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Beta Amyloid Definition

Amyloid beta (Aβ or Abeta) is a peptide of 39–43 amino acids that appear to be the main constituent of amyloid plaques in the brains of Alzheimer's disease patients. Read More

Research Overview 

Accumulation of b-amyloid protein (Ab) in the brain causes changes in neuritic processes in individuals with this disease. Steven Petratos et al., 2008 (1) have showed that Ab decreases neurite outgrowth from SH-SY5Y human neuroblastoma cells. To explore molecular pathways by which Ab alters neurite outgrowth, we examined the activation and localization of RhoA and Rac1 which regulate the level and phosphorylation of the collapsin response mediator protein-2 (CRMP-2). Ab increased the levels of the GTP-bound (active) form of RhoA in SH-SY5Ycells.This increase in GTP-RhoA correlatedwith an increase in an alternatively spliced formof CRMP-2 (CRMP-2A) and its threonine phosphorylated form. Both a constitutively active form of Rac1 (CA-Rac1) and the Rho kinase inhibitor, Y27632, decreased levels of the CRMP-2A variant and decreased threonine phosphorylation caused by Ab stimulation. The amount of tubulin bound to CRMP-2 was decreased in the presence of Ab but Y27632 increased the levels of tubulin bound to CRMP-2. Increased levels of both RhoA and CRMP-2 were found in neurons surrounding amyloid plaques in the cerebral cortex of the APP(Swe) Tg2576 mice.We found that there was an increase in threonine phosphorylation of CRMP-2 inTg2576 mice and the increase correlated with a decrease in the ability of CRMP-2 to bind tubulin.The results suggest that Ab-induced neurite outgrowth inhibition may be initiated through a mechanism in which Ab causes an increase in Rho GTPase activity which, in turn, phosphorylates CRMP-2 to interfere with tubulin assembly in neurites.

In another research theory reduced brain insulin signalling and low CSF-to-plasma insulin ratios have been observed in patients with Alzheimer disease (AD). Furthermore, intracerebroventricular or IV insulin administration improve memory, alter evoked potentials, and modulate neurotransmitters, possibly by augmenting low brain levels. After intranasal administration, insulin-like peptides follow extracellular pathways to the brain within 15 minutes.

Beta amyloid and Alzheimer’s disease:

The b-amyloid protein of Alzheimer’s disease increases neuronal CRMP-2 phosphorylation by a Rho-GTP mechanism:

The generation of Ab and the deposition of amyloid is associated with neuronal dysfunction and loss of functional synapses caused by changes to neurite morphology (2 &3). The neuritic changes in neurons of the frontal and temporal cortices may initially lead to a mild cognitive impairment (MCI), that is followed by more severe memory loss as the disease progresses (4). There is strong evidence for the involvement of extracellular Ab in the disruption of the integrated neuronal circuitry (5) It has been examined the effects of Ab on neurite outgrowth and to determine the activation of downstream signalling mechanisms which mediate these effects. In human neuroblastoma SH-SY5Y cells, Ab can reduce the length of neuritis by inactivating the neurite outgrowth signalling molecule Rac1, and that this Ab-mediated reduction in neurite length can be abrogated by the Rho Kinase inhibitor, Y27632. Furthermore, researchers have showed that the Ab-mediated decrease in neurite length involves the induction of a threonine phosphorylation of CRMP-2A, conferring a reduced binding capacity to tubulin, both of which can be reversed by inhibiting RhoA activity. Importantly, evidence validated that this mechanism also operates in the Tg2576 mouse model of AD, where RhoA and CRMP-2 are increased in the immediate vicinity of amyloid plaques and CRMP-2-bound tubulin is reduced. The data suggest that Ab-mediated neurite outgrowth inhibition is initiated through the activity of RhoA-GTP and through the dysregulation of CRMP-2 to bind tubulin for neurite outgrowth.

As Ab deposition is associated with neuritic abnormalities in the AD brain (6), authors investigated the effect of different Ab peptides on neurite outgrowth. Freshly added or ‘aged’ (aggregated) Ab40 and Ab42 (1 mM) were added to RA-differentiated SH-SY5Y cells. Ab40 caused a significant reduction in neurite length over a 24-h incubation period (Fig. 1). Ab40 induced an approximate 40% reduction in neurite length when added fresh (not aged) (Fig. 1A and C). When Ab40 that had been incubated for 7 days at 37C (aged) was used, a reduction in neurite length of ~25% was observed (Fig. 1B and D). There was no significant reduction in neurite length of SH-SY5Y cells incubated with Ab40 or Ab42 peptides containing a scrambled sequence of amino acids (scrambled Ab40 and Ab42) (Fig. 1A and B). Fresh Ab42 (1 mM) also produced a reduction in neurite length of ~25% (Fig. 1A). Surprisingly, after ageing Ab42, there was a decrease in the level of neurite outgrowth inhibition with only a 10–15% reduction in neurite length (Fig. 1B). It was speculated that because the Ab peptides were incubated in the medium for 24 h before the neurite outgrowth effects were observed, it was likely that the peptides had already aggregated to some extent and further ‘ageing’ of Ab42 may have produced high molecular weight aggregates that were no longer toxic.

 

Fig.1 Effect of fresh and aged Ab on neurite length in differentiated human SH-SY5Yneuroblastoma cell cultures. Ab peptides (1 mM) where freshly prepared (A,C) or aged for 7 days inDMEM/F12 and10% FCS at 37C (B,D). Peptides were added to cells for 24 h. Panels A and B show quantitation of neurite outgrowth. Panels C and D are representative photomicrographs of SH-SY5Ycells incubated with Ab40 and Ab42 peptides (bar=50 mm). (A, _#+P<0.001, ^P<0.05; B, _#^ P<0.001).

 

Role of the familial Dutch mutation E22Q in the folding and aggregation of the 15–28 fragment of the Alzheimer amyloid-b protein

The presence of amyloid fibrils in the brain is a clinical hallmark of Alzheimer‘s disease (AD). The fibrils consist of large ordered aggregates of amyloid-b (Ab) peptides, proteolytic by-products of the enzymatic cleavage of the Alzheimer amyloid precursor protein (APP). AD can be sporadic (occurring in elderly patients and characterized by a slow progression) or familial (a hereditary form characterized by early onset of the disease and aggravated severity). The majority of familial forms of AD involve single-point mutations in the 22–23 segment of the Abpeptide. The focus of this work is on the Dutch form of AD, which involves a mutation encoded by a point substitution G to C at codon 693 of the amyloid precursor protein (APP). The result is the production of an Ab peptide in which residue E22 is mutated to Q (E22Q mutation). Patients who have this mutation are at risk of developing cerebral amyloid angiopathy that typically leads to cerebral hemorrhage and stroke. Many in vitro experiments show that the AbE22Q peptide aggregates much more readily than its wild-type (WT) counterpart (7-9). As a free monomer in solution, the WT Abpeptide is for the most part unstructured with residual structure observed locally in a few regions of the primary sequence (10). The Ab peptide can populate a variety of oligomeric species, some of which may be toxic (such as transmembrane pores) (11), on route to the fibrillar state. Once bound to the fibril, the peptide adopts a b-sheet conformation (12), implying that fibrillization is accompanied by a major structural reorganization. In the case of the E22Q mutation, experiments by Maggio et al. (13) indicate that the rate of Ab monomer deposition onto fibrils is enhanced compared with the WT. The refolding of monomeric Ab into a conformation commensurate with the fibril presents a free energy barrier to fibril growth. Researchers are aiming of the twofold work: (i) to investigate how the E22Q mutation affects monomer Ab conformations and (ii) to investigate how the E22Q mutation affects the deposition reaction of monomers onto fibrils. In aim i, the focus on the effect of the E22Q mutation on two critical regions implicated in Ab monomer folding and aggregation, the E22–K28 region and the L17–A21 central hydrophobic cluster (CHC). NMR and molecular dynamics simulations have shown that the 22–28 region adopts a bend structure in the context of the proteolytically resistant 21–30 fragment of A Ab (14–17) and is likely the most structured region of the full-length Ab40 and Ab42 peptides (14). Further simulations in our group found this same bend region in fragments Ab12–28 (18) and Ab10–35 (19). The CHC, on the other hand, appears to serve a s a recognition site for monomer binding to the fibril. Mutations in this segment (such as F19T) completely abrogate fibril growth (20). It was considered that two model peptides: fragments 21–30 and 15–28 of the Ab peptide. The former will enable us to study the effect of the E22Q mutation on the structured bend motif while the latter will let researchers probe the effects of this mutation on the structure of the CHC region. Although we are not considering fragments that include residues distant in sequence space from residue 22, nonetheless expect to capture the essential effect of the E22Q mutation. Indeed, experiments on Ab12–28 (21) and Ab10–35 (7) have shown that the E22Q mutation only induces local changes to the folding properties of the peptide that do not extend beyond neighboring residues of E22. The simulations involved explicit solvent and the replica exchange protocol to achieve a thorough sampling of conformational space. In aim ii, investigated how the E22Q mutation affects deposition rates onto preexisting amyloid fibrils by identifying the transition state (TS) ensemble for the fibril elongation reaction and calculating the activation free energy for monomer deposition onto preexisting fibrils for the WT and mutant cases.

References:

1. Petratos, S., Li, Q-X., Amee, J., Xu Hou, G., Kerr, M. L.. Unabia, S. E., Hatzinisiriou, I., Marie-Isabel, D. M., and Small, D. H. 2008. The b-amyloid protein of Alzheimer’s disease increases neuronal CRMP-2 phosphorylation by a Rho-GTP mechanism. Brain 131, 90-108.

2. Tsai, J., Grutzendler, J., Duff, K., Gan, W.B. 2004. Fibrillar amyloid deposition leads to local synaptic abnormalities and breakage of neuronal branches. Nat Neurosci 7: 1181–3.

3. Spires, T.L., Meyer-Luehmann, M., Stern, E.A., McLean, P.J., Skoch, J., Nguyen, P.T., et al. 2005. Dendritic spine abnormalities in amyloid precursor protein transgenic mice demonstrated by gene transfer and intravital multiphoton microscopy. J Neurosci 25: 7278–87.

4. Naslund, J., Haroutunian, V., Mohs, R., Davis, K.L., Davies, P., Greengard, P., et al. 2000. Correlation between elevated levels of amyloid beta-peptide in the brain and cognitive decline. JAMA 283: 1571–7.

5. Masliah, E., Hansen, L., Albright, T., Mallory, M., Terry, R.D. 1991. Immunoelectron microscopic study of synaptic pathology in Alzheimer’s disease. Acta Neuropathol (Berl) 81: 428–33.

6. Knowles, R.B., Wyart, C., Buldyrev, S.V., Cruz, L., Urbanc, B., Hasselmo, M.E., et al. 1999. Plaque-induced neurite abnormalities: implications for disruption of neural networks in Alzheimer’s disease. Proc Natl Acad Sci USA 96: 5274–9.

7. Melchor JP, Mcvoy L, Van Nostrand WE (2000) Charge alterations of E22 enhance the pathogenic properties of the amyloid b -protein. J Neurochem 74:2209–2212.

8. Van Nostrand WE, Melchor JP, Cho HS, Greenberg SM, Rebeck GW (2001) Pathogenic effects of D23N Iowa mutant amyloid b -protein. J Biol Chem 276:32860–32866.

9. Murakami K, et al. (2003) Neurotoxicity and physicochemical properties of Ab  mutant peptides from cerebral amyloid angiopathy Ab Implication for the pathogenesis of cerebral amyloid angiopathy and Alzheimer’s disease. J Biol Chem 278:46179–46187.

10. Hou L, et al. (2004) Solution NMR studies of the ab (1–40) and ab (1–42) peptides establish that the met35 oxidation state affects the mechanism of amyloid formation. J Am Chem Soc 126:1992–2005.

11. Jang H, Zheng J, Nussinov R (2007) Models of b-amyloid ion channels in the membrane suggest that channel formation in the bilayer is a dynamic process. Biophys J 93:1938–1949.

12. Petkova A, Yau W-M, Tycko R (2006) Experimental constraints on quaternary structure in Alzheimer’s b-amyloid fibrils. Biochemistry 45:498–512.

13. Esler WP, et al. (2000) Activation barriers to structural transition determine deposition rates of Alzheimer’s disease Ab  amyloid. J Struct Biol 130:174–183.

14.  Lazo ND, Grant MA, Condron MC, Rigby AC, Teplow DB (2005) On the nucleation of amyloid b-protein monomer folding. Protein Sci 14:1581–1596.

15.  Baumketner A, et al. (2006) Structure of the 21–30 fragment of amyloid b-protein

Protein Sci 15:1239–1249.

16. Chen W, Mousseau N, Derreumaux P (2006) The conformations of the amyloid-_ (21–30) fragment can be described by three families in solution. J Chem Phys 125:084911.

17. Borreguero JM, et al. (2005) Folding events in the 21–30 region of amyloid-b-protein

(Ab) studied in silico. Proc Natl Acad Sci USA 102:6015–6020.

18. Baumketner A, Shea J-E (2006) Folding landscapes of the Alzheimer Ab 12–28 peptide. J Mol Biol 362:567–579.

19. Baumketner A, Shea J-E (2007) Structure of Alzheimer’s disease 10–35 amyloid b

peptide studied by molecular dynamics simulations in explicit water. J Mol Biol 366:275–285.

20. Esler WP, et al. (1996) Point substitutions in the central hydrophobic cluster of a human b-amyloid congener disrupts peptide folding and abolishes plaque competence. Biochemistry 35:13914–13921.

21. Zhang SS, Casey N, Lee JP (1998) Residual structure in the Alzheimer’s disease peptide: Probing the origin of a central hydrophobic cluster. Fold Des 3:413–422.

 

 




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