Category Archives: Neuro

Breaking Neuronal Symmetry by Chromatin Memories

The asymmetric fates of two bilaterally symmetrical neurons are determined by a two-step activation program at a miRNA locus. Very low levels of transcription ‘prime’ the locus many cell generations before the final fate determination is imposed by a bilateral ‘boost’.

Animal nervous systems are generally bilaterally symmetrical anatomically, whilst displaying many functionally important left-right asymmetries. How is asymmetry imposed on a bilaterally symmetrical ground plan? The nematode C. elegans with its invariant cell lineage and tractable genetics offers a powerful model system in which to tackle this issue. Cochella and Hobert have published an elegant new paper describing how a distinct chromatin state at a microRNA locus serves as a molecular mark encoding a memory of a cell’s ancestry in an asymmetric lineage.  After many cell generations, this mark engenders a different response to terminal differentiation from its’ bilaterally symmetric partner cell.

The bilaterally symmetrical pair of gustatory neurons ASEL(eft) and ASER(ight) express different repertoires of chemoreceptors. This functional asymmetry is underpinned by the differential expression of two transcription factors. DIE-1 is expressed in ASEL and COG-1 in ASER. Together with a microRNA, lsy-6, which represses COG-1 expression, they form a bistable feedback loop responsible for determining the asymmetric fates of ASE neurons. Loss of any of these three factors results in conversion of one ASE to the other. However, the asymmetric expression of die-1 and cog-1 only occurs within the post-mitotic neurons themselves. How then is this asymmetry established?

A schematic representation of the features of asymmetric ASE specification. Note the original asymmetry in the lineages is determined at the 4 cell stage, tbx-37/38 expression in the great-granddaughters of ABa, and lsy-6 in the loop in ASEL.

A schematic representation of the features of asymmetric ASE specification. Note the original asymmetry in the lineages is determined at the 4 cell stage, tbx-37/38 expression in the great-granddaughters of ABa, and lsy-6 in the loop in ASEL.

The two ASEs are derived from different cell lineages that diverge at the 4-cell stage. The two daughters of the 2-cell stage blastomere AB, ABa and ABp, differentiate from each other due to signalling from one of the other 4-cell stage blastomeres to ABp. This signalling event represses the expression in the ABp lineage of a pair of redundant transcription factors, TBX-37 and TBX-38, which are transiently expressed in the 8 great-granddaughter cells derived from ABa. The expression of these TBX proteins is crucial to the asymmetric fate specification of ASE neurons as in tbx-37/38 double mutants ASEL is converted into ASER. However, TBX-37 and 38 are only expressed in the lineage giving rise to ASEL six cell generations before it’s birth. This large gap between the different stages of asymmetric ASE determination lead researchers to postulate the existence of a ‘memory mark’ linking TBX-37/38 action to the expression of the asymmetry defining feedback loop.

During this hiatus between TBX-37/38 expression and terminal ASE determination, the lineages giving rise to the two neurons become symmetric. A number of left/right pairs of neuronal precursors expressing the proneural gene hlh-14 develop from the two lineages, but only the pair of ASE mother cells express the ‘terminal selector’ transcription factor CHE-1. CHE-1 drives the expression of many ASE-expressed genes and activates expression of the asymmetric loop components, lsy-6, die-1, and cog-1. It is at this point that the TBX-37/38-dependent memory mark must integrate into the bilateral activity of CHE-1 generating the asymmetric expression of the loop components.

To try to discover the nature of the memory mark Cochella and Hobert performed a detailed analysis of the expression of the loop components. lsy-6 was suggested to act upstream of die-1 and cog-1 by genetic experiments, and the researchers found that it was the first of the loop components to be expressed.  It is expressed asymmetrically from the start in the ASEL mother cell. Deletion of lsy-6 results in conversion of ASEL to ASER. A construct of lsy-6 in combination with 932 bp of upstream sequence is able to rescue this effect, but sometimes leads to the conversion of ASER to ASEL. This suggested that the ‘upstream element’ construct drove ectopic expression in ASER as well as ASEL. Indeed, the upstream element contains CHE-1 binding motifs causing expression in both the ASE neurons. Cochella and Hobert therefore assayed other lsy-6 surrounding sequence for cis-regulatory information limiting its expression to ASEL. In fact, a construct including the upstream element, lsy-6, and 300 bp of downstream sequence completely rescued lsy-6 null alleles, eliminated ectopic ASER conversion, and was expressed identically to the endogenous miRNA. Normal lsy-6 expression is therefore regulated by both the upstream and downstream elements.

When the downstream element was used alone to drive expression of a reporter gene, it produced a very different pattern. Expression started early in a few ABa-derived blastomeres, one cell division after the expression of tbx-37/38, continuing in the progenitive lineage of ASEL until its’ birth.It never drove expression in ABp derived lineages. Expression from the downstream element was completely lost in tbx-37/38 double mutants, whilst mis-expression of TBX-37/38 in ABp derived cells lead to ectopic expression of the downstream element reporter. The downstream element contains a predicted binding-site for T-box proteins, directly linking the lineage–dependent expression of tbx-37/38 with theasymmetry-defining loop.

The expression pattern driven by the downstream reporter suggested that lsy-6 may be expressed far earlier than previously observed. The researchers therefore used a very sensitive technique to image potential lsy-6 transcripts. This showed that a few lsy-6 RNAs were present in cells in the lineage giving rise to ASEL five generations before strong expression is observed in the ASEL mother cell.

Broadly therefore, lsy-6 expression occurs in two phases; a very low level of activation early, dependent on the downstream element, and a second upstream element-dependent higher level of expression in the ASEL mother cell. However, deletion of the downstream element within large genomic constructs abrogated expression at all stages, and failed to rescue lsy-6 null alleles. This contrasted with earlier observations in which the upstream element alone could drive expression and rescue. The difference between these observations suggested  that, within a normal genomic context, the upstream element can only function in combination with the downstream element.

The authors therefore posited a model in which early downstream element/tbx-37/38– dependent transcription may ‘prime’ the locus in some way, rendering it competent to respond to the later transcriptional ‘boost’ mediated by CHE-1 acting on the upstream element.

Cochella and Hobert tested their model by substituting priming via tbx-37/38/downstream element for priming via ectopic CHE-1. In worms with the downstream element deleted, they drove early expression of CHE-1 from a heat-shock promoter approximately 4 cell generations before its’ normal time of expression. This caused low levels of lsy-6 transcription, rescuing the priming phase and allowing later ASEL expression and determination. Priming is therefore not dependent on a specific transcription factor acting on the downstream element, rather as long as low levels of transcription occur at the locus, it is primed.

This suggested that the memory mark causing the different response of the lsy-6 locus may be a lineage-specific transcription-dependent chromatin state. Using a cunning technique to visualise the level of chromatin compaction on transgenic arrays containing the lsy-6 locus, they observed chromatin decompaction of thelocus in the ASEL progenitive lineage 1 cell division after tbx-37/38 expression. Chromatin decompaction is associated with active genes; in the absence of early transcription the locus becomes compacted and refractory to CHE-1 activation later. In tbx-37/38 double mutants this lineage-specific decompaction was never observed, nor was it seen when the downstream element was deleted.

The memory mark is therefore chromatin decompaction at a miRNA locus linked to very low levels of transcription imposed within a cell lineage at an early stage of development. This primed state relays asymmetric information into an otherwise bilaterally symmetrical developmental program, facilitating diversification of neuronal cell fates. The timing of the priming mechanism fits in with earlier evidence that C. elegans embryos are relatively developmentally plastic until the 64-128 cell stage when developmental genes become compacted and refractory to ectopic activation.

Although I find this paper very elegant and convincing, I do have a few qualms about the most crucial experiment: the early ectopic activation by CHE-1. It seems like a slightly dirty experiment and I think I would’ve preferred to see ectopic induction of lsy-6 transcription via an unrelated mechanism. Perhaps experiments such as these would’ve had their own problems and my doubts are unfounded. I would also have liked to see the compaction assay performed with the ectopic CHE-1 induced activation.

The demonstration of a chromatin-based lineage specific prepattern facilitating differential responses to more generic inputs later in embryogenesis has wide implications, not just for asymmetries in the worm nervous system, but for the way we understand development in many animals. Firstly a technical point; to visualise early lsy-6 transcription the authors had to use a very labour intensive and hi-tech form of in situ hybridisation. The transcription they found, of just a few individual RNA molecules per cell, had massive developmental significance. Generally the techniques used to judge expression in developmental studies is nowhere near as sensitive, implying that we may be missing a lot of important information. Secondly, a more general point; A cell or tissues’ ‘competence’ to respond to developmental signalling, a concept derived from experimental embryology, and perhaps disdained in more genetical perspectives is relevant here. Molecular memories encoded by chromatin states may be a very widespread mode for imposing pre-pattern or developmental competence during embryogenesis. It seems to me that these types of understandings can begin to blend together the two different meanings of epigenetics; namely the derivation of the word by Waddington from epigenesis (meaning the increase in complexity during development), with the more current usage of epigenetics as describing a diverse collection of non-genetic inherited information.

Cochella, L., & Hobert, O. (2012). Embryonic Priming of a miRNA Locus Predetermines Postmitotic Neuronal Left/Right Asymmetry in C. elegans Cell, 151 (6), 1229-1242 DOI: 10.1016/j.cell.2012.10.049

Barcoding the Brain

A new DNA-sequencing based idea for mapping the connectome is presented in Plos biology.

The defining purpose of neurons is information transmission and processing within a network. Hence, to appreciate neural function we must look at the interactions between neurons; understand their connections; know which other neurons they synapse with.

The idea of documenting all the connections within brains – mapping the ‘connectome’ – is receiving a lot of attention (and money) at the moment. However, the only comprehensive technique so far available is the painful reconstruction of the synaptic map from electron micrographs of serially sectioned brains. Seeing as the human brain is estimated to contain 85 billion neurons making 1014/1015 synaptic connections, one does have to wonder whether even if we could accurately document the connectome and afford it, would we really appreciate it?

The only organism to have had it’s connectome documented in this way is the nematode worm, C. elegans. Mapping its’ 302 neurons and their 7000 connections required over 50 person-years of labour. Despite the utility of this information, and the intrinsic glory of the knowledge, it should be noted that it most certainly can’t be said that ‘we understand the C. elegans nervous system’.

Another, very elegant technique being developed, with the capacity to map connectomes at the mesoscopic level, is the use of rabies-type viruses. These viruses can be transferred across synapses, but engineered to only do so once. Carrying fluorescent protein encoding reporter genes, they can be used to track the connecting neurons within a network. As one can engineer these viruses to carry allsorts of genetic trickery, the mapping of networks can go hand in hand with functional experimentation.

The trans-synaptic transfer of rabies viruses, along with the use of randomising recombination to create hundreds of different combinations of fluorophores in ‘brainbow’ neuronal imaging, are major inspirations underlying a new theoretical connectome mapping technique laid out by Zador et al.

The technique, termed BOINC (barcoding of individual neuronal connections) converts the mapping of the connectome from an anatomical problem into one that can be tackled by DNA sequencing. As the costs of sequencing are currently dropping through the floor, BOINC would make mapping the connectome a repeatable assay rather than a one-off mega-mission.

The method can be divided into three phases. In the first stage, each neuron is labelled with a unique DNA sequence – a barcode. The authors calculate that a random sequence of 20 nucleotides would be sufficient to individually label the entire neuronal complement of the mouse brain (1012 possible sequences to <108 neurons). Zador et al. are sketchy about the specifics of how this could be achieved; suffice to say that it’s conceptually similar to the generation of antibody diversity by recombination.

The second stage is the association of barcodes from synaptically connected neurons. This would be achieved by the single transynaptic spread of rabies-like viruses. The barcode would therefore be carried within the virus genome. In the third phase, the barcodes must be joined together. Each neuron will therefore contain its’ own barcode combined with barcodes from every cell that it synapses with. These tags would then be sequenced – yielding a connectivity matrix.

This term perhaps clarifies the biggest shortcoming of the technique; it creates a matrix, devoid of anatomical or functional detail. However, these dimensions would surely become gradually coupled with repeated experimentation.

In short, BOINC is a very clever idea. If they pull it off, it will be a great advance, allowing cheap, repeated screening of the brain’s circuitry. Obviously, it’s unlikely to be much help for studying the human brain, but providing the technical hurdles are surmountable it could revolutionize the neurobiology of model systems such as the mouse.

Zador, A.M., Dubnau, J., Oyibo, H.K., Zhan, H., Cao, G.,Peikon, I.D. (2012). Sequencing the Connectome Plos Biology, 10 (10) : 10.1371/journal.pbio.1001411

The original paper is available here

Check out some excellent blogposts by Mo Costandi at Neurophilosophy; A book review discussing the connectome concept (with special bilious email from Andrew Lumsden), and descriptions of work using Rabies viruses for mapping connectivity and functional neurobiology.

piRNAs in the brain: epigenetics and memory

An exciting new paper in Cell, links Piwi-interacting RNAs (piRNAs) to long-term memory via the epigenetic regulation of gene expression by DNA methylation.

Two different novel findings are especially important: piRNAs had been thought to be a germline specific mode of genetic control, specifically a type of genetic immunity against the mobilisation of transposable elements. Rajasethupathy et al demonstrate that in the sea hare, Aplysia, piRNAs are expressed in the CNS and other somatic tissues. Secondly, this paper demonstrates specific dynamic de novo methylation of the promoter of a gene regulating neuronal plasticity in response to neurotransmitter- mediated excitation. This provides an epigenetic mechanism by which memories can persist by molecular encoding.

To investigate microRNAs expressed in the Aplysia CNS, Rajasethupathy et al had constructed a small RNA library. Surprisingly, they found that ~20% of the sequence reads from this library were ~28nt long, and showed a bias towards having 5′ uridine residue. This fitted with them being Piwi-interacting RNAs (piRNAs) rather than microRNAs. When they were mapped to the Aplysia genome, it was clear that the piRNAs were generated from piRNA clusters (see previous introductory post). After constructing more libraries and deep sequencing, Rajasethupathy et al. identified 372 distinct Aplysia piRNA clusters. Certain piRNAs are found far more commonly than surrounding piRNAs from the same cluster, indicating that an amplification process is occurring during piRNA biogenesis. Although overall piRNA content was highest in the ovotestes (ie the germline and associated somatic tissues), various piRNAs were found to be enriched in the CNS, as well as other somatic tissues analysed.

Consistent with the presence of piRNAs in the CNS, Rajasethupathy et al. were able to clone a full-length cDNA for Piwi protein from Aplysia CNS. Using an antibody against Piwi, they were then able to co-immunoprecipitate piRNAs with Piwi protein from the CNS. By separating cell nuclei from cytoplasm and then western and northern blotting against Piwi and piRNAs respectively, they showed that both were primarily found in nuclei.

To briefly comment on the piRNA aspect of this paper; a number of outstanding questions arise. In the best characterised model systems, piRNA amplification occurs by the ‘ping-pong’ mechanism in which reciprocal recognition and cleavage reactions between sense and antisense piRNAs complexed with the (cytoplasmic) Piwi-related proteins Aubergine and AGO3 (Drosophila terminology), leads to selective amplification of piRNAs with a tell-tale 10nt offset. Neither of these proteins, nor the 10nt offset, nor the ratio between sense and antisense piRNAs are mentioned by Rajasethupathy et al. I imagine that these questions would’ve been looked into and mentioned if found, therefore it seems that the mechanism of piRNA amplification in the Aplysia CNS is potentially novel.

To explore possible functions of piRNAs in the Aplysia CNS, the researchers used a co-culture system that monitors changes in synaptic plasticity in response to stimulation by the neurotransmitter serotonin (5HT). In this assay, two sensory neurons synapse with a motor neuron. Changes in the strength of one sensory-motor synapse are monitored by electrophysiological recording from the motor neuron. This system measures long-term facilitation (LTF): ie. changes in synaptic strength in response to 5HT stimulation. LTF is considered to be a memory-related phenomenon, however, it is contentious just how well it serves as a paradigm for long-term memory.

Knockdown of Piwi (and hence of complexed piRNAs), by the injection of antisense oligonucleotides into one of the sensory neurons, significantly impaired LTF, whilst overexpression of Piwi enhanced it. To investigate how these Piwi effects on LTF were mediated, Rajasethupathy et al. looked at the expression of proteins known to be regulating synaptic plasticity in response to Piwi knockdown. Only one of the assayed proteins was responsive: CREB2, a transcriptional repressor, known to be a major inhibitory constraint on LTF, was upregulated in response to Piwi knockdown. Interestingly, an even greater increase in CREB2 mRNA was observed.

The fact that Piwi knockdown led to an increase in CREB2 mRNA, and it’s nuclear localisation, suggested that rather than acting post-transcriptionally (ie by degrading mRNAs as in Drosophila), Piwi/piRNA complexes appeared to be inhibiting CREB2 gene expression at the DNA level.  It is known that in mice Piwi/piRNA complexes act to silence transcription by facilitating DNA methylation. Rajasethupathy et al therefore asked whether CREB2 regulation by Piwi occurred via DNA methylation.

The enzyme responsible for methylation of cytosine residues in CG dinucleotides, DNA methyltransferase (DNMT), was known to be expressed in the Aplysia CNS. Inhibition of DNMT activity (using a pharmacological reagent) led to a strong increase in the level of CREB2. In the normal LTF experiments, CREB2 levels are reduced 12 hours after exposure to 5HT and remain low until 48hrs. This downregulation of CREB2 was abolished when DNMT activity was inhibited, as was 5HT-dependent LTF. This led the researchers to search for CpG islands in the promoter region of CREB2 that could be sites for DNA methylation mediated transcriptional control. Indeed, they identified a CpG island in the CREB2 promoter region that normally exists in both methylated and unmethylated states. After 5HT exposure, this CpG island is almost entirely methylated, whilst in the presence of the DNMT inhibitor it becomes almost entirely unmethylated. This 5HT-dependent methylation of the CREB2 CpG island requires Piwi, as it was abolished when Piwi was inhibited. The authors then went on to search for candidate piRNAs that could be responsible for mediating this effect, by searching for those with complementarity to the CREB2 promoter. They identified 4, one of which, aca-piR-F, when knocked down caused an increse in CREB2 expression. Notably, Rajasethupathy et al. did not demonstrate the expected result that aca-piR-F knockdown would lead to demethylation of the CREB2 CpG island, although this experiment was surely attempted.

In conclusion, this paper offers a broad outline for a mechanism of memory encoding; it connects neurotransmitter synaptic stimulation with the stable epigenetic marking of the transcription state of an important regulator of neuronal plasticity, via the action of Piwi/piRNA complexes. It should be noted that ‘epigenetic’ is used in this context in a loose definition with reference only to a stable marking of cellular state. Strictly ‘epigenetic’ should refer to heritable non-genetic changes, but as neurons do not divide that is inapplicable. In this case DNA methylation is a relatively long-lasting mark. However, for instance the change in CREB2 expression with respect to 5HT-stimulated long term facilitation only lasts a couple of days – does this correspond to the level of methylation in the promoter? In which case, one gets an impression that DNA methylation and demethylation are highly dynamic processes in the Aplysia CNS. Currently there are three modes proposed to resolve the difficult question of how memories can persist for a long time, whilst the cellular components that must mediate them have a high rate of turnover: Prion-like synaptic marks, autoregulatory loops that can maintain a cell state whilst their components come and go, and epigenetic mechanisms that can alter gene expression in a long term manner. This paper shows a clear example of the latter mode, but the apparent dynamism of DNA methylation in this system suggests a lack of permanence.

Although I like the way this paper has ranged over a large terrain and connected so many disparate elements, by necessity it raises many questions and leaves many aspects of the work unmentioned. I’ve already mentioned some questions about Aplysia piRNAs; no doubt a fully annotated Aplysia genome will answer some of them. A few other questions and future directions spring to mind: The authors haven’t quite shown that DNA methylation is responsible for the transcriptional silencing at the CREB2 promoter, only correlated it. Likewise the mode by which Piwi/piRNA complexes act to promote DNA methylation is unclear. A wider question is the nature of DNA methylation in Aplysia and other invertebrates. Some invertebrates show virtually no DNA methylation (C. elegans, Drosophila) whilst the majority display mosaic patterns quite different from those found in vertebrates. This suggests functional differences, and without deeper knowledge of the role of DNA methylation in Aplysia it is difficult to guess how widely applicable these findings are in other systems. Likewise the finding that piRNAs are acting at the level of DNA methylation, previously only found in mammals, raises questions about the state of affairs in other invertebrate model systems. Also, do Aplysia piRNAs only act on DNA methylation, or post-transcriptionally aswell.? Future studies will no doubt also look at how this type of regulation corresponds to histone marks, and try to synthesise the different levels of regulation. Perhaps the most important take home message is that piRNAs are more than a germline specific immunity against tranposons. Just how widespread these other roles are is an open question.

Rajasethupathy, P., Antonov, I., Sheridan, R., Frey, S., Sander, C., Tuschl, T., & Kandel, E. (2012). A Role for Neuronal piRNAs in the Epigenetic Control of Memory-Related Synaptic Plasticity Cell, 149 (3), 693-707 DOI: 10.1016/j.cell.2012.02.057

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Amyloid-like oligomers and long-term memory.

A new paper in Cell, shows that the formation of amyloid-like oligomers of a RNA-binding protein in synaptic membranes is necessary for the persistence of memories in Drosophila.

If memory is the maintenance of learning associated changes in synaptic efficacy and number, and these synaptic modifications are associated with changes in protein composition, how do they persist for years when individual protein molecules are turned over in days?

Cytoplasmic polyadenylation element-binding proteins (CPEBs) are a family of RNA-binding proteins that have been shown to regulate activity-dependent protein synthesis at synapses. A Drosophila CPEB, Orb2, has been shown to be required for the long-term persistence of memory (Keleman et al.). Previous studies on a CPEB from the sea slug Aplysia, had shown that it had prion-like properties, in that it could exist in two conformational states: a monomeric form and a self-perpetuating multimeric form with amyloid-like properties. Synaptic stimulation leads to the conversion of Aplysia CPEB into the multimeric form, and the formation of a self-sustaining synaptic mark. Blocking the activity of multimeric ApCPEB inhibited the long-term facilitation of a synaptic response (Si et al.).  Based on these Aplysia studies, Majumdar et al. hypothesised that Orb2’s role in Drosophila long-term memory could also be mediated by activity-dependent formation of amyloid-like oligomers.

Purifying Orb2 from Drosophila head extracts, Majumdar et al. show that it either exists in a monomeric form, or in oligomers consisting of between two and six Orb2 molecules. Orb2 oligomers are resistant to denaturation by heat or detergents, showing that they have amyloid-like characteristics. The oligomeric form of Orb2 appears to be localised to synaptic membranes, whilst the monomeric form is mainly found in cell bodies. Stimulation of neurons involved in memory formation increased the level of oligomeric Orb2.

Majumdar et al. go on to show that there are two different isoforms of Orb2 involved in the formation of Orb2 oligomers, Orb2A and Orb2B. Although the Orb2 oligomers found in vivo were mainly composed of Orb2B, the researchers found Orb2 oligomerisation requires Orb2A. Both isoforms contain a glutamine rich unstructured prion-like domain that acts as a substrate for oligomerisation, but a short 8 amino acid N-terminal domain of Orb2A catalyses the oligomerisation reaction. Orb2A appears to regulate the formation of amyloid-like Orb2 oligomers.

The previous study (Keleman et al.) that showed that Orb2 was involved in long-term memory stabilisation had used a technique that reduced the Orb2 concentration. By generating flies that carried a single point mutation in the Orb2A N-terminal domain, specifically impairing Orb2 oligomerisation, Majumdar et al. went on to show Orb2 oligomerisation is necessary for long-term memory persistence.

This work is exciting on two different fronts. It shows a mechanism for the generation of stable synaptic marks by the formation of amyloid-like oligomers. This could be a more widespread phenomenon underlying memory formation and persistence. More broadly, this study yields insight into potential physiological roles for amyloid formation and prion-like mechanisms.

Insoluble amyloid-like aggregations are a common pathological characteristic in human neurodegenerative conditions such as Alzheimer’s disease and Parkinson’s disease. In the case of prion diseases such as Creutzfeld-Jacob disease, amyloidogenic forms of Prion-Protein propagate the disease by seeding the conformational changes to the non-pathogenic form. The authors of the paper do not call the Orb2 oligomers prions, as the mechanisms underlying oligomerisation are not understood. However, in the case of ApCPEB, multimerisation was shown to be self-sustaining, and hence prion-like.

Is there a link between the preponderance of amyloid associated neurodegenerative diseases and the role of amyloid-like oligomers in memory? It is probably unwise to extrapolate too far from these results. However, one could postulate that if this is a widespread mechanism of memory formation, amyloid associated diseases could be more likely to affect the brain. Likewise, if amyloid-like protein multimerisation has normal physiological roles in the brain, it could be a more permissive environment for amyloid formation than other tissues.

Majumdar, A., Cesario, W., White-Grindley, E., Jiang, H., Ren, F., Khan, M., Li, L., Choi, E., Kannan, K., Guo, F., Unruh, J., Slaughter, B., & Si, K. (2012). Critical Role of Amyloid-like Oligomers of Drosophila Orb2 in the Persistence of Memory Cell, 148 (3), 515-529 DOI: 10.1016/j.cell.2012.01.004

Si, K., Choi, Y., White-Grindley, E., Majumdar, A., & Kandel, E. (2010). Aplysia CPEB Can Form Prion-like Multimers in Sensory Neurons that Contribute to Long-Term Facilitation Cell, 140 (3), 421-435 DOI: 10.1016/j.cell.2010.01.008

Keleman, K., Krüttner, S., Alenius, M., & Dickson, B. (2007). Function of the Drosophila CPEB protein Orb2 in long-term courtship memory Nature Neuroscience, 10 (12), 1587-1593 DOI: 10.1038/nn1996