Abstract: Background: What is neural memory? Content Access Key:. The Molecular Basis of Neural Memory.
The Molecular Basis of Neural Memory. Part 4. “Binary” Computation vs “Multinary” Mentation
Volume 4 , Issue 1 , Journal Home. Graphical Abstract: Abstract: Background: What is neural memory? Close Print this page. Content: Citation Only. Citation and Abstract. Article Metrics PDF: Related Journals. Related eBooks. D Residues 88 to are sufficient for recruitment into pre-existing oligomers. Full-length Orb2A from D. To further explore the conformational variability in the monomer population and to rule out the possibility that resistant monomers were not simply oligomers that have dissociated, we obtained protein fractions containing primarily monomeric Orb2 by differential centrifugation.
PLDs are often organized such that two distinct regions mediate the initiation of aggregation and recruitment to the aggregate for self-perpetuation. To determine whether the Orb2A PLD has this organization, we studied the recruitment of red-labelled proteins into EGFP-labelled preformed aggregates using a cell fusion-based assay [ 39 ]. Surprisingly, the Orb2A protein from D. However, Orb2A from D. One surprising feature of PLDs is that although the domains can substitute each other, the amino acid sequence of the various PLDs are distinct.
Therefore, it remains unclear to what extent individual amino acids at various positions contribute to the prion-like properties.
Intriguingly, previously we found that a F to Y substitution at the 5th position of the Orb2A PLD—representing the addition of a single hydroxyl group—dramatically reduced Orb2 oligomerization and impaired memory consolidation [ 11 ]. This prompted us to determine the role of the 5th residue in amyloid formation and what aspects of amyloid formation e. To address these issues, the F5 residue was substituted for 18 different residues Fig 3A , and Orb2A puncta stability was quantified using fluorescence recovery after photobleaching FRAP.
To determine whether this 5th residue might also play an important role in oligomer packing, in addition to affecting the rate of Orb2A oligomerization, we performed fluorescence resonance energy transfer FRET assay. FRET efficiency depends on the distance between the fluorophores, which in turn depends on the orientation and packing of the protein subunits in the homo-oligomer. Thus, the 5th residue of Orb2A seems to mediate a key intramolecular interaction important for the oligomerization process [ 40 ]. A Schematic representation of the experimental design and the positions analyzed by the indicated methods.
E A single residue substitution in Orb2A, F5Y, dramatically reduced the ability to adopt the prion-like state. F Orb2A harbouring the F5Y mutation partitions less into the pellet fraction, and it forms less SDS-resistant oligomers than the wild type form. We then asked whether this perturbation in oligomer packing and stability affected the prion-like behaviour of Orb2A see Fig 1H. Taken together, these results corroborate the critical role of the F5 residue of Orb2A for its self-sustaining recruitment and amyloidogenic properties reported to be necessary for memory consolidation [ 11 ].
These results also suggest that there are inherent structural features determined by specific amino acid residues that control the Orb2A amyloid formation. The monomeric species of amyloidogenic proteins represent the starting point and a key determinant of the amyloidogenic cascade. The conformational changes that occur in the monomeric protein are difficult to study with bulk structural techniques because of the coexistence of many conformational states in a dynamic equilibrium.
However, single-molecule techniques, like atomic force microscopy-based single molecule force spectroscopy AFM-SMFS , allow the determination of the conformational diversity of these monomers [ 41 ]. We focused on the disordered region comprising the first amino acid residues of Orb2A-PLD S5A Fig as the sequence that is necessary and sufficient for prion-like behaviour and amyloid formation, as well as for memory consolidation [ 21 ].
As a single-molecule reporter, we constructed a fusion protein whereby the Orb2A PLD was mechanically protected inside the fold of a reference protein carrier , rendering it amenable to unequivocal single-molecule analysis S5B Fig. Furthermore, turbidimetry analysis, CR binding, and negative-staining TEM experiments corroborate that the PLD region maintains its amyloidogenic properties when protected inside the fold of the carrier S6 Fig.
Within the M class, a variety of monomeric conformers with different mechanical stabilities were observed. Like in other pathological amyloids [ 41 ], we observed extremely stable M conformers whose unfolding forces approached the values needed to break a covalent bond [ 44 ]. Intriguingly, it must be noted that some of these M conformers might be related to the prion conformers stabilized by strong noncovalent forces thought to account for prion inheritance and transmission [ 37 ].
This reduction is in agreement with the decreased ability of the F5Y variant to form self-perpetuating amyloids Fig 3 and S4 Fig and the failure to stabilize memory beyond 48 h [ 11 ]. B Immunodot blot showed that like toxic oligomeric intermediates of other amyloidogenic proteins, both full-length Orb2A, and to a lesser extent Orb2B, as well as their isolated PLDs, are recognized by the A11 antibody [ 45 ].
C A representative electron micrograph of aged Orb2A PLD shows the formation oligomers asterisks and typical unbranched amyloid fibers arrows resembling those of the full-length Orb2A see Fig 1G and pathological amyloids. Like pathological amyloids, those species are recognized by the fiber-specific OC monoclonal antibody [ 46 ].
The molecular basis of memory.
Furthermore, Orb2 shares additional features with pathological amyloids at later stages. Taken together, these data suggest that the Orb2A PLD monomer samples a wide conformational space, consistent with our TEV protease analysis Fig 2B and S3 Fig , and that Orb2A follows an amyloidogenic pathway reminiscent of pathological amyloids [ 41 ].
The similarities in the amyloidogenic cascade between Orb2A and pathological amyloids led us to wonder why, despite these common features, Orb2A is functional rather than toxic to the cell. One possibility is that the lifetime of the toxic oligomers formed by Orb2A and pathological amyloids is different. A Structural features during Orb2A assembly probed with conformational antibodies showed the formation of species reactive to A11 and OC antibodies.
E Survival curves of COS-7 cells microinjected with several samples the Areactivity of which is indicated. F Fluorescence micrographs of COS-7 cells microinjected with different samples and fluorescein-labelled dextran at 0 and 24 h after microinjection. Microinjection of the Orb2A:AmB complex resulted in a marked drop in the number of live cells at 24 h compared to those at 0 h.
Notably, the A11 antibody rescued cells from apoptosis. Orb2A and HttQ coaggregates. In the light of these data, and to determine whether the transient Orb2 oligomers are indeed cytotoxic, we used conformational trapping with two small inhibitors Fig 5C : EGCG [ 24 , 47 ] and Amphotericin B AmB [ 48 ]. We found that AmB slowed down Orb2A assembly and arrested it in the Areactive conformation, while EGCG seemed to direct the protein to the fibril state through a pathway that does not involve the formation of toxic Areactive oligomers Fig 5D.
Notably, incubation of the Orb2A:AmB complex with a very low concentration of the conformational A11 antibody complex:A11, attenuated this toxicity, indicating that toxicity was indeed due to the A11 specific conformation acquired by Orb2A Fig 5E and 5F , S9 Fig. To determine whether these differences in kinetic parameters are inherent features of nontoxic and toxic amyloids, we performed a domain swapping analysis between Orb2A and a disease-associated version of Huntingtin Htt exon 1 ex1HttQ Taken together, these results suggest that Orb2A can form a toxic conformer, which structurally resembles that of toxic amyloids.
However, intrinsic structural features in Orb2A-PLD renders this toxic conformation rare and transient; furthermore, it appears that Orb2A may have evolved to form mature amyloidogenic oligomers much more efficiently than pathological amyloids. Our observations suggest that in solution, Orb2 and polypeptides that form pathological amyloids sample similar conformational ensembles. We wondered whether in spite of being transient and rare, because of their structural similarity, these Orb2 transient toxic conformations could be hijacked by similar, yet longer-lived conformers from neurotoxic amyloids.
HttQ, unlike Orb2A, is neurotoxic, and its chronic expression produces lethality [ 50 ].
These results suggest that pathological amyloids, when present in excess, can nucleate Orb2A aggregation, presumably by capturing the transient toxic conformers of Orb2 proteins. The conformational similarity at the monomer level between Orb2 and HttQ prompted us to further examine whether QBP1 could inhibit Orb2A amyloid formation. Together, these results indicate that QBP1 interferes with Orb2 amyloid formation in vitro and suggest that early in amyloidogenesis, Orb2 and some pathological amyloid-forming polypeptides adopt similar conformations that are recognized and blocked by QBP1.
Based on the fact that QBP1 inhibits the transition from the monomeric Orb2A state to conformations that lead to amyloid formation, we tested in D. To this end, we used the male courtship suppression memory paradigm, in which a virgin male repeatedly exposed to unreceptive females learns to suppress courtship towards them, and this learned suppression persisted for days. QBP1 expression in the nervous system, or that of the control SCR, did not affect fly development, although normal courtship behaviour was slightly dampened S13 Fig. Since QBP1 is a low affinity peptide that can only block early stages of oligomerization but not already formed oligomer, we also investigated the consequence of transient expression of QBP1 in courtship suppression memory.
In this operant conditioning paradigm, flies learn to avoid one side of an otherwise symmetrical chamber [ 56 ].
Intriguingly, the heat box paradigm produces robust short-term memory, but the memory does not persist beyond an hour or two. Taken together, these results suggest that chronic QBP1 expression can interfere with some form of long-term memory and the effects of QBP1 in memory may be partly mediated through Orb2. Pan-neuronal expression of QBP1 disrupts long-term male courtship suppression memory, while it does not interfere with short-term memory. Amyloids were discovered and primarily studied in the context of disease or nonfunctional states of proteins.
The unique structural features of amyloids set them apart, endowing them with extraordinary stability and, in some cases, the unique capacity to self-perpetuate in a dominant manner [ 37 ]. The recent discovery of functional amyloids raises some fundamental questions as to what makes some amyloids pathological and others beneficial, how similar the structures of pathological and functional amyloids are, and how they are formed.
Here we have analyzed the structural states of Orb2A, an amyloidogenic protein that is important for memory consolidation. We find that Orb2A not only forms self-perpetuating amyloids but it does so extremely quickly and efficiently. Surprisingly, Orb2A has several structural features and properties that are similar to those of pathological amyloids Fig 8. First, the Orb2A monomer shows a rich conformational polymorphism with mechanical stabilities similar to those of the well-characterized pathological amyloids [ 41 ].
Second, Orb2 binds to the conformational antibody A11, which detects the toxic oligomeric species of pathological amyloids [ 45 ]. Third, like pathological amyloids, Orb2A can form unbranched amyloid fibers and pre-fibrillar oligomers that react against the conformational antibody OC [ 46 , 57 — 60 ]. Finally, Orb2A amyloid formation is inhibited by QBP1, a peptide capable of inhibiting amyloid formation in expanded polyQ tracts [ 41 , 51 ].
In Drosophila , the Orb2 protein top is a functional amyloid with self-sustaining prion-like properties that follows an amyloidogenic pathway resembling that of pathological amyloids bottom. During the assembly pathway, Orb2 and pathological amyloids can form Areactive toxic oligomers, and Orb2 can be sequestered by pathological amyloids to form hetero-oligomers. However, intrinsic structural features make Orb2 toxic oligomers rare and transient, suggesting that this functional amyloid appears to have been honed by evolution in adopting a self-sustaining amyloid-like state much more efficiently than pathological amyloids in order to avoid cytotoxicity and perform its role in memory consolidation.
This function is supported by the amyloid state of Orb2, since its inhibition by an anti-amyloidogenic peptide QBP1 selectively interferes with the long-term persistence of memory. Orb2 amyloid-like oligomers are formed in response to neuronal stimulation to support memory, which indicates the existence of a highly regulated mechanism in the cell to drive Orb2 amyloid formation and most likely to avoid the toxicity of its Areactive oligomers.
One such regulation is the tight control of the Orb2A protein level via phosphorylation [ 20 ] and possibly other mechanisms. Here we show the existence of additional intrinsic kinetic factors in Orb2A, i. We postulate that although the early events in Orb2 amyloid formation are similar to those of pathological amyloids, in subsequent steps it acquires structural features to rapidly evolve to a nontoxic amyloid-like state Fig 8. For Orb2 proteostasis, these intrinsic structural features could be further enhanced in the cell by association with other components or by the direct modification of proteins, such as phosphorylation [ 20 ].
Both the presence of Areactive oligomers and the ability to form amyloids efficiently have been also reported for the intracellular nonpathological amyloids of the yeast Sup35 prion [ 48 ], which suggests that this may be a general mechanism for some amyloids to avoid cytotoxicity. This distinction is fundamental for two reasons: first, the nervous system is particularly susceptible to amyloid-based disease and; second, amyloids are known to interfere with cognitive abilities including memory.
The persistence of memories over months and years raises the fundamental question of how memory is protected against molecular turnover [ 61 ]. Structural changes in individual proteins and in supramolecular protein assemblies have been proposed to play an important role in long-term memory [ 62 , 63 ]. The prion features of the specific neuronal CPEB isoform, such as Orb2 in Drosophila [ 11 ], or CPEB3 in mouse [ 9 , 12 ] that can adopt a self-sustaining state at synapses very rapidly, provide a plausible solution as to how memories can remain stable in the face of constant molecular turnover [ 10 , 16 , 64 ].
Although exactly to what extent the self-sustaining amyloidogenic properties observed in vitro reflect the in vivo properties remains unclear, accumulating evidence reinforces the hypothesis that an amyloid-like state of neuronal CPEB is involved in long-lasting memory. Similarly, the expression of QBP1, an amyloid-blocking peptide, impairs memory consolidation Fig 8. However, we are not sure whether the memory-disrupting effect of QBP1 is applicable for all forms of long-term memory or whether the effects of QBP1 in memory are mediated exclusively through the Orb2 protein.
Furthermore, in the absence of atomic-level 3D structural analysis of endogenous CPEB protein in the amyloid-like state, it remains unclear exactly to what extent they are similar to toxic amyloids. Our observations also highlight certain features of functional amyloids that may be relevant for neuropathologies of amyloid-based diseases. First, the short life span of Orb2 toxic oligomers supports a therapeutic approximation based on speeding up the conversion of toxic oligomers into stable amyloids [ 65 ]. Third, anti-amyloidogenic compounds that target early stages of amyloidogenic pathways might inadvertently interfere with functional amyloid formation, impairing their associated function.
Finally, the polyvalent amyloid blocker QBP1, or improved analogues based on its recently determined structure [ 66 ], could be used in the future to block newly formed traumatic memories Fig 8 [ 67 ]. However, we are cognisant of the inherent limitations of our ideas since none of our studies are carried out with human proteins in the human nervous system. These anticipated connections of our in vitro findings with memory need to be explored in detail. The ThT binding assay was performed essentially as described previously [ 68 ].
The reaction mix was excited at nm, and the emission at nm was measured using a Fluoromax-4 spectrofluorimeter, and three independent measurements were taken for each sample. The instrument was corrected for ATR penetration. Spectra were collected at 2 cm -1 resolution and averaged over 64 scans. Pellets were spread on the ATR and dried with a stream of air before each measurement. Spectra were corrected for water vapor by subtracting the background collected immediately after each scan scaled to fit the local shape of the spectrum in the 1,—1, cm -1 region.
The liquid water spectrum was subtracted by matching the signal at 2, cm -1 to a liquid water spectrum via linear least squares. A linear background between 1, and 1, cm -1 was subtracted, and the spectrum in this region was normalized to obtain the final spectrum.
The resulting spectra were fit to four Gauasian peaks with nonlinear least squares. Finally, the 1, cm -1 band with a width fixed at 13 cm -1 was assigned to turns. For the chimeric yeast prion assay, we have used the methods developed previously [ 69 ]. Selected colonies were grown in YP-glycerol plates to avoid petites. To discard that phenotypes are not due to mutation in the Orb2-Sup35C, the plasmid was isolated from both cell types and sequenced, and white phenotypes were rescued by transforming the cells with plasmid expressing just the C-terminal domain of Sup35 protein.
To determine the heritability of the prion-like state, individual red or white colonies were streaked for multiple times. For western blotting, 7. For TEV protease digestion, approximately four times the volume of the pellet fraction was used compared to the supernatant fraction to normalize for the amount of Orb2A and Orb2B present. All anti-Orb2 antibodies were raised against recombinant six histidine-tagged full-length Orb2A proteins.
The antibody was raised in rabbits Covance , and antibody was raised in guinea pigs Pocono.
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All antibodies were affinity purified against Orb2A protein. For western blots, the antibodies were used at the following dilutions: Ab, , dilution; Ab, , dilution; and Ab, , dilution. For all biochemical analyses, freshly prepared head extracts were used unless mentioned otherwise. The antibody-antigen interaction was visualized by chemiluminescence using HRP-coupled anti-rabbit for Ab or anti-guinea pig Ab secondary antibodies Pierce. After 2 h, the lysates were incubated with protein A beads Repligen for a further 2 h, and the beads were washed five times with the PBS buffer I.
The protein bound to the beads was analyzed in western blots as described above. Pre-equilibrated Streptavidin Paramagnetic Beads Roche were added to each binding reaction, and the mixture was incubated for a further 40 min. The supernatant and pellet fractions were prepared as described above. The cotransfected cells were plated the next day on concanavalin-A-coated glass-bottom dishes Mattek and analysed by confocal microscopy.
The Orb2 constructs were cloned into the Pac5. One day later, the cotransfected cells were mixed, and fusion was initiated by adding CuSO 4 to the media, thereby inducing EFF-1 expression. After 16 to 18 h, the cells were plated on concanavalin-A Sigma -coated mattek glass-bottom dishes, and the cells containing both GFP and Cherry were detected manually by confocal microscopy.
Orb2A PLD sequence was also analyzed by applying the Rosetta-Profile algorithm, which identifies sequence segments that form the steric-zipper spines of amyloid fibrils [ 71 ]. The day before performing microinjections, cells were plated on a 35 mm dish at a density of 1 x 10 5 cells per dish. Just before single-cell microinjection, Orb2A protein 2.
Over the following 3 d, the number of fluorescein-positive cells was counted every 24 h to calculate the cell survival rate. The proteins were equilibrated in PBS pH 7. The apparent heat of reaction for each injection was obtained by integration of the peak area. The heat developed with the protein or peptide dilutions was determined in separate runs, loading the sample cell or the injection syringe with buffer in the conditions used for the binding experiments.
The complexity of the system and the lack of precise information on the distribution of the Orb2A conformations before and after complex formation precluded the quantitative analysis of the titration curves. Various genetic combinations were made by standard genetic crosses.
Flies expressing HttQ were previously described [ 50 ]. For the behavioural assays, the flies were maintained using standard fly husbandry. All controls and transgenic lines carried one copy of the mini white gene known to influence fly behaviour. The male courtship conditioning assay was modified from that described previously [ 73 ].
A five-d-old male virgin was paired with a freshly mated female for three sessions of 2 h each, with a 30 min rest period in between. Memory performance was tested with a fresh-mated female 5 min and 24 h after the three sessions of spaced training. A courtship index CI was measured as the fraction of time the tested male spent chasing the female in a 10 min interval. The Memory Index was calculated as: , where CI Naive and CI Trained are the mean courtship indices for independent samples of naive and trained males, respectively.
GraphPad Prism version 4. The heat box apparatus was built in the workshop of Konrad Ochsner at the Universitiy of Wuerzburg and is a modified version of the one used by [ 74 ]. In brief, the heat chamber system consists of an array of 16 chambers length, 29 mm; width, 4 mm; height, 2 mm operated in parallel. Upper and counter ceilings are equipped with Peltier elements that control the temperature in the chamber.
Glass side walls allow the transmission and detection of infrared light from an LED source invisible to the flies. When a fly walks along the length of the chamber, it casts a shadow on a bar code reader light gate array , and this signal is sent to a computer. The fly position signal from the bar code reader is sent to the computer with a frequency of 10 Hz. All experiments were conducted in complete darkness. Measurements are performed on at least three different days to avoid effects of daily variability. The experiment consists of a preference test, training and memory test.
A computer controls the experiment in all three phases by continuously monitoring the time and direction of transition at the light gate. One half of the chamber is defined as the punished side and the other half as the unpunished side. These designations are switched for every experiment to avoid aftereffects of previous experiments e.
The performance index PI is calculated as the difference between the time the fly spent in the unpunished versus that in the punished side of the chamber divided by the total time. The PI is a measure of heat avoidance and memory score during the training and memory test, respectively. To yield a measure of general activity, the sum of position changes over time is calculated. The time spent on a given side is measured, and heat avoidance indices are calculated as above PI.
All animals that do not show substantial motor activity or do not experience punishment were excluded from the study. A complete list of the oligonucleotides used here can be found in S2 Table. A Nine purified recombinant proteins were used as controls to assess the specificity of the ThT binding assay under our experimental conditions.
Recombinant Orb2A was purified under denaturing conditions, and it was then allowed to refold in the presence of EGCG, rottlerin, or chloroquinone. Surprisingly, chloroquinone, which is effective against mammalian PrP amyloid had almost no effect on Orb2A oligomerization. Orb2 colonies were grown to mid log phase, and the supernatant S and pellet P fractions were analyzed in 1. B Similar distribution of Orb2A top and Orb2B bottom , with or without the TEV protease recognition sequence, in the supernatant top and pellet bottom fractions. The expression of the TEV protease was induced by adding 0.
The studies of adult brain lysates suggested that the monomeric Orb2 proteins may also have distinct protease accessibilities. The N-terminal fragment of Orb2A red derived from the pellet fraction is not recognized by Ab middle panel but it is recognized by Ab, whereas both antibodies recognize the same fragment that partitions to the supernatant. Unlike Orb2A, in Orb2B the N-terminal fragment derived from both the pellet and supernatant fractions is recognized by Ab and Ab C The F5Y mutation reduces amyloid formation, as assessed by measuring the concentration of bound CR dye.
D A representative TEM micrograph shows that the presence of oligomers and amyloid fibers was drastically reduced by the F5Y mutation. Compared to the spectrum of I27 that carries a small loop resulting from the translation of the multicloning site IMCS, black , in the IOrb2A PLD spectrum blue the signals of native I27 are retained but somewhat broadened due to the slower tumbling, indicating that I27 remains folded while Orb2A PLD is still disordered. Some resonances are labelled that are representative of folded I27, such as Ala 82 and Leu 84 in the "mechanical clamp," or Trp 34 and Ile 23 in the hydrophobic core.
A Aggregation of both isolated and carrier-fused Orb2A PLD over the incubation period monitored by turbidimetry at nm. The polyprotein encoded by the pFS-2 vector also contains a random coil region a fragment of titin N2B, in grey that acts as a spacer to avoid the noisy proximal region of the force-extension recordings, a major problem in AFM-SMFS [ 77 ]. The elasticity of the stretched proteins was analyzed using the WLC model of polymer elasticity [ 78 ].
A The average number of force peaks structures per molecule is shown, where 0 represents the NM conformations. The M conformers usually show more than one force peak per Orb2A PLD molecule, suggesting the formation of several mechanical barriers at different positions of the molecule. Therefore, it is reasonable to assume that its F value would be higher than the detachment force indicated by the arrow. The spectrum shows the force peaks due to the unfolding of the I27 carrier and a region of the Orb2A PLD that is shorter than the expected full-length protein 61 nm versus F detachment versus of putative events analysis bottom suggests that the population of M conformers actually observed in our experiments is an underestimation.
The presence of putative data i. Putative data were not included in our sample size n. D The average number of force peaks structures per molecule is decreased in the F5Y variant. F Putative incomplete recordings are fewer and of lower mechanical stability in the presence of the F5Y mutation suggesting the formation of fewer, weaker M conformers.
A Negative controls do not cause cell death after microinjection. C Survival curves of COS-7 cells microinjected with the chimeric proteins their Areactivity at microinjection time is indicated in an inset. B Incomplete recordings of putative M events decrease in the presence of QBP1 inhibitor peptide, which suggests the formation of fewer, and weaker, M conformers.
The genetic controls for each group were tested simultaneously, and the number of flies used in each group is indicated. Following drug feeding the flies were trained in the male courtship suppression paradigm and memory was measured at 24 h. Both control -RU and experimental groups showed significant courtship suppression at 24 h, suggesting that long-term memory was not affected by the transient expression of QBP1.
The number of flies tested in various groups are indicated. Schematic of the single training protocol in the heat box operant conditioning paradigm. In this operant conditioning paradigm, individual flies were conditioned to avoid one side of a uniform chamber [ 74 ]. Following training, the flies learn to avoid the punish side even in the absence of punishment. Memory is measured as the duration of the preferred response for unpunished side. Flies are either trained in a single trial protocol or multiple trial protocol where the single training protocol is repeated successively the indicted times.
The avoidance index after each training session. With repeated training, the avoidance index increases, and after six training sessions the performance plateaus. There was no significant difference in the performance between wild type flies and flies expressing QBP1 chronically in the nervous system. NM, Nonmechanostable. M, mechanostable. The values are only shown in the NM column since they are the same as for the M states.
The restriction sites introduced by PCR into the amplified sequences are in italics. The extra sequences added to each restriction site were recommended by New England Biolabs to enhance the digestion efficiency of linear DNA sequences. The vectors and strategy used are the same as those described previously [ 41 ]. We thank J. Pons, J. Oroz, and the members of M. We are grateful to M. Valpuesta for the access to his glow-discharge apparatus, and to J.
Molecular Basis of Neural Memory— Reviewing 'Neuro-Mimetic' Technologies
Lucas for providing us with the HttQ72 clone. Abstract Amyloids are ordered protein aggregates that are typically associated with neurodegenerative diseases and cognitive impairment. Author Summary Amyloids are ordered protein aggregates typically associated with neurodegenerative diseases, such as Alzheimer disease and Parkinson disease, which usually result in cognitive impairment. Results Orb2 Forms Canonical Amyloids Very Efficiently Both the endogenous and the recombinant Orb2 protein form sodium dodecyl sulphate SDS and urea-resistant oligomers that are characteristic of amyloids [ 11 ].
Download: PPT. Prion-like Properties of Orb2A In order to determine whether Orb2A amyloid possesses the self-sustaining properties of prion-like proteins, we took advantage of the well-characterized yeast prion Sup35, a translation termination factor that can exist in two states: as an active monomeric state nonprion and as a less active amyloid state prion [ 26 ]. Dissection of the Orb2A PLD PLDs are often organized such that two distinct regions mediate the initiation of aggregation and recruitment to the aggregate for self-perpetuation.
Fig 3. The F5 residue in Orb2A is critical for Orb2 amyloid-like oligomer formation and self-sustaining prion-like properties. Fig 4. Orb2 shares common features with the pathological amyloidogenic pathway. Orb2 Forms Transient Toxic Oligomers The similarities in the amyloidogenic cascade between Orb2A and pathological amyloids led us to wonder why, despite these common features, Orb2A is functional rather than toxic to the cell.