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The model organism in this review is Caenorhabditis elegans, which is used to review recent relevant findings that focus on Alzheimer's disease and its worm homolog gene, amyloid precursor-like protein (APL-1). In humans, alzheimer's disease (AD) is a neurodegenerative disorder characterized by the buildup of amyloid plaques in the brain. Mutations in APP will result in the development of Familial Alzheimer's Disease (FAD). C. elegans are used because these worms contain only one APP related gene,Â apl-1 while the mammals have two APP and related proteins with related functions, making it complicated to comprehend the role of APP in the pathogenesis of AD. In the article titiled, The expression of Alzheimer's Amyloid Precursor Protein-like gene is regulated by developmental timing microRNAs and their targets in Caenorhabditis elegans the research mainly focused on the genetic interaction of apl-1 with let-7 microRNAs and let-7-targeted genes. In this study Ryusuke et al.,( 2008) concluded that apl-1 works with and is under the control of molecules regulating developmental progression. In the second article that is being reviewed, , Intracellular Trafficking and Synaptic Function of APL-1 in Caenorhabditis elegans points out that the RNAi knock down of apl-1 leads to critical molting defects and early larval lethality. From this study, Wiese et al. 2010 also provides information on how the larval molting and lethality can be recovered by full length APL-1 C-terminal mutations and C-terminal truncations. In both of these articles, RNAi was performed in order to conduct the research. Both articles reveal new insights on apl-1 and Alzheimer's disease.
Alzheimer's disease (AD) is named after Alois Alzheimer, German neuropathologist, who described the disease first. AD is an irreversible, progressive neurodegenerative disorder characterized by the accumulation of Î² amyloid plaques and intracellular neurofibrillary tangles within the brain. Previous studies have supported the hypothesis that amyloid precursor protein (APP) is critical for AD development. But on the other hand, the development of this deposition is thought to occur due to age related changes with unidentified mechanism. The cleavage product of APP is Î² amyloid (Ryusuke et al., 2008). Earlier studies have suggested that APP is not limited to having a single function; instead it plays a role in cell adhesion, apoptosis, cell migration, synaptogeneis and also in the formation of the neuromuscular junctions (Wiese et al., 2008).For both these review articles, Caenorhabditis elegans, a nematode was an ideal model because they have amyloid precursor- like protein (APL-1) which is a single APP related gene, apl-1 on the X chromosome while mammals have two APP related genes. Multiple cell types express apl-1 gene and it is required for several developmental process which includes proper molting and morphogenesis. Larval lethality which is caused by the loss of apl-1 can be rescued by the expression of the extracellular domain of APL-1. In addition, the over expression of this gene can cause defects in brood size, movement and larval viability (Marakaki et al., 2012).
Ryusuke et al., 2008 suggested that apl-1 could function as a receptor receiving a ligand to control the development of seam cells and molting. In order to better understand the functions and biochemical properties of this gene in C. elegans more enhanced studies will need to be conducted. In the study done by Wiese et al., the RNAi knock down of the APP worm homolog followed by testing on the acetylcholinesterase inhibitor aldicarb demonstrated that loss of apl-1 resulted in hypersensitivity to aldicarb which indicated a fault in the synaptic function. The hypersensitivity to aldicrab could be reversed by full length apl-1 in a dose dependent manner. kinesins UNC-104/KIF-1A and UNC-116/kinesin-1 are some of the affirmative regulators of apl-1 expression in the brain. Wiese et al., 2010 proposed that RNAi knock-down of the small GTPase rab-5 resulted in a significant reduction in the apl-1 expression in neuron. This suggests that the interaction of the cell membrane to the early endosome is significant for apl-1 to function properly.
Discussion and Experiments:
Knock down of apl-1and its genetic rescue
In the research conducted by Wiese et al., (2012) knocked down apl-1 using the RNAi sensitive rrf-3(pk1426) strain resulted in defective molting which started in the L3/L4 molt and continued in the L4/YA molt. Several different molting phenotypes were observed, which varied from internal pinching posterior to its head, loose cuticle around the tail and the head or degradation around the mouth area. The gene knock out also delayed development; majority of the treated worm was still in L4 stage after 2 days while the control worms had completed their development and reached adulthood. C. elegans that are homozygous for apl-1 (tm385) deletion shows signs of degradation, internal vacuolization and are also L1 lethal. These characterizations are similar to the null mutations which indicate that the tm385 lesion can also be null. The researchers endeavored to rescue the lethality by using constructs which contained full length apl-1 mutations in the conserved C-terminal domain (CTD). All these constructs were fused to a C-terminal GFP which was induced by the apl-1 promoter. A deletion of the conserved YENPTY motif was included in the rescue constructs. This motif binds to various mutations of the conserved Thr658 residue which has kinase activity that can control what the APP can bind and localize. Alanine (T658A) or Glutamic acid (T658E) was used to mutate the tyrosine site to model the dephosphorylated and phosphorylated protein respectively. Additionally, in order to prevent any possible complications with the C-terminal GFP fusion, the researchers tracked the APL-1 expression, and carried out parallel experiments with full length apl-1 which lacked the GFP tag. After carrying out the experiment, researchers concluded that full length apl-1 and all the C-terminal mutation constructs were successful in rescuing the lethality and molting defects resulted from the tm385 deletion. Although the injection of apl-1 Î”IC at 10Î¼g/Âµl was able to rescue the lethality and molting but the construct was unsuccessful in rescuing the defects at a much higher concentration of 20ng/Âµl. The reason behind this dose dependent rescue is not understood yet. In order to determine if the mammalian homologs could work as a functional homolog to apl-1, the researchers individually injected each of these genes expressed by the apl-1 promoter into tm385 to test for the rescue of the lethality. The results indicated that none of the mammalian genes were able to rescue the tm385 lethality.
Neurotransmission defects are a result of loss of apl-1
The loss of apl-1 expression in C. elegans resulted in neurotransmission defects like in the mammals. A potential pathway apl-1 utilize to initiate synaptic transmission is the EGL-30 G-protein coupled receptor pathway, which can alter cholinergic signaling. In both apl-1 and APP, the Go protein binding was conserved on its C-terminus. Although the C- terminus on apl-1 might not be necessary for carrying out appropriate molting, the researchers couldn't eliminate a significant function for this domain in neurotransmission since they couldn't generate a strain that saves the hypersensitivity of aldicrab using the DIC construct. The loss of apl-1 resulted in improved pharyngeal pumping. This supports one of the regulatory functions through the EGL-30 pathway pumping like pharyngeal pumping, which is a function modified by the G-protein. Wiese et al.,., 2010 have suggested that the control of neurotransmission by apl-1 doesn't seem to be associated to its control over molting. A defect in molting didn't occur in any of the rescue strains, or intentionally prevented by carrying out apl-1 RNAi knock-down in adults, but the hypersensitivity of aldicarb was observed in these worms. The mechanism of dual regulation could not be analyzed by the simple removal of the c terminal domain, or YENPTY motif, due to the fact that full length apl-1 rescue of lethality at lower expression levels was not sufficient in rescuing aldicarb hypersensitivity. These data from the study suggests that the roles of apl-1 in molting are not linked to its function in neurotransmission. Both these functions are independent of each other.
apl-1 transport and localization
In the study, Wiese et al.,., 2010 established that the localization of the worm gene, apl-1 in neurons are controlled through the activity of the kinesins UNC-104/KIF1A, UNC-116/kinesin-1, small GTPases RAB-5 and UNC-108/Rab2. As a membrane protein, apl-1 moves through the endosomal pathway that uses RAB-5. A decrease in RAB-5 can also decrease the level of apl-1 gene inside the neurons. After knocking out the RAB-5 using RNAi, the researchers concluded that the apl-1 gene becomes tethered on the surface of that cell, where it is exposed to the proteases on the membrane. Because the localization of apl-1 depends on the presence of UNC-108 and other proteins, a potential function of apl-1 could be within the dense core vesicles (DCVs) or play a role in the maturation of the DCV. A DCV is a discrete vesicular containing neuropeptides and peptide hormones. This could be another possible explanation for the capability of apl-1 to control synaptic transmission as DSV cargos can modify cholinergic signaling. UNC-108 has the ability to modify the localization of apl-1 this denotes a unique process by which apl-1 is controlled in the cell. Principally, Wiese et al., suggests that the transport of apl-1 in the neuron allows apl-1 to accurately carry out its several roles by admitting the proteins to molecules which can cleave and control the release of important N-terminal part of the protein. This has significance for the natural science of APP and its homologs where the N termini of these proteins could also function as ligands to initiate the downstream pathways that modify neurotransmission.
hbl-1, lin-41 and lin-42 regulates apl-1 transcription
In the nematode Caenorhabditis elegans, the genes that control the timing during development are known as the herterochronic genes. These genes regulate the sequential execution of processes of cell division that are stage specific. Heterochronic genes also function in the differentiation through the larval stages of C. elegans. Let-7 is a heterochronic gene that is expressed in the late larval stage and assists in the progression from L4 to adult. Let-7 encodes for a micro RNA and binds with the defective complementary 3â€² UTR of its targets, like lin-41 and hbl-1, to block their translation. In this study, the researchers describe that the C. elegans apl-1 gene works with and is regulated by the heterochronic gene, miRNA let-7. apl-1 genetically interacts with let-7 miRNAs and their target genes such as hbl-,1 lin-42 and lin-41.
Downstream of let-7 are heterochronic regulators hbl-1, lin-42 and lin-41. The researchers observed a precocious development of apl-1 at the L3 molt in hbl-1, lin-41 and lin-42 mutant seam cells. This suggests that the heterochronic genes generally suppress the advanced expression of apl-1 during the last larval stage of the worm. In addition, hbl-1, lin-42 and lin-41 are temporally responsible for sustaining the apl-1 expression later on in the development when the amount of worms showing the proper seam cell expression in the late larval stage is reduced drastically in hbl-1, lin-42 or lin-41 mutants. From this study, the researchers concluded that these genes could affect apl-1 expression positively in the late L4 stage but negatively in the early larval stages.
Based on the information presented in both these scientific articles, Caenorhabditis elegans homolog of APP, APP-like-1 (apl-1), associates with and is under the regulatory control of molecules controlling the developmental progression. Ryuseuke et al., (2008) mainly focused his study on heterochronic genes such as let-7m which controlled the timing of the cell fate determination. This study could possibly shed light on the time dependent progression of Alzheimer's disease since heterochronic genes are engaged in the aging process. In the research conducted by Wiese et al.,(2010) suggested that apl-1 controls neurotransmission and molting process independently and the hypersensitivity to aldicarb could be rescued with full length APL-1 in a dose dependent manner. Moreover, more detailed researches could be conducted in order to learn more about this gene. A good understanding of the homolog of APP could possibly help make advances in the therapeutic sector. And one day, scientists will have a solution to control this progressive neurodegenerative disease in humans!
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