The A-to-T SNP rs7794745 in the CNTNAP2 gene was found to be associated with increased risk of autism (see Arking et al., 2008).  Specifically, the TT genotype, found in about 15% of individuals, increases these folks’ risk by about 1.2-1.7-fold.  Sure enough, when I checked my 23andMe profile, I found that I’m one of these TT risk-bearing individuals.  Interesting, although not alarming since me and my kids are beyond the age where one typically worries about autism.  Still, one can wonder if such a risk factor might have exerted some influence on the development of my brain?

The recent paper by Tan et al., “Normal variation in fronto-occipital circuitry and cerebellar structure with an autism-associated polymorphism of CNTNAP2” [doi:10.1016/j.neuroimage.2010.02.018 ] suggests there may be subtle, but still profound influences of the TT genotype on brain development in healthy individuals.  According to the authors, “homozygotes for the risk allele showed significant reductions in grey and white matter volume and fractional anisotropy in several regions that have already been implicated in ASD, including the cerebellum, fusiform gyrus, occipital and frontal cortices. Male homozygotes for the risk alleles showed greater reductions in grey matter in the right frontal pole and in FA in the right rostral fronto-occipital fasciculus compared to their female counterparts who showed greater reductions in FA of the anterior thalamic radiation.”

The FA (fractional anisotropy – a measurement of white-matter or myelination) results are consistent with a role of CNTNAP2 in the establishment of synaptic contacts and other cell-cell contacts especially at Nodes of Ranvier – which are critical for proper function of white-matter tracts that support rapid, long-range neural transmission.  Indeed, more severe mutations in CNTNAP2  have been associated with cortical dysplasia and focal epilepsy (Strauss et al., 2006).

Subtle changes perhaps influencing long-range information flow in my brain – wow!

More on CNTNAP2 … its evolutionary history and role in language development.

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.

So what are these genes that, in the case of Down syndrome, can alter the course of brain development?  Well, it is widely known that individuals with Down syndrome have an extra copy of chromosome 21.  However, the disorder does not necessarily depend on having an extra copy of each and every gene on chromosome 21.   Rare partial trisomies of only 5.4 million base-pairs on 21q22 can produce the same developmental outcomes as the full chromosome trisomy.  Also, it turns out that mice have a large chunk of mouse chromosome 16 that has the very same linear array of genes (synteny) found on human chromosome 21 (see the figure here).  In mice that have an extra copy of about 104 genes, (the Ts65Dn segment above) many of the developmental traits related to brain structure and physiology are observed.  In mice that have an extra copy of about 81 genes, this is also the case (the Ts1Cje segment).

To focus this line of research even further, the recent paper by Belichenko et al., “The “Down Syndrome Critical Region” Is Sufficient in the Mouse Model to Confer Behavioral, Neurophysiological, and Synaptic Phenotypes Characteristic of Down Syndrome” [DOI:10.1523/JNEUROSCI.1547-09.2009]  examine brain structure, physiology and behavior in a line of mice that carry an extra copy of just 33 genes (this is the Ts1Rhr segment seen in the figure above).  Interestingly, these mice display many of the various traits (admittedly mouse versions) that have been associated with Down syndrome – thus greatly narrowing the search from a whole chromosome to a small number of genes.  20 out of 48 Down syndrome-related traits such as enlargement of dendritic spines, reductions of dendritic spines, brain morphology and various behaviors were  observed.  The authors suggest that 2 genes in this Ts1Rhr segment, in particular, look like intriguing candidates.  DYRK1A a gene, that when over-expressed can lead to hippocampal-dependent learning deficits, and KCNJ6, a potassium channel which could readily drive neurons to hyperpolarize if over-expressed.

One of the difficult aspects of understanding mental illness, is separating the real causes of the illness from what might be secondary or tertiary consequences of having the illness.  If you think about a car whose engine is not running normally, there may be many observable things going wrong (pinging sound, stalling, smoke, vibration, overheating, loss of power, etc.) – but, what is the real cause of the problem?  What should be done to fix the car? – a faulty sparkplug or timing belt perhaps?  Such is often the problem in medicine, where a fundamental problem can lead to a complex, hard-to-disentangle, etiology of symptoms.  Ideally, you would fix the core problem and then expect the secondary and tertiary consequences to normalize.

This inherent difficulty, particularly in mental illness, is one of the reasons that genetic research is of such interest.  Presumably, the genetic risk factors are deeper and more fundamentally involved in the root causes of the illness – and hence – are preferable targets for treatment.  The recent paper, “Widespread Reductions of Cortical Thickness in Schizophrenia and Spectrum Disorders and Evidence of Heritability” [Arch Gen Psychiatry. 2009;66(5):467-477] seeks to ascertain whether one aspect of schizophrenia – a widespread and well-documented thinning of the neocortex – is due to genetic risk (hence something that is closer to a primary cause) or – rather – if cortical thinning is not due to genetics, and so more of a secondary consequence of things that go wrong earlier in the development of the illness.

To explore this idea, the team of Goldman et al., did something novel.  Rather than examine the differences in cortical thickness between patients and control subjects, the team evaluated the cortical thickness of 59 patients and 72 unaffected siblings as well as 196 unrelated, matched control participants.  If the cortical thickness of the siblings (who share 50% of their genetic variation) was more similar to the patients, then it would suggest that the cortical thinning of the patients was under genetic control and hence – perhaps – a biological trait that is more of a primary cause.  On the other hand, if the cortical thickness of the siblings (who share 0% of their genetic variation) was more similar to that of the healthy control participants, then it would suggest that cortical thinning was – perhaps more of a secondary consequence of some earlier deficit.

The high-resolution structural neuroimaging allowed the team to carefully assess cortical thickness - which is normally between a mere 2 and 4 millimeters – across different areas of the cortex.  The team reports that, for the most part, the cortical thickness measures of the siblings were more similar to the unrelated controls – thus suggesting that cortical thickness may not be a direct component of the genetic risk architecture for schizophrenia.  Still, the paper discusses several candidate mechanisms which could lead to cortical thinning in the illness – some of which might be assessed in the future using other imaging modalities in the context of their patient/sibling/control experimental design.

It turns out that astrocytes not only seem to care for dendritic spines, but also provide the essential signal that initiates the sprouting of neuronal spines in the first place.  As covered in their recent paper, “Gabapentin Receptor α2δ-1 Is a Neuronal Thrombospondin Receptor Responsible for Excitatory CNS Synaptogenesis” [doi:10.1016/j.cell.2009.09.025] Eroglu and colleagues report the discovery – in mice – of CACNA2D1 the alpha-2/delta-1 subunit of the voltage-dependent calcium channel complex encodes a protein that binds to thrombospondins (humans have THBS1 and THBS2) which are adhesive glycoproteins that mediate cell-to-cell and cell-to-matrix interactions – and are required for the formation of new dendritic spines.  When neurons are cultured in the absence of thrombospondins, they fail to produce new spines and mice that do not make thrombospondins do not make very many excitatory synaptic spines.

The interesting twist to me is that thrombospondins are secreted solely by astrocytes! The newly identified CACNA2D1 receptor – as revealed by Eroglu et al., – binds to the EGF-repeats of thrombospondin and initiates a signalling cascade that results in the sprouting of new – silent – dendritic spines.  Gabapentin, a drug that is prescribed for seizures, pain, methamphetamine addiction and many other mental health conditions appears to bind to CACNA2D1 and interfere with the binding of thrombospondin and also inhibits the formation of new spines in vitro as well during the development of somatotopic maps in the mouse whisker barrel cortex.

This seems to be an important discovery in the understanding of how cognitive development unfolds since much of the expression of thrombospondin and its effects on synaptogenesis occur in the early postnatal stages of development.  I will follow this thread in the months to come.

“A devil, a born devil, on whose nature
Nurture can never stick; on whom my pains,
Humanely taken, all, all lost, quite lost
And as with age his body uglier grows,
So his mind cankers.”

So says the wizard Prospero about the wretched Caliban in Shakespeare’s The Tempest (Act IV, Scene I, lines 188 – 192).  Although Shakespeare was not a neuroscientist (more to his credit!), his poignant phrase, “on whose nature, Nurture can never stick“  strikes the very core of the modern debates on the role of genes and personal genomes, and perhaps reminds us that our human experience is delicately balanced amidst the interaction of genes and environment.

Among the some 20,500 genes in the human genome (yes, this is the latest estimate from Eric Lander this past weekend) one particularly amazing gene stands out.   CACNA2D1 the alpha-2/delta-1 subunit of the voltage-dependent calcium channel complex (which also binds to the widely-prescribed drug Gabapentin) encodes a protein who, in conjunction with other related subunits, forms a calcium channel to mediate the influx of calcium ions into neurons when membrane polarization occurs.  In the recent article, “Gabapentin Receptor α2δ-1 Is a Neuronal Thrombospondin Receptor Responsible for Excitatory CNS Synaptogenesis” [doi:10.1016/j.cell.2009.09.025] Eroglu and colleagues reveal that this single gene – initiates the development of synapses – the dynamic structures whose ever changing interconnections make us who we are – that allow “nurture to stick” as it were.

More on the biology of CACNA2D1 and its interactions with its ligand – Thrombospondins – to come.

Joseph LeDoux‘s book, “Synaptic Self: How Our Brains Become Who We Are” opens with his recounting of an incidental glance at a t-shirt, “I don’t know, so maybe I’m not” (a play on Descartes’ “cogito ergo sum“) that prompted him to explore how our brain encodes memory and how that leads to our sense of self.  More vividly, Elizabeth Wurtzel, in “Prozac Nation” recounts, “Nothing in my life ever seemed to fade away or take its rightful place among the pantheon of experiences that constituted my eighteen years. It was all still with me, the storage space in my brain crammed with vivid memories, packed and piled like photographs and old dresses in my grandmother’s bureau. I wasn’t just the madwoman in the attic — I was the attic itself. The past was all over me, all under me, all inside me.” Both authors, like many others, have shared their personal reflections on the fact that – to put it far less eloquently than LeDoux and Wurtzl – “we” or “you” are encoded in your memories, which are “saved” in the form of synaptic connections that strengthen and weaken and morph through age and experience.  Furthermore, such synaptic connections and the myriad biochemical machinery that constitute them, are forever modulated by mood, motivation and your pharmacological concoction du jour.

Well, given that my “self” or “who I think of as myself” or ” who I’m aware of at the moment writing this blog post” … you get the neuro-philosophical dilemma here … hangs ever so tenuously on the biochemical function of a bunch of tiny little proteins that make up my synaptic connections – perhaps I should get to know these little buggers a bit better.

OK, how about a gene known as kalirin – which is named after the multiple-handed Hindu goddess Kali whose name, coincidentally, means “force of time (kala)” and is today considered the goddess of time and change (whoa, very fitting for a memory gene huh?).  The imaginative biochemists who dubbed kalirin recognized that the protein was multi-handed and able to interact with lots of other proteins.  In biochemical terms, kalirin is known as a “guanine nucleotide exchange factor” – basically, just a helper protein who helps to activate someone known as a Rho GTPase (by helping to exchange the spent GDP for a new, energy-laden GTP) who can then use the GTP to induce changes in neuronal shape through effects on the actin cytoskeleton.  Thus, kalirin, by performing its GDP-GTP exchange function, helps the actin cytoskeleton to grow.  The video below, shows how the actin cytoskeleton grows and contracts – very dynamically – in dendrites that carry synaptic spines – whose connectivity is the very essence of “self”.  Indeed, there is a lot of continuing action at the level of the synapse and its connection to other synapses, and kalirin is just one of many proteins that work in this dynamic, ever-changing biochemical reaction that makes up our synaptic connections.

In their paper”Kalirin regulates cortical spine morphogenesis and disease-related behavioral phenotypes” [doi: 10.1073/pnas.0904636106] Michael Cahill and colleagues put this biochemical model of kalirin to the test, by examining a mouse whose version of kalirin has been inactivated.  Although the mice born with this inactivated form are able to live, eat and breed, they do have significantly less dense patterns of dendritic spines in layer V of the frontal cortex (not in the hippocampus however, even though kalirin is expressed there).  Amazingly, the deficits in spine density could be rescued by subsequent over-expression of kalirin!  Hmm, perhaps a kalirin medication in the future? Further behavior analyses revealed deficits in memory that are dependent on the frontal cortex (working memory) but not hippocampus (reference memory) which seems consistent with the synaptic spine density findings.

Lastly, the authors point out that human kalirin gene expression and variation has been associated with several neuro-psychiatric conditions such as schizophrenia, ADHD and Alzheimer’s Disease.   All of these disorders are particularly cruel in the way they can deprive a person of their own self-perception, self-identity and dignity.  It seems that kalirin is a goddess I plan on getting to know better.  I hope she treats “me” well in the years to come.

If you like gardening, the doldrums of winter can be dreary indeed. Although I’d never admit to it, my neighbors might swear to having seen me outside strangely (pathetically) counting the number of branches on my icicle-laden roses and rhododendrons.  In any case, I do admit to spending way too much time forlornly staring at my garden from the window while fantasizing about all the things I’ll plant come springtime.

Each new branch brings a new burst of color and fragrance and concomitant joy.  A good thing right ?  Similarly, each neuron in the brain – which look just like little trees with branches – should also strive to send out as many new branches and make new synaptic connections.  Afterall, there are brain disorders associated with a loss of or fewer dendrites, such as Down’s syndrome and schizophrenia. More branches, more synapses, more brain power and concomitant joy ? Well, perhaps not quite.

A gene known as seizure-related gene 6 (sez6) which is expressed in the developing brain as well as in response to environmental stimulation, seems to play a role in limiting the the number of branches that a neuron can send out.  Gunnersen and colleagues [doi: 10.1016/j.neuron.2007.09.018] show that mice that carry an inactivated version of sez6 show more dendritic branches (implying that the normal function of the active gene is to inhibit branch formation), and that this is definitely not a good thing.  These sez6(-/-) mice, while looking rather indistinguishable from their normal littermates, did not perform as well on tasks involving motor control, memory and emotional sensitivity (implying that having too many branches may not be so beneficial).  In humans, a frameshift mutation involving an insertion of a C residue at position 1435 of the cDNA is associated with febrile seizures, similarly suggesting that dendritic overload can have negative effects on human brain function.

Clearly, the human brain seeks a balance between too many and too few dendritic branches.  I suppose most experienced gardeners would also agree that too many branches is not desirable.  Perhaps they are right.  However, I don’t think I’d mind much if plants came with an analogous sez6 mutation !