will shed light on how these metals affect neuoroprotection, and conversely, neurodegeneration. Of particular interest is the role that the AD-associated presenilins have on protein trafficking and degradation, as metal homeostasis is dependent on the efficient trafficking and recycling of specific metal transporters. This review summarizes what is currently known about presenilin-dependent protein trafficking and the role of presenilin in protein turnover, particularly via the autophagy-lysosomal system.
Topics: Alzheimer Disease; Animals; Homeostasis; Humans; Metals; Presenilins; Protein Transport; Proteolysis
PubMed: 27558108
DOI: 10.1007/s12031-016-0826-4 -
Acta Biochimica Et Biophysica Sinica Jan 2020Dementia, a devastating neurological disorder commonly found in the elderly, is characterized by severe cognitive and memory impairment. Ample clinical and... (Review)
Review
Dementia, a devastating neurological disorder commonly found in the elderly, is characterized by severe cognitive and memory impairment. Ample clinical and epidemiological evidence has depicted a close association between dementia and heart failure. While cerebral blood under perfusion and neurohormonal activation due to the dampened cardiac pump function contribute to the loss of nutrient supply and neuronal injury, Alzheimer's disease (AD), the most common type of dementia, also provokes cardiovascular function impairment, in particular impairment of diastolic function. Aggregation of amyloid-β proteins and mutations of Presenilin (PSEN) genes are believed to participate in the pathological changes in the heart although it is still debatable with regards to the pathological cue of cardiac anomalies in AD process. In consequence, reduced cerebral blood flow triggered by cardiac dysfunction further deteriorates vascular dementia and AD pathology. Patients with atrial fibrillation, heart failure, and other cardiac anomalies are at a higher risk for cognitive decline and dementia. Conclusion: Due to the increased incidence of dementia and cardiovascular diseases, the coexistence of the two will cause more threat to public health, warranting much more attention. Here, we will update recent reports on dementia, AD, and cardiovascular diseases and discuss the causal relationship between dementia and heart dysfunction.
Topics: Adult; Aged; Aged, 80 and over; Aging; Alzheimer Disease; Amyloid beta-Peptides; Animals; Cardiovascular Diseases; Comorbidity; Disease Models, Animal; Humans; Mice; Middle Aged; Mutation; Presenilins; Risk Factors
PubMed: 31897470
DOI: 10.1093/abbs/gmz115 -
Advances in Experimental Medicine and... 2020Alzheimer's disease (AD) is the most common type of dementia caused by severe neurodegeneration in the hippocampus and neocortical regions of the brain. In addition to... (Review)
Review
Alzheimer's disease (AD) is the most common type of dementia caused by severe neurodegeneration in the hippocampus and neocortical regions of the brain. In addition to neurodegeneration, AD brains contain high levels of amyloid plaques (APs) and neurofibrillary tangles (NFTs) which are used as neuropathological hallmarks of the disorder. Despite intense research efforts, the mechanism(s) of the AD neurodegeneration are imperfectly understood, hampering efforts for the development of efficient therapeutics. Furthermore, failure of clinical trials to benefit AD patients suggests that AD hallmarks are poor therapeutic targets and supports the suggestion that these hallmarks are sequelae of neurodegeneration. Although genetic evidence seem to support the amyloid theory of AD, additional empirical observations and experimental data are inconsistent with the amyloid/Aβ theories of AD [Robakis and Neve (1998), TINS vol. 21 pp.15-19; Robakis (2011) NBA vol. 32, pp 372-379]. This possibility is further supported by data that amyloid plaques and neurofibrillary tangles are found in a number of distinct neurodegenerative disorders and that animal models expressing high levels of AD pathological structures show little neuronal loss. Furthermore, genetic evidence linking genetic loci to disease reveal little about the molecular mechanisms involved. Mutants of APP, PS1, and PS2 cause familial AD (FAD) suggesting these mutants can be used as models to study mechanisms of neurodegeneration. Recent reports show that the ability of efnB1 and BDNF (factors) to rescue neurons from excitotoxicity depends on PS1 but is independent of γ-secretase. Interestingly, PS1 FAD mutations block the ability of factors to protect neurons from toxicity suggesting that FAD mutants may increase neuronal death by blocking neuroprotective activities of brain neurotrophins. Other reports also suggest that proteins involved in FAD have Aβ-/γ-secretase-independent functions that can play important roles in AD. Furthermore, non-neuronal brain cells like microglia are implicated in AD pathology.
Topics: Alzheimer Disease; Amyloid Precursor Protein Secretases; Amyloid beta-Peptides; Amyloid beta-Protein Precursor; Animals; Brain; Clinical Trials as Topic; Disease Models, Animal; Humans; Plaque, Amyloid; Presenilin-1; Presenilin-2; Treatment Failure
PubMed: 32468473
DOI: 10.1007/978-3-030-32633-3_23 -
CNS Neuroscience & Therapeutics Feb 2024Alzheimer's disease (AD) is the most common form of dementia. Depression is one of the most critical psychiatric complications of AD, and 20%-30% of patients with AD...
INTRODUCTION
Alzheimer's disease (AD) is the most common form of dementia. Depression is one of the most critical psychiatric complications of AD, and 20%-30% of patients with AD experience symptoms of depression. Phospho-glycogen synthase kinase-3 beta (GSK3β) is known to be associated with AD and depression. Furthermore, the role of disheveled (DVL) is known to regulate GSK3β. Moreover, presenilin-2 (PS2) and DVL have cross-talk with each other. Also, it is widely hypothesized that stress leads to hypersecretion of cortisol and is thus associated with depression. Dickkopf WNT signaling pathway inhibitor-1 (DKK-1) is a crucial factor regulating depression and both amyloid beta (Aβ) and phosphorylation of tau are widely known as a biomarker of AD.
METHODS
To investigate the relationship between AD and depression, and possible pathways connecting the two diseases, we examined memory function and depression-related behavior test results in PS2 knock-in AD mice (PS2 MT). Next, we confirmed that there are relationships between DVL, depression, and cognitive disease through the comparative toxicogenomics database (https://ctdbase.org) and STRING (https://string-db.org) database.
RESULTS
PS2 knock-in mice showed much more severe memory impairment and depression than PS2 wild-type mice (PS2 WT). In AD-related behavioral experiments, PS2 MT mice showed more memory dysfunction compared with PS2 WT group mice. Moreover, Aβ and phosphorylation of tau showed higher expression in PS2 MT mice than in PS2 WT mice. Depression-related behavioral tests showed that PS2 MT mice exhibited more depressive behaviors than PS2 WT mice. Furthermore, both higher cortisol levels and higher expression of DKK-1 were found in PS2 MT mice relative to PS2 WT mice. The results indicated that there is a relationship between DVL and the release of AD-related mediators and expression of the depression-related glucocorticoid receptor and DKK-1. In the PS2 knock-in group, DVL was significantly decreased compared with the PS2 WT group.
CONCLUSION
Depression increases the risk of developing AD and other forms of dementia. Recent evidence indicates that depression symptoms could trigger changes in memory and thinking over time. However, it is recognized that there are no drugs to facilitate a full recovery for both AD and depression. However, our results suggest that AD and depression could be associated, and DVL could be a significant target for the association between AD and depression.
Topics: Animals; Mice; Alzheimer Disease; Amyloid beta-Peptides; Dishevelled Proteins; Down-Regulation; Glycogen Synthase Kinase 3 beta; Hydrocortisone; Mice, Transgenic; Presenilin-1; Presenilin-2
PubMed: 37501340
DOI: 10.1111/cns.14370 -
Proceedings of the National Academy of... Oct 2023Mutations in the ( and ) genes are the major cause of early-onset familial Alzheimer's disease (FAD). Presenilin (PS) is the catalytic subunit of the γ-secretase...
Mutations in the ( and ) genes are the major cause of early-onset familial Alzheimer's disease (FAD). Presenilin (PS) is the catalytic subunit of the γ-secretase complex, which cleaves type I transmembrane proteins, such as Notch and the amyloid precursor protein (APP), and plays an evolutionarily conserved role in the protection of neuronal survival during aging. FAD mutations exhibit impaired γ-secretase activity in cell culture, in vitro, and knockin (KI) mouse brains, and the L435F mutation is the most severe in reducing γ-secretase activity and is located closest to the active site of γ-secretase. Here, we report that introduction of the codon-optimized wild-type human cDNA by adeno-associated virus 9 (AAV9) results in broadly distributed, sustained, low to moderate levels of human PS1 (hPS1) expression and rescues impaired γ-secretase activity in the cerebral cortex of mutant mice either lacking PS or expressing the L435F KI allele, as evaluated by endogenous γ-secretase substrates of APP and recombinant γ-secretase products of Notch intracellular domain and Aβ peptides. Furthermore, introduction of hPS1 by AAV9 alleviates impairments of synaptic plasticity and learning and memory in mutant mice. Importantly, AAV9 delivery of hPS1 ameliorates neurodegeneration in the cerebral cortex of aged mutant mice, as shown by the reversal of age-dependent loss of cortical neurons and elevated microgliosis and astrogliosis. These results together show that moderate hPS1 expression by AAV9 is sufficient to rescue impaired γ-secretase activity, synaptic and memory deficits, and neurodegeneration caused by mutations in mouse models.
Topics: Humans; Mice; Animals; Aged; Amyloid Precursor Protein Secretases; Presenilin-1; Alzheimer Disease; Amyloid beta-Protein Precursor; Mutation; Memory Disorders; Presenilin-2; Amyloid beta-Peptides
PubMed: 37816062
DOI: 10.1073/pnas.2306714120 -
International Journal of Molecular... Jan 2020Alzheimer's disease (AD) is the most common form of dementia. Even though most AD cases are sporadic, a small percentage is familial due to autosomal dominant mutations... (Review)
Review
Alzheimer's disease (AD) is the most common form of dementia. Even though most AD cases are sporadic, a small percentage is familial due to autosomal dominant mutations in amyloid precursor protein (APP), presenilin-1 (PSEN1), and presenilin-2 (PSEN2) genes. AD mutations contribute to the generation of toxic amyloid β (Aβ) peptides and the formation of cerebral plaques, leading to the formulation of the amyloid cascade hypothesis for AD pathogenesis. Many drugs have been developed to inhibit this pathway but all these approaches currently failed, raising the need to find additional pathogenic mechanisms. Alterations in cellular calcium (Ca) signaling have also been reported as causative of neurodegeneration. Interestingly, Aβ peptides, mutated presenilin-1 (PS1), and presenilin-2 (PS2) variously lead to modifications in Ca homeostasis. In this contribution, we focus on PS2, summarizing how AD-linked PS2 mutants alter multiple Ca pathways and the functional consequences of this Ca dysregulation in AD pathogenesis.
Topics: Alzheimer Disease; Amyloid beta-Protein Precursor; Calcium Signaling; Humans; Presenilin-1; Presenilin-2
PubMed: 31991578
DOI: 10.3390/ijms21030770 -
The FEBS Journal Feb 2020The accumulation of amyloid-β (Aβ) peptides is a key histopathological feature of the Alzheimer's brain. Defective clearance mechanisms result in toxic levels of... (Review)
Review
The accumulation of amyloid-β (Aβ) peptides is a key histopathological feature of the Alzheimer's brain. Defective clearance mechanisms result in toxic levels of soluble Aβ and Aβ oligomers, leading to impaired synaptic function, neurodegeneration and cognitive decline. Growing evidence points to the involvement of P-glycoprotein (P-gp or ABCB1), an ATP-binding cassette transporter highly expressed on the luminal side of the blood-brain barrier, in facilitating the clearance of Aβ from the brain. In this review, we summarise evidence from human, animal and in vitro studies examining the contribution of P-gp to Aβ clearance, and discuss the potential for P-gp as a novel pharmacological target in Alzheimer's disease (AD). P-gp expression and activity in the brain are inversely correlated with ageing, Aβ deposition and AD. Moreover, Aβ itself has been found to compromise the expression of P-gp, thereby exacerbating Aβ deposition and disease. Despite decades of research, the pathophysiology of AD remains elusive. Understanding the normal versus impaired processing and clearance mechanisms affecting Aβ peptides will assist the development of more effective therapeutic agents to combat this progressive neurodegenerative condition that continues to devastate millions of patients globally.
Topics: ATP Binding Cassette Transporter, Subfamily B; Alzheimer Disease; Amyloid beta-Peptides; Animals; Blood-Brain Barrier; Brain; Disease Models, Animal; Gene Expression Regulation; Humans; Neurons; Peptide Fragments; Presenilin-1; Presenilin-2; Protein Aggregates; Protein Transport; Proteolysis; Signal Transduction
PubMed: 31750987
DOI: 10.1111/febs.15148