As far as science movies go, the new movie, “To Age or Not To Age” seems like a lot of fun.  The interview with Dr. Leonard Guarente suggests that the sirtuin genes play a starring role in the film.  Certainly,  an NAD+ dependent histone deacetylase – makes for a sexy movie star – especially when it is able to sense diet and metabolism and establish the overall lifespan of an organism.

One comment in the movie trailer, by Aubrey de Grey, suggests that humans may someday be able to push the physiology of aging to extreme ends.  That studies of transgenic mice over-expressing SIRT1 showed physiological properties of calorie-restricted (long lived) mice – even when fed ad libitum – suggests that something similar might be possible in humans.

Pop a pill and live it up at your local Denny’s for the next 100 years?  Sounds nice (& a lot like grad school).

Just a few twists to the plot here.  It turns out that – in the brain – SIRT1 may not function as it does in the body.  Here’s a quote from a research article “Neuronal SIRT1 regulates endocrine and behavioral responses to calorie restriction” that inactivated SIRT1 just in the brain:

Our findings suggest that CR triggers a reduction in Sirt1 activity in hypothalamic neurons governing somatotropic signaling to lower this axis, in contrast with the activation of Sirt1 by CR in many other tissues. Sirt1 may have evolved to positively regulate the somatotropic axis, as it does insulin production in β cells, to control mammalian health span and life span in an overarching way. However, the fact that Sirt1 is a positive regulator of the somatotropic axis may complicate attempts to increase murine life span by whole-body activation of this sirtuin.

To a limited extent, it seems that – in the brain – SIRT1 has the normal function of promoting aging.  Therefore, developing “pills” that are activators of SIRT1 would be good for the body, but somehow might be counteracted by what the brain would do.  Who’s in charge anyway?  Mother Nature will not make it easy to cheat her! Another paper published recently also examined the role of SIRT1 in the brain and found that – normally – SIRT1 enhances neuronal plasticity (by blocking the expression of a  micro-RNA miR-134 that binds to the mRNA of, and inhibits the translation of, synaptic plasticity proteins such as CREB).

So, I won’t be first to line up for SIRT1 “activator” pills (such as Resveratrol), but I might pop a few if I’m trying to learn something new.

The current buzz about about GWAS  and longevity and GWAS in general has stirred up many longstanding inconvenient issues that arise when trying to interpret the results of very large, expensive and worthwhile genetic studies.  Its seems that Mother Nature does not give up her secrets without a fight.

One of the most common “inconvenient issues” is the fact that so many of the SNPs that come out of these studies are located far away from protein-encoding exons.  This ubiquitous observation is almost always followed with, “well, maybe its in linkage disequilibrium with a more functional SNP” or something along these lines – wherein the authors get an automatic pass.  OK by me.

Another “inconvenient issue” is the fact that many of these SNPs are of minimal effect and don’t exactly add up or interact to account for the expected heritability.  This problem of “missing heritability” is a big one (see some new insights in the latest issue of Nature Genetics) leading many to suspect that the effects of genes are dependent on complex interactions with each other and the environment.

A recent paper, “A map of open chromatin in human pancreatic islets” [doi:10.1038/ng.530] by Gaulton and colleagues caught my eye because it seems to shed light on both of these particular inconvenient issues.  The authors find that the diabetes risk variant rs7903146 in the TCF7L2 gene is both located in an intron and subject to epigenetic regulation (our sedentary, high-fat, high-stress lives can potentially interact with the genome by causing epigenetic change).

It appears that the T-allele of the intronic rs7903146 is correlated with a more open, transcription-prone form of DNA/chromatin than is the C-allele. The authors confirmed this using both chromatin mapping and gene expression assays on pancreatic islet cells harvested from non-diabetic donors and islet cell-lines.  The results suggest that the risk-conferring T-allele of this intronic SNP may be driving expression (gain-of-function) of the TCF7L2 gene.  What types of environmental stimuli might also impact the opening and closing of chromatin at this location?

This type of interplay of environment, genome and epigenome is probably rampant in the area of brain and behavior – so perhaps the study of diabetes will provide some clues to the many GWAS SNPs that are far away from exons. More on the genetics of epigenetics here.

pointer to the NOVA program on epigenetics “Ghost in Your Genes” (YouTube link here).  Fantastic footage.  Great intro to epigenetics and so-called trans-generational effects and the inheritance of epigenetic marks – which, in some cases – are left by adverse or stressful experience.  A weird, wild, game-changing concept indeed – that my grandchildren could inherit epigenetic changes induced in my genome by adverse experience.

In an earlier post on Williams Syndrome, we delved into the notion that sometimes a genetic variant can lead to enhanced function – such as certain social behaviors in the case of WS.  A mechanism that is thought to underlie this phenomenon has to do with the way in which information processing in the brain is widely distributed and that sometimes a gene variant can impact one processing pathway, while leaving another pathway intact, or even upregulated.  In the case of Williams Syndrome a relatively intact ventral stream (“what”) processing but disrupted dorsal stream (“where”) processing leads to weaker projections to the frontal cortex and amygdala which may facilitate gregarious and prosocial (a lack of fear and inhibition) behavior.  Other developmental disabilities may differentially disrupt these 2 visual information processing pathways.  For instance, developmental dyspraxia contrasts with WS as it differentially disrupts the ventral stream processing pathway.

A recent paper by Woodcock and colleagues in their article, “Dorsal and ventral stream mediated visual processing in genetic subtypes of Prader–Willi syndrome” [doi:10.1016/j.neuropsychologia.2008.09.019] ask how another developmental disability – Prader-Willi syndrome – might differentially influence the development of these information processing pathways.  PWS arises from the lack of expression (via deletion or uniparental disomy) of a cluster of paternally expressed genes in the 15q11-13 region (normally the gene on the maternally inherited chromosome is silent, or imprinted – related post here).  By comparing PWS children to matched controls, the team reports evidence showing that PWS children who carry the deletion are slightly more impaired in a task that depends on the dorsal “where” pathway whilst some sparing or relative strength in the ventral “what” pathway.

I’m wondering how much physical and/or social stress is enough to cause changes in the epigenome?  Does the concern about epigenetics only apply to exposure to severe stress?  or run of the mill forms of stress?  How much do we know about this?

This year, I hope to explore this line of inquiry further.  For starters, I came across a fantastic paper by Fraga et al., entitled, “Epigenetic differences arise during the lifetime of monozygotic twins” [doi:10.1073/pnas.0500398102].   The group carries out a remarkably straightforward and time honored approach – a twin study – to ask how much identical twins differ at the epigenetic level.  Since identical twins have the same genome sequence, any differences in their physiology, behavior etc. are, strictly speaking, due to the way in which the environment (from the uterus to adulthood) shapes their development.  Hence, the team of Fraga et al., can compare the amount and location of methyl (CH3) and acetyl (OCCH3) groups to see whether the environment has differentially shaped the epigenome.

An analysis of some 40 identical twin pairs from ages 3-74 years old showed that – YES – the environment, over time, does seem to shape the epigenome (in this case of lymphocytes).  The most compelling evidence for me was seen in Figure 4 where the team used a method known as Restriction Landmark Genomic Scanning (RLGS) to compare patterns of methylation in a genome-wide manner.  Using this analysis, the team found that older twin pairs had about 2.5 times as many differences as did the epigenomes of the youngest twin pairs.  These methylation differences also correlated with gene expression differences (older pairs also had more gene expression differences) and they found that the individual who showed the lowest levels of methylation also had the highest levels of gene expression.  Furthermore, the team finds that twin pairs who lived apart and had more differences in life history were more likely to have epigenetic differences.  Finally, measures of histone acetylation seemed consistent with the gradient of epigenetic change over time and life-history distance.

Thus it seems that, as everyday life progresses, the epigenome changes too.  So, perhaps, one does not need extreme forms of stress to leave long-lasting epigenetic marks on the genome?  Is this true during early life (where the team did not see many differences between pairs)?  and in the brain (the team focused mainly on lymphocytes)?  Are the differences between twins due to the creation of new environmentally-mediated marks or the faulty passage of existing marks from dividing cell-to-cell over time?  Will be fun to seek out information on this.

The genome is just the A,G,T,C bases that encode proteins and other mRNA molecules.  The “epi”genome are various modification to the DNA – such as methylation (at C residues) – and acetylation of histone proteins.   These changes help the DNA form various secondary and tertiary structures that can facilitate or block the interaction of DNA with the transcriptional machinery.

When DNA is highly methylated, it generally is less accessible for transcription and hence gene expression is reduced.  When histone proteins (purple blobs that help DNA coil into a compact shape) are acetylated, the DNA is much more accessible and gene expression goes up.

We know that proper epigenetic regulation is critical for cognitive development because mutations in MeCP2 – a protein that binds to methylated C residues – leads to Rett syndrome.  MeCP2 is normally responsible for binding to methylated DNA and recruiting histone de-acetylases (HDACs) to help DNA coil and condense into a closed form that is inaccessible for gene expression (related post here).

When DNA is accessible for gene expression, then it appears that – during brain development – there are relatively more synaptic spines produced (related post here).  Is this a good thing? Rett syndrome would suggest that – NO – too many synaptic spines and too much excitatory activity during brain development may not be optimal.  Neither is too little excitatory (too much inhibitory) activity and too few synaptic spines.  It is likely that you need just the right balance (related post here). Some have argued (here) that autism & schizophrenia are consequences of too many & too few synapses during development.

The sketch above illustrates a theoretical conjecture – not a scenario that has been verified by extensive scientific study. It tries to explain why epigenetic effects can, in practice, be difficult to disentangle from true (changes in the A,G,T,C sequence) genetic effects.  This is because – for one reason – a mother’s experience (extreme stress, malnutrition, chemical toxins) can – based on some evidence – exert an effect on the methylation of her child’s genome.  Keep in mind, that methylation is normal and widespread throughout the genome during development.  However, in this scenario, if the daughter’s behavior or physiology were to be influenced by such methylation, then she could, in theory, when reaching reproductive age, expose her developing child to an environment that leads to altered methylation (shown here of the grandaughter’s genome).  Thus, an epigenetic change would look much like there is a genetic variant being passed from one generation to the next, but such a genetic variant need not exist (related post here, here) – as its an epigenetic phenomenon.  Genes such as BDNF have been the focus of many genetic/epigenetic studies (here, here) – however, much, much more work remains to determine and understand just how much stress/malnutrition/toxin exposure is enough to cause such multi-generational effects.  Disentangling the interaction of genetics with the environment (and its influence on the epigenome) is a complex task, and it is very difficult to prove the conjecture/model above, so be sure to read the literature and popular press on these topics carefully.

What the heck is an “in”sensitive period then?   Well, whereas visual input is clearly a “good thing” for the sensitive period of visual development, perhaps some inputs are “bad” and it may be useful to shield or protect the brain from exposure.  Maybe some environmental inputs are “bad” and one would not want the developing brain to be exposed to them and say, “OK, this (bad stuff) is normal“.  As a parent, I am constantly telling my children that the traffic-filled street is a “bad place” and, like all parents, I would not want my children to think that it was OK to wander into the street.  Clearly, I want my child to recognize the car-filled street as a “bad thing”.

In the developing brain, it turns out that there are some “bad things” that one would NOT like (the brain) to get accustomed to.  Long-term exposure to glucocorticoids is one example – well-known to cause a type of neuronal remodelling in the hippocampus, that is associated with poor cognitive performance (visit Bruce McEwen’s lab at Rockefeller University to learn more about this).  Perhaps an “in”sensitive period – where the brain is insensitive to glucocorticoids – is one way to teach the brain that glucocorticoids are “bad” and DO NOT get too familiar with them (such a period does actually occur during early post-natal mammalian development).  Of course, we do need our brains to mount an acute stress response, if and when, we are being threatened, but it is also very important that the brain learn to TURN-OFF the acute stress response when the threat has passed – an extensive literature on the deleterious effects of chronic exposure to stress bears this out.  Hence, the brain needs to learn to recognize the flow of glucocorticoids as something that needs to be shut down.

OK, so our developing brain needs to learn what/who is “good vs. bad”.  Perhaps sensitive and insensitive periods help to reinforce this learning – and also – to cement learning into the system in a sort of permanent way (I’m really not sure if this is the consensus view, but I’ll try and podcast interview some of the experts here asap).  In any case, in the case of the visual system, it is clear that the lack of visual input during the sensitive period has long lasting consequences.  In the case of the stress response, it is also clear that if there is untoward stress early in development, one can be (somewhat) destined to endure a lifetime of emotional difficulty.  Previous posts here, here, here cover research on behavioral/genomic correlates of early life stress.

Genes meet environment in the epigenome during sensitive and insensitive periods?

As stated at the outset – genes are not destiny.  The DNA cannot encode a system that knows who/what is good vs. bad, but rather can only encode a system of molecular parts that can assemble to learn these contingencies on the fly.  During sensitive periods in the visual system, cells in the visual system are more active and fire more profusely during the sensitive period. This extra firing leads to changes in gene expression in ways that (somewhat) permanently set the connectivity, strength and sensitivity of visual synapses.  The expression of neuroligins, neurexins, integrins and all manner of extracellular proteins that stabilize synaptic connections are well-known tagets of activity-induced gene expression.  Hence the environment “interacts” with the genome via neuronal firing which induces gene expression which – in turn – feeds back and modulates neuronal firing.  Environment –> neuronal firing –> gene expression –> modified neuronal firing.  OK.

Similarly, in the stress response system, the environment induces changes in the firing of cells in the hypothalamus which leads (through a series of intermediates) to the release of glucocorticoids.  Genes induced during the firing of hypothalamic cells and by the release of glucocorticoid can modify the organism’s subsequent response to stressful events.  Environment –> neuronal firing –> gene expression –> modified neuronal firing.  OK.

Digging deeper into the mechanism by which neuronal firing induces gene expression, we find an interesting twist.   Certainly there is a well-studied mechanism wherein neuronal firing causes Ca++ release which activates gene expression of neuroligins, neurexins, integrins and all manner of extracellular proteins that stabilize synaptic connections – for many decades.  There is another mechanism that can permanently mark certain genes and alter their levels of expression – in a long-lasting manner.  These are so-called epigenetic mechanisms such as DNA methylation and acetylation.  As covered here and here, for instance, Michael Meaney’s lab has shown that DNA CpG methylation of various genes can vary in response to early-life stress and/or maternal care. In some cases, females who were poorly cared for, may, in turn, be rather lousy mothers themselves as a consequence of these epigenetic markings.

A new research article, “Dynamic DNA methylation programs persistent adverse effects of early-life stress” by Chris Murgatroyd and colleagues [doi:10.1038/nn.2436] explores these mechanisms in great detail.  The team explored the expression of the arginine vasopressin (AVP) peptide – a gene which is important for healthy social interaction and social-stress responsivity.  Among many other interesting results, the team reports that early life stress (using a mouse model) leads to lower levels of methylation in the 3rd CpG island which is located downstream in a distal gene-expression-enhancer region.  In short, more early-life stress was correlated with less methylation, more AVP expression which is known to potentiate the release of glucocorticoids (a bad thing).   The team reports that the methyl binding MeCP2 protein, encoded by the gene that underlies Rett syndrome, acts as a repressor of AVP expression – which would normally be a good thing since it would keep AVP levels (and hence glucocorticoid levels) down.  But unfortunately, early-life stress removes the very methyl groups to which MeCP2 binds and also the team reports that parvocelluar neuronal depolarization leads to phosphorylation (on serine residue #438) of MeCP2 – a form of MeCP2 that is less accessible to its targets.  So, in  a manner similar to other examples, early life stress can have long-lasting effects on gene expression via an epigenetic mechanism – and disables an otherwise protective mechanism that would shield the organism from the effects of stress.  Much like in the case of Rett syndrome (as covered here) it seems that when MeCP2 is bound – then it silences gene expression – which would seem to be a good thing when it comes to the case of AVP.

So who puts these epigenetic marks on chromosomes and why?

I’ll try and explore this further in the weeks ahead.  One intriguing idea about why methylation has been co-opted among mammals, has to do with the idea of parent-offspring conflict.  According to David Haig, one of the experts on this topic, males have various incentives to cause their offspring to be large and fast growing, while females have incentive to combat the genomic tricks that males use, and to keep their offspring smaller and more manageable in size.  The literature clearly show that genes that are marked or methylated by fathers (paternally imprinted genes) tend to be growth promoting genes and that maternally imprinted genes tend to be growth inhibitors.  One might imagine that maternally methylated genes might have an impact on maternal care as well.

Lastly, the growth promoting/inhibiting effects of paternal/maternal genes and gene markings is now starting to be discussed somewhat in the context of autism/schizophrenia which have have been associated with synaptic under-/over-growth, respectively.

Building a brain is already tough enough – but to have to do it amidst an eons-old battle between maternal and paternal genomes.  Sheesh!  More on this to come.

Few events are as hard to understand as the loss of a loved one to suicide – a fatal confluence of factors that are oft scrutinized – but whose analysis can provide little comfort to family and friends.  To me, one frightening and vexing aspect of what is known about the biological roots of depression, anxiety, impulsivity and other mental traits and states associated with suicide, is the way in which early life (even prenatal) experience can influence events in later life.  As covered in this blog here and here, there appear to be very early interactions between emotional experience in early life and the methylation of specific points in the genome.  Such methylation – often referred to as epigenetic marks – can regulate the expression of genes that are important for synaptic plasticity and cognitive development.

The recent paper, “Alternative Splicing, Methylation State, and Expression Profile of Tropomyosin-Related Kinase B in the Frontal Cortex of Suicide Completers” is a recent example of a link between epigenetic marks and suicide.  The team of Ernst et al., examined gene expression profiles from the frontal cortex and cerebellum of 28 males lost to suicide and 11 control, ethnically-matched control participants.  Using a subject-by-subject comparison method described as “extreme value analysis” the team identified 2 Affymetrix probes: 221794_at and 221796_at – that are specific to NTRK2 (TRKB) gene – that showed significantly lower expression in several areas of the frontal cortex.  The team also found that these probes were specific to exon 16 – which is expressed only in the TRKB.T1 isoform that is expressed only in astrocytes.

Further analysis showed that there were no genetic differences in the promoter region of this gene that would explain the expression differences, but, however, that there were 2 methylation sites (epigenetic differences) whose methylation status correlated with expression levels (P=0.01 and 0.004).  As a control, the DNA-methylation at these sites was not correlated with TRKB.T1 expression when DNA and RNA was taken from the cerebellum (a control since the cerebellum is not thought to be directly involved in the regulation of mood).

In the case of TRKB.T1 expression, the team reports that more methylation at these 2 sites in the promoter region is associated with less TRKB.T1 expression in the frontal cortex.  Where and when are these marks laid down?  Are they reversible?  How can we know or suspect what is happening to our epigenome (you can’t measure this by spitting into a cup as with current genome sequencing methods)? To me, the team has identified an important clue from which such follow-up questions can be addressed.  Now that they have a biomarker, they can help us begin to better understand our complex and often difficult emotional lives within a broader biological context.

The team focused on the BDNF gene whose transcription can be initiated from any one of eight promoter sites (I-XIII).  These sites vary in activity depending on the phase of development and/or the tissue or type of cell – all of which make for a complex set of instructions able to turn the BDNF gene on and off in precise developmental and/or tissue-specific ways.  In the case of promoter IV, it appears to be triggered in the cortex in response to Ca++ release that occurs when neurons are firing – a phenomena called, “neuronal activity dependent transcription” – a top example of how the environment can influence gene expression.  Seeing as how BDNF promoter IV is important for this type of environment-induced gene expression, the team asked what happens when you remove this particular promoter?

To do this, the team constructed – keep in mind that these are – mice that contain mutations in several of the Calcium (Ca++) response elements in the promoter IV region.  They introduced point mutations so that the Ca++ sensitive protein CREB could not bind to the promoter and activate gene expression.  OK, so what happens?

Firstly, the team reports that the mutant mice are more or less indistinguishable from controls in appearance, gait, growth rate, brain size and can also reproduce and transmit the mutations.  WOW! Is that one strike AGAINST nurture? The team then shows that BDNF levels are more than 50% reduced in cultured neurons, but that levels of other immediate early genes are NOT affected (as expected).  In living animals, the effects were similar when they asked how much gene expression occurs in the sensory cortex when animals are exposed to light (after an extended period of darkness).  OK, so there are few effects, so far, other than lower levels of nurture-induced BDNF expression – hmmm. Looking more closely however, the team found that the mutant mice generated lower levels of inhibitory neuron activity – as measured by the firing of miniature inhibitory postsynaptic currents.  Follow-on results showed that the total number of inhibitory neurons (parvalbumin and NPY + GABAergic cells) was no different than controls and so it would seem that the activity dependence of BDNF is important for the maintenance of inhibitory synapses.

Hence, the team has found that what “nurture” does (via the BDNF promoter IV in this case) is to exert an effect on the connectivity of inhibitory neurons.  Wow, thanks mother nurture!  Although it may seem like an obscure role for something as important as THE environment, the team points out that the relative balance of excitation-to-inhibition (yin-yang as covered here for Rett syndrome) is crucial for proper cognitive development.

To explore the notion of inhibory/excitation balance further, check out this (TED link) video lecture, where Michael Merzenich describes this imbalance as a “signal-to-noise” problem wherein some children’s brains are rather noisy (due to any number of genetic/environmental reasons – such as, perhaps, poorly maintained inhibitory connections).  This can make it harder to develop and function in life.  Perhaps someday, the genetic/environment research like that of Hong and colleagues will inform the rehabilitative strategies developed by Merzenich.