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Are You Reelin in the Years? Not without Alternative Splicing

Article appears by special arrangement with Alzheimer Research Forum. See original article with additional links/commentary.

25 August 2005. Well, would you know a diamond if you held it in your hand? Perhaps not without long-term potentiation (LTP), a property that allows neurons to mount stronger and stronger responses as they are more frequently stimulated. LTP is important for building memories and the process can be prevented by, amongst other things, amyloid-β (see Alzheimer Research Forum related news story), the peptide that forms the insoluble plaques found in the brains of those with Alzheimer disease (AD). In last Thursday’s Neuron, Joachim Herz and colleagues reported how two other AD-linked proteins, Reelin and the receptor for apolipoprotein E (ApoER2), are involved in LTP. Together, in a process that depends on alternative splicing of ApoER2, the two induce phosphorylation of the N-methyl-D-aspartate (NMDA) form of glutamate receptors, which have been implicated many times in long-term potentiation and memory formation.

ApoER2, of course, binds ApoE, the ε4 variant of which is the strongest genetic risk factor for late-onset AD (see Alzheimer Research Forum ApoE primer for some more background). Reelin, another ligand for ApoER2, is essential for regulation of neuronal migration during development. But it is also expressed in the adult brain. In fact, recent evidence suggests that Reelin-producing Cajal-Retzius cells in the cortex may be some of the earliest hit in AD (see Alzheimer Research Forum related news story). So exactly what role does Reelin play in adults?

Recently, the authors showed that Reelin and ApoE receptors cooperate to enhance LTP (see Weeber et al., 2002), but exactly how the proteins achieved this was unclear. To shed some light on the mystery, first author Uwe Beffert and colleagues focused on an exon of the highly conserved ApoER2 that is only present in mammals.

The exon, number 19, is differentially spliced, a common feature of proteins involved in neurotransmission. So using knock-in vectors to replace the normal copies of ApoER2 in mice with unspliceable variants, Beffert and colleagues tested to see what role the exon assumes in the mammalian brain. They found that in the absence of exon 19, neuronal migration occurs normally, ruling out any role in development. Likewise, activation of tyrosine kinases triggered by binding of Reelin to the ApoE receptor was normal both in the presence or absence of exon 19, suggesting that the exon is not required for this particular set of signals to work properly, either. This led the authors to look for other interactions that might be influenced by the exon.

Beffert and colleagues examined hippocampal slices from the knock-ins for LTP effects. They found that there was no difference in basal transmission or LTP between mice expressing ApoER2 with exon 19 and those producing ApoER2 without the exon. However, stimulation of LTP by Reelin was another matter. Reelin-induced enhancement of LTP was totally abolished in mice lacking the exon 19. Next, to find out if Reelin enhances LTP by modifying transmission through NMDA receptors, Beffert and colleagues recorded postsynaptic potentials while blocking the other type of glutamate receptors, the AMPA receptors. Addition of Reelin increased transmission through normal neurons by about twofold, but in those expressing ApoER2 minus exon 19, transmission actually fell by about 50 percent.

“Taken together, these data indicate that LTP enhancement by Reelin involves a molecular mechanism that is dependent upon the alternatively spliced exon 19. In addition, this mechanism involves, at least in part, the modulation of NMDA receptor functions,” write the authors.

This LTP enhancement is not just restricted to hippocampal slices, either. The authors evaluated the knock-in mice in two commonly used memory tests—fear conditioning (a variation on the Pavlov dog, where, for example, the mouse learns to associate a stimulus, such as an audible tone, with a mild foot shock) and spatial learning (where mice in a water tank learn to find and swim to safety on a submerged platform). In both cases, the knock-in mice expressing ApoER2 minus exon 19 were statistically poorer performers. In the fear conditioning test, for example, mice lacking the exon only responded to the tone about 10 percent of the time, compared to 40 percent of the time for knock-ins with the exon. And in the water maze, mice lacking exon 19 spent about 20 percent less time searching in the correct quadrant for the platform.

What is even more intriguing is that alternative splicing of ApoER2 seems to be regulated by activity. In wild-type mice, expression of the exon 19-positive variant increased during periods of activity, for example, when the mice were feeding. This alternative splicing was not dependent on the time of day, indicating that it is truly activity controlled and not regulated by the endogenous circadian clock.

So how do Reelin and ApoER2 influence the NMDA receptors? In coimmunoprecipitation experiments using cultured cells and extracts from mouse brains, Beffert and colleagues found that ApoER2 and the NMDA receptor subunit 2A (NR2A) and 2B (NR2B) can interact with each other. Using the same strategy, the authors found that ApoER2 also interacts with postsynaptic density 95 (PSD95), a protein that binds to, and has been implicated in toxicity of, Aβ (see Lacor et al., 2004). Interestingly, this interaction only occurs if exon 19 is present in ApoER2. Furthermore, the authors found that all these components are present in synaptosomes and that Reelin leads to phosphorylation of NMDA receptors that are isolated in complex with ApoER2.

How ApoER2 Exon 19 Can Modulate NMDA Receptors and LTP
The only two classes of proteins known to bind to exon 19 of ApoER2 (purple) are PSD95 and the c-Jun N-terminal kinase (JNK) interacting proteins (JIP). The former can bind to SFKs and may recruit them to the NMDA receptor (see Tezuka et al., 1999), while JIPs could recruit JNK, which can modulate LTP (see Curran et al., 2003). How ApoE might affect Reelin signaling is unclear, but should be of great interest to AD researchers. [Image taken from Beffert et al., courtesy of Neuron and Cell Press]

All this evidence has led the authors to draw a better picture (see above) of how Reelin, ApoER2, PSD95, and the NMDA receptor interact. The role of ApoE is currently unclear, but as the authors point out, “ApoE, another ligand for ApoER2 and a major isoform-specific modifier of late-onset Alzheimer’s disease, could in principle alter ApoER2 signaling and NMDAR response by interfering with normal receptor activation (see figure). Such a mechanism might contribute to the role of ApoE in Alzheimer’s disease.”

The unequivocal demonstration that Reelin has a function in the adult brain is an important aspect of this work, writes Gabriella D’Arcangelo, Baylor College of Medicine, Houston, in a related Neuron Preview. She also suggests that future work should focus on whether Reelin, required for neuritic outgrowth in the developing brain, might also regulate synaptogenesis or synapse stability in the adult brain. Loss of synapses is, of course, a major part of AD pathology (see Alzheimer Research Forum symposium coverage), while in patients with schizophrenia, reduced expression of Reelin has recently been attributed to hypermethylation of DNA (see Grayson et al., 2005) documented in patients with schizophrenia. “It will be interesting to examine ApoER2 splicing in the brain of schizophrenia patients as well as in other patient populations affected by learning and memory defects, such as Alzheimer’s disease,” D’Arcangelo concludes.—Tom Fagan.

Beffert U, Weeber EJ, Durudas A, Qiu S, Masiulis I, Sweatt JD, Li W-P, Adelmann G, Frotscher M, Hammer RE, Herz J. Modulation of synaptic plasticity and memory by reelin involves differential splicing of the lipoprotein receptor ApoER2. Neuron. August 18, 2005;47:567-579. Abstract

D’Arcangelo G. ApoER2: A Reelin receptor to remember. Neuron. 2005 Aug 18;47(4):471-3. Abstract

Comments on Related News

Related News: On Again, Off Again—DNA Methylation, Genes, and Plasticity

Comment by:  David Yates
Submitted 18 April 2007
Posted 26 April 2007

Are these studies of relevance to the report from Israel that older men feed their mutations into the gene pool and this in part accounts for keeping the “schizophrenia gene” going despite poor fertility (Malaspina et al., 2002)? And might a comparison of the DNA of healthy siblings born before the mutations of an “older man” mutation with that of a sibling who got such a later mutation and developed schizophrenia reveal something of interest?

View all comments by David Yates

Related News: Gene Expression Study May Open Window on Brain Development

Comment by:  Barbara Lipska
Submitted 15 June 2009
Posted 15 June 2009

In this very important and innovative study, Sestan and colleagues report a transcriptome-wide survey across multiple brain regions of the fetal mid-gestation brain. They show dramatic differences in expressed transcripts, including alternative splice variants, between brain regions, and most surprisingly, between several cortical regions. The authors have undertaken an ambitious task of further characterizing differentially expressed genes by functional clustering and co-expression clustering and comparing the results with genes identified through neurobiological experiments. They have also performed extensive validation using several additional fetal brains. Most interestingly, the authors showed that differentially expressed genes are more frequently associated with human-specific evolution of putative cis-regulatory elements. For this, they have identified genes that are near highly conserved non-coding sequences (CNSs) and found that the genes that are differentially expressed between the regions are more frequently near human-specific accelerated evolution CNSs.

The weakness of the study is a very small number of fetal brains (four) and the fact that they range in age from 18 to 23 weeks of gestation. During these several weeks of fetal life, the brain undergoes dramatic developmental changes and expression of many genes, either increases or decreases steeply. Thus, it would be critical to fully characterize these changes across fetal age. It is also crucial to explore genetic influences on fetal gene expression as it appears that in adult brain both gene expression and splicing are strongly genetically regulated. The authors have made an important contribution to our understanding of development of human brain, and further research of this type will generate the data that would help in better understanding of human brain disorders. In particular, genetic-expression effects in human brain across the entire lifespan, including fetal period, may help identify molecular mechanisms whereby candidate genes increase risk for developing the disorder. Using expression levels of transcripts and their splicing characteristics as intermediate phenotypes may yield statistically positive associations and improved understanding of the mechanisms that lead to neurodevelopmental disorders such as autism and schizophrenia, as they are the most proximal phenotypes to the risk alleles.

View all comments by Barbara Lipska

Related News: Gene Expression Study May Open Window on Brain Development

Comment by:  Karoly Mirnics, SRF Advisor
Submitted 15 June 2009
Posted 15 June 2009

This outstanding study reinforces how much we still do not understand about human brain development and function! It is just mind-boggling that the mid-fetal human brain expresses more than three quarters of the human genome, and that region-specific splicing appears to be an absolutely critical feature of the developing brain. Interestingly, the structural and functional interhemispheric differences do not appear to be related to gene expression differences in mid-fetal life, but rather, either they develop independently of gene expression patterns, or they are developing at later stages of cortical maturation, perhaps in a postnatal activity-driven pattern.

So, how is this developmental expression machinery related to various neurodevelopmental disorders, such as schizophrenia? Is usage of an "inappropriate" splice variant sufficient to alter the neuronal phenotypic development to a degree that would predispose the brain to developing a disease? Are environmental insults capable of disrupting this finely tuned, region-specific splicing machinery? As this is a likely possibility, we must rethink the existing disease-related gene expression findings in the context of the present study, and accept that our previous gene expression measurements may have been too crude to uncover some of the most meaningful changes that are potentially hallmarks of various brain disorders. Furthermore, as the genes that show the most widespread regional use of splice variants can be essential for proper neuronal migration or connectivity, one can argue that these genes should be the primary targets for evaluation in the various regions of postmortem tissue of diseased individuals.

Finally, there is also a minor, cautionary note arising from this study. The fact that the Affymetrix U133 and the Exon array results showed a correlation of R2 >0.5 is encouraging, but underscores that platform-dependence of the findings remains a significant interpretational challenge. Some platforms will be better suited to identify certain gene expression changes, while others will have a greater power to reveal a different (but also potentially valid!) set of mRNA alterations.

View all comments by Karoly Mirnics