Epigenetics of BDNF in depression

Depression is the leading cause of disability worldwide, says the World Health Organization. The. The. I knew it was bad, but… ‘the’? More than 300 million people suffer from it worldwide and in many places fewer than 10% of these receive treatment. Lack of treatment is due to many things, from lack of access to healthcare to lack of proper diagnosis; and not in the least due to social stigma.

To complicate matters, the etiology of depression is still not fully elucidated, despite hundreds of thousand of experimental articles published out-there. Perhaps millions. But, because hundreds of thousands of experimental articles perhaps millions have been published, we know a helluva a lot about it than, say, 50 years ago. The enormous puzzle is being painstakingly assembled as we speak by scientists all over the world. I daresay we have a lot of pieces already, if not all at least 3 out of 4 corners, so we managed to build a not so foggy view of the general picture on the box lid. Here is one of the hottest pieces of the puzzle, one of those central pieces that bring the rabbit into focus.

Before I get to the rabbit, let me tell you about the corners. In the fifties people thought that depression is due to having too little neurotransmitters from the monoamine class in the brain. This thought did not arise willy-nilly, but from the observation that drugs that increase monoamine levels in the brain alleviate depression symptoms, and, correspondingly, drugs which deplete monoamines induce depression symptoms. A bit later on, the monoamine most culpable was found to be serotonin. All well and good, plenty of evidence, observational, correlational, causational, and mechanistic supporting the monoamine hypothesis of depression. But two more pieces of evidence kept nagging the researchers. The first one was that the monoamine enhancing drugs take days to weeks to start working. So, if low on serotonin is the case, then a selective serotonin reuptake inhibitor (SSRI) should elevate serotonin levels within maximum an hour of ingestion and lower symptom severity, so how come it takes weeks? The second was even more eyebrow raising: these monoamine-enhancing drugs work in about 50 % of the cases. Why not all? Or, more pragmatically put, why not most of all if the underlying cause is the same?

It took decades to address these problems. The problem of having to wait weeks until some beneficial effects of antidepressants show up has been explained away, at least partly, by issues in the serotonin regulation in the brain (e.g. autoreceptors senzitization, serotonin transporter abnormalities). As for the second problem, the most parsimonious answer is that that archeological site called DSM (Diagnostic and Statistical Manual of Mental Disorders), which psychologists, psychiatrists, and scientists all over the world have to use to make a diagnosis is nothing but a garbage bag of last century relics with little to no resemblance of this century’s understanding of the brain and its disorders. In other words, what DSM calls major depressive disorder (MDD) may as well be more than one disorder and then no wonder the antidepressants work only in half of the people diagnosed with it. As Goldberg put it in 2011, “the DSM diagnosis of major depression is made when a patient has any 5 out of 9 symptoms, several of which are opposites [emphasis added]”! He was referring to DSM-4, not that the 5 is much different. I mean, paraphrasing Goldberg, you really don’t need much of a degree other than some basic intro class in the physiology of whatever, anything really, to suspect that someone who’s sleeping a lot, gains weight, has increased appetite, appears tired or slow to others, and feels worthless might have a different cause for these symptoms than someone who has daily insomnias, lost weight recently, has decreased appetite, is hyperagitated, irritable, and feels excessive guilt. Imagine how much more understanding we would have about depression if scientists didn’t use the DSM for research. No wonder that there’s a lot of head scratching when your hypothesis, which is logically correct, paradigmatically coherent, internally consistent, flawlessly tested, turns out to be correct only sometimes because you’re ‘depressed’ subjects are as a homogeneous group as a pack of Trail Mix.

I got sidetracked again. This time ranting against DSM. No matter, I’m back on track. So. The good thing about the work done trying to figure out how antidepressants work and psychiatrists’ minds work (DSM is written overwhelmingly by psychiatrists), scientists uncovered other things about depression. Some of the findings became clumped under the name ‘the neurotrophic hypothesis of depression’ in the early naughts. It stems from the finding that some chemicals needed by neurons for their cellular happiness are in low amount in depression. Almost two decades later, the hypothesis became mainstream theory as it explains away some other findings in depression, and is not incompatible with the monoamines’ behavior. Another piece of the puzzle found.

One of these neurotrophins is called brain-derived neurotrophic factor (BDNF), which promotes cell survival and growth. Crucially, it also regulates synaptic plasticity, without which there would be no learning and no memory. The idea is that exposure to adverse events generates stress. Stress is differently managed by different people, largely due to genetic factors. In those not so lucky at the genetic lottery (how hard they take a stressor, how they deal with it), and in those lucky enough at genetics but not so lucky in life (intense and/or many stressors hit the organism hard regardless how well you take it or how good you are at it), stress kills a lot of neurons, literally, prevents new ones from being born, and prevents the remaining ones from learning well. Including learning on how to deal with the stressors, present and future, so the next time an adverse event happens, even if it is a minor stressor, the person is way more drastically affected. in other words, stress makes you more vulnerable to stressors. One of the ways stress is doing all these is by suppressing BDNF synthesis. Without BDNF, the individual exposed to stress that is exacerbated either by genes or environment ends up unable to self-regulate mood successfully. The more that mood is not regulated, the worse the brain becomes at self-regulating because the elements required for self-regulation, which include learning from experience, are busted. And so the vicious circle continues.

Maintaining this vicious circle is the ability of stressors to change the patterns of DNA expression and, not surprisingly, one of the most common findings is that the BDNF gene is hypermethylated in depression. Hypermethylation is an epigenetic change (a change around the DNA, not in the DNA itself), meaning that the gene in question is less expressed. This means lower amounts of BDNF are produced in depression.

After this long introduction, the today’s paper is a systematic review of one of epigenetic changes in depression: methylation. The 67 articles that investigated the role of methylation in depression were too heterogeneous to make a meta-analysis out of them, so Li et al. (2019) made a systematic review.

The main finding was that, overall, depression is associated with DNA methylation modifications. Two genes stood out as being hypermethylated: our friend BDNF and SLC6A4, a gene involved in the serotonin cycle. Now the question is who causes who: is stress methylating your DNA or does your methylated DNA make you more vulnerable to stress? There’s evidence both ways. Vicious circle, as I said. I doubt that for the sufferer it matters who started it first, but for the researchers it does.

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A little disclaimer: the picture I painted above offers a non-exclusive view on the causes of depression(s). There’s more. There’s always more. Gut microbes are in the picture too. And circulatory problems. And more. But the picture is more than half done, I daresay. Continuing my puzzle metaphor, we got the rabbit by the ears. Now what to do with it…

Well, one thing we can do with it, even with only half-rabbit done, is shout loud and clear that depression is a physical disease. And those who claim it can be cured by a positive attitude and blame the sufferers for not ‘trying hard enough’ or not ‘smiling more’ or not ‘being more positive’ can bloody well shut up and crawl back in the medieval cave they came from.

REFERENCES:

1. Li M, D’Arcy C, Li X, Zhang T, Joober R, & Meng X (4 Feb 2019). What do DNA methylation studies tell us about depression? A systematic review. Translational Psychiatry, 9(1):68. PMID: 30718449, PMCID: PMC6362194, DOI: 10.1038/s41398-019-0412-y. ARTICLE | FREE FULLTEXT PDF

2. Goldberg D (Oct 2011). The heterogeneity of “major depression”. World Psychiatry, 10(3):226-8. PMID: 21991283, PMCID: PMC3188778. ARTICLE | FREE FULLTEXT PDF

3. World Health Organization Depression Fact Sheet

By Neuronicus, 23 April 2019

Pic of the day: Dopamine from a non-dopamine place

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Reference: Beas BS, Wright BJ, Skirzewski M, Leng Y, Hyun JH, Koita O, Ringelberg N, Kwon HB, Buonanno A, & Penzo MA (Jul 2018, Epub 18 Jun 2018). The locus coeruleus drives disinhibition in the midline thalamus via a dopaminergic mechanism. Nature Neuroscience,21(7):963-973. PMID: 29915192, PMCID: PMC6035776 [Available on 2018-12-18], DOI:10.1038/s41593-018-0167-4. ARTICLE

Locus Coeruleus in mania

From all the mental disorders, bipolar disorder, a.k.a. manic-depressive disorder, has the highest risk for suicide attempt and completion. If the thought of suicide crosses your mind, stop reading this, it’s not that important; what’s important is for you to call the toll-free National Suicide Prevention Lifeline at 1-800-273-TALK (8255).

The bipolar disorder is defined by alternating manic episodes of elevated mood, activity, excitation, and energy with episodes of depression characterized by feelings of deep sadness, hopelessness, worthlessness, low energy, and decreased activity. It is also a more common disease than people usually expect, affecting about 1% or more of the world population. That means almost 80 million people! Therefore, it’s imperative to find out what’s causing it so we can treat it.

Unfortunately, the disease is very complex, with many brain parts, brain chemicals, and genes involved in its pathology. We don’t even fully comprehend how the best medication we have to lower the risk of suicide, lithium, works. The good news is the neuroscientists haven’t given up, they are grinding at it, and with every study we get closer to subduing this monster.

One such study freshly published last month, Cao et al. (2018), looked at a semi-obscure membrane protein, ErbB4. The protein is a tyrosine kinase receptor, which is a bit unfortunate because this means is involved in ubiquitous cellular signaling, making it harder to find its exact role in a specific disorder. Indeed, ErbB4 has been found to play a role in neural development, schizophrenia, epilepsy, even ALS (Lou Gehrig’s disease).

Given that ErbB4 is found in some neurons that are involved in bipolar and mutations in its gene are also found in some people with bipolar, Cao et al. (2018) sought to find out more about it.

First, they produced mice that lacked the gene coding for ErbB4 in neurons from locus coeruleus, the part of the brain that produces norepinephrine out of dopamine, better known for the European audience as nor-adrenaline. The mutant mice had a lot more norepinephrine and dopamine in their brains, which correlated with mania-like behaviors. You might have noticed that the term used was ‘manic-like’ and not ‘manic’ because we don’t know for sure how the mice feel; instead, we can see how they behave and from that infer how they feel. So the researchers put the mice thorough a battery of behavioral tests and observed that the mutant mice were hyperactive, showed less anxious and depressed behaviors, and they liked their sugary drink more than their normal counterparts, which, taken together, are indices of mania.

Next, through a series of electrophysiological experiments, the scientists found that the mechanism through which the absence of ErbB4 leads to mania is making another receptor, called NMDA, in that brain region more active. When this receptor is hyperactive, it causes neurons to fire, releasing their norepinephrine. But if given lithium, the mutant mice behaved like normal mice. Correspondingly, they also had a normal-behaving NMDA receptor, which led to normal firing of the noradrenergic neurons.

So the mechanism looks like this (Jargon alert!):

No ErbB4 –> ↑ NR2B NMDAR subunit –> hyperactive NMDAR –> ↑ neuron firing –> ↑ catecholamines –> mania.

In conclusion, another piece of the bipolar puzzle has been uncovered. The next obvious step will be for the researchers to figure out a medicine that targets ErbB4 and see if it could treat bipolar disorder. Good paper!

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P.S. If you’re not familiar with the journal eLife, go and check it out. The journal offers for every study a half-page summary of the findings destined for the lay audience, called eLife digest. I’ve seen this practice in other journals, but this one is generally very well written and truly for the lay audience and the non-specialist. Something of what I try to do here, minus the personal remarks and in parenthesis metacognitions that you’ll find in most of my posts. In short, the eLife digest is masterly done. As my continuous struggles on this blog show, it is tremendously difficult for a scientist to write concisely, precisely, and jargonless at the same time. But eLife is doing it. Check it out. Plus, if you care to take a look on how science is done and published, eLife publishes all the editor’s rejection notes, all the reviewers’ comments, and all the author responses for a particular paper. Reading those is truly a teaching moment.

REFERENCE: Cao SX, Zhang Y, Hu XY, Hong B, Sun P, He HY, Geng HY, Bao AM, Duan SM, Yang JM, Gao TM, Lian H, Li XM (4 Sept 2018). ErbB4 deletion in noradrenergic neurons in the locus coeruleus induces mania-like behavior via elevated catecholamines. Elife, 7. pii: e39907. doi: 10.7554/eLife.39907. PMID: 30179154 ARTICLE | FREE FULLTEXT PDF

By Neuronicus, 14 October 2018

Arc: mRNA & protein from one neuron to another

EDIT 1 [Jan 17, 2018]: I promised four days ago that I will post this, while it was still hot, but my Internet was down, thanks to the only behemoth provider in USA. And rated the worst company in the Nation, too. You definitely know by now about whom I’m talking about. Grrrr…  Anyway, here is the paper:

As promised, today’s paper talks about mRNA transfer between neurons.

Pastuzyn et al. (2018) looked at the gene Arc in neurons because they thought its Gag sequence looks suspiciously similar to some retroviruses. Could it be possible that it also behaves like a virus?

Arc is heavily involved in the immune system, is essential for the formation of long-term memories, and is involved in all sorts of diseases, like schizophrenia and Alzheimer’s, among other things.

Pastuzyn et al. (2018) is a relatively long and dense paper, albeit well written. So, I thought that this time, instead of giving you a summary of their research it would be better to give you the authors’ story directly in their own words written as subtitles in the Results section (bold letters – the authors words, normal font – mine). Warning: this is a much more jargon-dense blog post than my previous one on the same topic and, because it is so much material, I will not explain every term.

  • Fly and Tetrapod (us) Arc Genes Independently Originated from Distinct Lineages of Ty3/gypsy Retrotransposons, the phylogenomic analyses tell us, meaning the authors have done a lot of computer-assisted comparisons of similar forms of the gene in hundreds of species.
  • Arc Proteins Self-Assemble into Virus-like Capsids. Arc likes to oligomerize spontaneously (dimers and trimers). The oligomers resemble virus-like capsids, similar to HIV.
  • Arc Binds and Encapsulates RNA. Although it loves its own RNA about 10 times more than other RNAs, it’s a promiscuous protein (doesn’t care which RNA as long as it follows the rules of stoichiometry). Arc capsids encapsulate both the Arc protein (maybe other proteins too?), its mRNA, and whatever mRNA happened to be in the vicinity at the time of encapsulation. Arc capsids are able to protect the mRNA from RNAases.
  • Arc Capsid Assembly Requires RNA. If there is no RNA around, the capsids are few and poorly formed.
  • Arc Protein and Arc mRNA Are Released by Neurons in Extracellular Vesicles. Arc capsid packages Arc protein & Arc mRNA into extracellular vesicles (EV). The size of these EVs is < 100nm, putting them in the exosome category. This exosome, which the authors gave the unfortunate name of ACBAR (Arc Capsid Bearing Any RNA), is being expelled from cortical neurons in an activity-dependent manner. In other words, when neurons are stimulated, they release ACBARs.
  • Arc Mediates Intercellular Transfer of mRNA in Extracellular Vesicles. ACBARs dock to the host cell and then undergo clathrin-dependent endocytosis, meaning they expel their cargo in the host cell. The levels of Arc protein and Arc mRNA peaks in a host hippocampal cell in four hours from incubation. The ACBARs tend to congregate around donor cell’s dendrites.
  • Transferred Arc mRNA Can Undergo Activity-Dependent Translation. Activating the group 1 metabotropic glutamate receptor (mGluR1/5) by application of the agonist DHPG induces a significant increase of the amount of Arc protein in the host neurons.

This is a veritable tour de force paper. The Results section has 7 sub-sections, each with multiple experiments to dot every i and cross every t. I’m eyeballing about 40 experiments. It is true that there are 13 authors on the paper from different institutions – yeay for collaboration! – but c’mon! Is this what you need to get in Cell these days? Apparently so. Don’t get me wrong, this is an outstanding paper. But in the end it is still only one paper, which means only one first author. The rest are there for the ride because for a tenure track application nobody cares about your papers in CNS (Cell, Nature, Science = The Central Nervous System of the scientific community, har, har) if you’re not the first author. It looks like the increasing amount of work you need to be published in top tier journals these days is becoming a pet peeve of mine as I keep mentioning it (for example, here).

My pet peeves aside, Pastuzyn et al. (2018) is an excellent paper that opens interesting practical (drug delivery) and theoretical (biological repurpose of ancient invaders) gates. Kudos!

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REFERENCE: Pastuzyn ED, Day CE, Kearns RB, Kyrke-Smith M, Taibi AV, McCormick J, Yoder N, Belnap DM, Erlendsson S, Morado DR, Briggs JAG, Feschotte C, & Shepherd JD. (11 Jan 2018). The Neuronal Gene Arc Encodes a Repurposed Retrotransposon Gag Protein that Mediates Intercellular RNA Transfer. Cell, 172(1-2):275-288.e18. PMID: 29328916. doi: 10.1016/j.cell.2017.12.024. ARTICLE | FULLTEXT PDF via ResearchGate

P.S. I said that ACBAR is an unfortunate acronym because I don’t know about you but I for one wouldn’t want my discovery to be linked either with a religion or with terrorist cries, even if that link is done only by a small fraction of the population. Although I can totally see the naming-by-committee going: “ACBAR! Our exosome is the greatest! Yeay!” or “Arc Acbar! Our Arc is the greatest. Double yeay!”. On a second thought, it’s kindda nerdy geeky neat. I still wouldn’t have done it though…

By Neuronicus, 14 January 2018

EDIT 2 [Jan 22, 2018]: There is another paper that discovered that Arc forms capsids that encapsulate RNA and then shuttles it across the neuromuscular junction in Drosophila (fly). To their credit, Cell published both these papers back-to-back so no researcher gets scooped of their discovery. From what I can see, the discovery really happened simultaneously, so I modified my infopic to reflect that (both papers were submitted in January 2017, received in revised version on August 15, 2017 and published in the same issue on January 11, 2018). Here is the reference to the other article:

Ashley J, Cordy B, Lucia D, Fradkin LG, Budnik V, & Thomson T (11 Jan 2018). Retrovirus-like Gag Protein Arc1 Binds RNA and Traffics across Synaptic Boutons, Cell. 172(1-2): 262-274.e11. PMID: 29328915. doi: 10.1016/j.cell.2017.12.022. ARTICLE

EDIT 3 [Jan 29, 2018]: Dr. Shepherd, the last author of the paper I featured, was kind enough to answer a few of my questions about the implications of his and his team’s findings, answers which you will find here.

By Neuronicus, 22 January 2018

The third eye

The pineal gland has held fascination since Descartes’ nefarious claim that it is the seat of the soul. There is no evidence of that; he said it might be where the soul resides because he thought the pineal gland was the only solitaire structure in the brain so it must be special. By ‘solitaire’ I mean that all other brain structures come in doublets: 2 amygdalae, 2 hippocampi, 2 thalami, 2 hemispheres etc. He was wrong about that as well, in that there are some other singletons in the brain besides the pineal, like the anterior or posterior commissure, the cerebellar vermis, some deep brainstem and medullary structures etc.

Descartes’ dualism was the only escape route the mystics at the time had from the demands for evidence by the budding natural philosophers later known as scientists. So when some scientists noted that some lizards have a third eye on top of their head connected to the pineal gland, the mystics and, later, the conspiracy theorists went nuts. Here, see, if the seat of the soul is linked with the third eye, then the awakening of this eye in people would surely result in heightened awareness, closeness to the Divinity, oneness with Universe and other similar rubbish that can otherwise easily and reliably be achieved by a good dollop of magic mushrooms. Cheaper, too.

Back to the lizards. Yes, you read right: some lizards and frogs have a third eye. This eye is not exactly like the other two, but it has cells sensitive to light, even if they are not perceiving light in the same way the retinal cells from the lateral eyes are. It is located on the top of the skull, so sometimes it is called the parietal organ (because it is in-between the parietal skull bones, see pic).

Anolis_carolinensis_parietal_eye.CC BY-SA 3.0jpg
Dorsal view of the head of the adult Carolina anole (Anolis carolinensis) clearly showing the parietal eye (small gray/clear oval) at the top of its head. Photo by TheAlphaWolf. License: CC BY-SA 3.0, courtesy of Wikipedia.

It is believed to be a vestigial organ, meaning that primitive vertebrates might have had it as a matter of course but it disappeared in the more recently evolved animals. Importantly, birds and mammals don’t have it. Not at all, not a bit, not atrophied, not able to be “awakened” no matter what your favorite “lemme see your chakras” guru says. Go on, touch your top of the skull and see if you have some peeking soft tissue there. And no, the soft tissue that babies are born with right there on the top of the skull is not a third eye; it’s a fontanelle that allows for the rapid expansion of the brain during the first year of life.

The parietal organ’s anatomical connection to the pineal gland is not surprising at all for scientists because the pineal’s role in every single animal that has it is the regulation of some circadian rhythms by the production of melatonin. In humans, the eyes tell the pineal that is day or night and the pineal adjusts the melatonin production accordingly, i.e. less melatonin produced during the day and more during the night. Likewise, the lizards’ third eye’s main role is to provide information to the pineal about the ambient light for thermoregulatory purposes.

After this long introduction, here is the point: almost twenty years ago Xiong et al. (1998) looked at how this third eye perceives light. In the human eye, light hitting the rods and cones in the retina (reception) launches a biochemical cascade (transduction) that results in seeing (coding of the stimulus in the brain). Briefly, transduction goes thusly: the photon(s) cause(s) a special protein sensitive to light (e.g. rhodopsin) in the photoreceptor cells in the retina to split into its components (photobleaching), one of these components (11-cis-retinal) changes its conformation (photoisomerization), then activates a G-protein (transducin), which then activates the enzyme phosphodiesterase (PDE), which then destroys a nucleotide called cyclic guanosine monophosphate (cGMP), which results in the closing of the cell’s sodium ion channels, which leads to less neurotransmitter released (glutamate), which causes the nearby cells (bipolar cells) to release the same neurotransmitter, which now has the opposite effect, meaning it increases the firing rate of another set of cells (ganglion cells) and from there to the brain we go. Phew, visual transduction IS difficult. And this is the brief version.

It turns out that the third eye’s retina doesn’t have all the types of cells that the normal eyes have. Specifically, it misses the bipolar, horizontal and amacrine cells, having only ganglion and photoreception cells. So how goes the phototransduction in the third eye’s retina, if at all?

Xiong et al. (1998) isolated photoreceptor cells from the third eyes of the lizard Uta stansburiana. And then they did a bunch of electrophysiological recording on those cells under different illumination and chemical conditions.

They found that the phototransduction in the third eye is different from the lateral eyes in that when they expected to see hyperpolarization of the cell, they observed depolarization instead. Also, when they expected the PDE to break down cGMP they found that PDE is inhibited thereby increasing the amount of cGMP.  The fact that G-protein can inhibit PDE was totally unexpected and showed a novel way of cellular signaling. Moreover, they speculate that their results can make sense only if not one, but two G-proteins with opposite actions work in tandem.

A probably dumb technical question though: the human rhodopsin takes about 30 minutes to restore itself from photobleaching. Xiong et al. (1998) let the cells adapt to dark for 10 minutes before recordings. So I wonder if the results would have been slightly different if they allowed the cell more time to adapt? But I’m not an expert in retina science, you’ve seen how difficult it is, right? Maybe the lizard proteins are different or rhodopsin adaptation time has little or nothing to do with their experiments? After all, later research has shown that the third eye has its own unique opsins, like the green-sensitive parietopsin discovered by Su et al. (2006).

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REFERENCE:  Xiong WH, Solessio EC, & Yau KW (Sep 1998). An unusual cGMP pathway underlying depolarizing light response of the vertebrate parietal-eye photoreceptor. Nature Neuroscience, 1(5): 359-365. PMID: 10196524, DOI: 10.1038/1570. ARTICLE

Additional bibliography: Su CY, Luo DG, Terakita A, Shichida Y, Liao HW, Kazmi MA, Sakmar TP, Yau KW (17 Mar 2006). Parietal-eye phototransduction components and their potential evolutionary implications. Science, 311(5767): 1617-1621. PMID: 16543463, DOI: 10.1126/science.1123802. ARTICLE

By Neuronicus, 30 March 2017

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Aging and its 11 hippocampal genes

Aging is being quite extensively studied these days and here is another advance in the field. Pardo et al. (2017) looked at what happens in the hippocampus of 2-months old (young) and 28-months old (old) female rats. Hippocampus is a seahorse shaped structure no more than 7 cm in length and 4 g in weight situated at the level of your temples, deep in the brain, and absolutely necessary for memory.

First the researchers tested the rats in a classical maze test (Barnes maze) designed to assess their spatial memory performance. Not surprisingly, the old performed worse than the young.

Then, they dissected the hippocampi and looked at neurogenesis and they saw that the young rats had more newborn neurons than the old. Also, the old rats had more reactive microglia, a sign of inflammation. Microglia are small cells in the brain that are not neurons but serve very important functions.

After that, the researchers looked at the hippocampal transcriptome, meaning they looked at what proteins are being expressed there (I know, transcription is not translation, but the general assumption of transcriptome studies is that the amount of protein X corresponds to the amount of the RNA X). They found 210 genes that were differentially expressed in the old, 81 were upregulated and 129 were downregulated. Most of these genes are to be found in human too, 170 to be exact.

But after looking at male versus female data, at human and mouse aging data, the authors came up with 11 genes that are de-regulated (7 up- and 4 down-) in the aging hippocampus, regardless of species or gender. These genes are involved in the immune response to inflammation. More detailed, immune system activates microglia, which stays activated and this “prolonged microglial activation leads to the release of pro-inflammatory cytokines that exacerbate neuroinflammation, contributing to neuronal loss and impairment of cognitive function” (p. 17). Moreover, these 11 genes have been associated with neurodegenerative diseases and brain cancers.

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These are the 11 genes: C3 (up), Cd74  (up), Cd4 (up), Gpr183 (up), Clec7a (up), Gpr34 (down), Gapt (down), Itgam (down), Itgb2 (up), Tyrobp (up), Pld4 (down).”Up” and “down” indicate the direction of deregulation: upregulation or downregulation.

I wish the above sentence was as explicitly stated in the paper as I wrote it so I don’t have to comb through their supplemental Excel files to figure it out. Other than that, good paper, good work. Gets us closer to unraveling and maybe undoing some of the burdens of aging, because, as the actress Bette Davis said, “growing old isn’t for the sissies”.

Reference: Pardo J, Abba MC, Lacunza E, Francelle L, Morel GR, Outeiro TF, Goya RG. (13 Jan 2017, Epub ahead of print). Identification of a conserved gene signature associated with an exacerbated inflammatory environment in the hippocampus of aging rats. Hippocampus, doi: 10.1002/hipo.22703. ARTICLE

By Neuronicus, 25 January 2017

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How do you remember?

Memory processes like formation, maintenance and consolidation have been the subjects of extensive research and, as a result, we know quite a bit about them. And just when we thought that we are getting a pretty clear picture of the memory tableau and all that is left is a little bit of dusting around the edges and getting rid of the pink elephant in the middle of the room, here comes a new player that muddies the waters again.

DNA methylation. The attaching of a methyl group (CH3) to the DNA’s cytosine by a DNA methyltransferase (Dnmt) was considered until very recently a process reserved for the immature cells in helping them meet their final fate. In other words, DNA methylation plays a role in cell differentiation by suppressing gene expression. It has other roles in X-chromosome inactivation and cancer, but it was not suspected to play a role in memory until this decade.

Oliveira (2016) gives us a nice review of the role(s) of DNA methylation in memory formation and maintenance. First, we encounter the pharmacological studies that found that injecting Dnmt inhibitors in various parts of the brain in various species disrupted memory formation or maintenance. Next, we see the genetic studies, where mice Dnmt knock-downs and knock-outs also show impaired memory formation and maintenance. Finally, knowing which genes’ transcription is essential for memory, the researcher takes us through several papers that examine the DNA de novo methylation and demethylation of these genes in response to learning events and its role in alternative splicing.

Based on these here available data, the author proposes that activity induced DNA methylation serves two roles in memory: to “on the one hand, generate a primed and more permissive epigenome state that could facilitate future transcriptional responses and on the other hand, directly regulate the expression of genes that set the strength of the neuronal network connectivity, this way altering the probability of reactivation of the same network” (p. 590).

Here you go; another morsel of actual science brought to your fingertips by yours truly.

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Reference: Oliveira AM (Oct 2016, Epub 15 Sep 2016). DNA methylation: a permissive mark in memory formation and maintenance. Learning & Memory,  23(10): 587-593. PMID: 27634149, DOI: 10.1101/lm.042739.116. ARTICLE

By Neuronicus, 22 September 2016

Who invented optogenetics?

Wayne State University. Ever heard of it? Probably not. How about Zhuo-Hua Pan? No? No bell ringing? Let’s try a different approach: ever heard of Stanford University? Why, yes, it’s one of the most prestigious and famous universities in the world. And now the last question: do you know who Karl Deisseroth is? If you’re not a neuroscientist, probably not. But if you are, then you would know him as the father of optogenetics.

Optogenetics is the newest tool in the biology kit that allows you to control the way a cell behaves by shining a light on it (that’s the opto part). Prior to that, the cell in question must be made to express a protein that is sensitive to light (i.e. rhodopsin) either by injecting a virus or breeding genetically modified animals that express that protein (that’s the genetics part).

If you’re watching the Nobel Prizes for Medicine, then you would also be familiar with Deisseroth’s name as he may be awarded the Nobel soon for inventing optogenetics. Only that, strictly speaking, he did not. Or, to be fair and precise at the same time, he did, but he was not the first one. Dr. Pan from Wayne State University was. And he got scooped.98.png

The story is at length imparted to us by Anna Vlasits in STAT and republished in Scientific American. In short, Dr. Pan, an obscure name in an obscure university from an ill-famed city (Detroit), does research for years in an unglamorous field of retina and blindness. He figured, quite reasonably, that restoring the proteins which sense light in the human eye (i.e. photoreceptor proteins) could restore vision in the congenitally blind. The problem is that human photoreceptor proteins are very complicated and efforts to introduce them into retinas of blind people have proven unsuccessful. But, in 2003, a paper was published showing how an algae protein that senses light, called channelrhodopsin (ChR), can be expressed into mammalian cells without loss of function.

So, in 2004, Pan got a colleague from Salus University (if Wayne State University is a medium-sized research university, then Salus is a really tiny, tiny little place) to engineer a ChR into a virus which Pan then injected in rodent retinal neurons, in vivo. After 3-4 weeks he obtained the expression of the protein and the expression was stable for at least 1 year, showing that the virus works nicely. Then his group did a bunch of electrophysiological recordings (whole cell patch-clamp and voltage clamp) to see if shining light on those neurons makes them fire. It did. Then, they wanted to see if ChR is for sure responsible for this firing and not some other proteins so they increased the intensity of the blue light that their ChR is known to sense and observed that the cell responded with increased firing. Now that they saw the ChR works in normal rodents, next they expressed the ChR by virally infecting mice who were congenitally blind and repeated their experiments. The electrophysiological experiments showed that it worked. But you see with your brain, not with your retina, so the researchers looked to see if these cells that express ChR project from the retina to the brain and they found their axons in lateral geniculate and superior colliculus, two major brain areas important for vision. Then, they recorded from these areas and the brain responded when blue light, but not yellow or other colors, was shone on the retina. The brain of congenitally blind mice without ChR does not respond regardless of the type of light shone on their retinas. But does that mean the mouse was able to see? That remains to be seen (har har) in future experiments. But the Pan group did demonstrate – without question or doubt – that they can control neurons by light.

All in all, a groundbreaking paper. So the Pan group was not off the mark when they submitted it to Nature on November 25, 2004. As Anna Vlasits reports in the Exclusive, Nature told Pan to submit to a more specialized journal, like Nature Neuroscience, which then rejected it. Pan submitted then to the Journal of Neuroscience, which also rejected it. He submitted it then to Neuron on November 29, 2005, which finally accepted it on February 23, 2006. Got published on April 6, 2006. Deisseroth’s paper was submitted to Nature Neuroscience on May 12, 2005, accepted on July, and published on August 14, 2005… His group infected rat hippocampal neurons cultured in a Petri dish with a virus carrying the ChR and then they did some electrophysiological recordings on those neurons while shining lights of different wavelengths on them, showing that these cells can be controlled by light.

There’s more on the saga with patent filings and a conference where Pan showed the ChR data in May 2005 and so on, you can read all about it in Scientific American. The magazine is just hinting to what I will say outright, loud and clear: Pan didn’t get published because of his and his institution’s lack of fame. Deisseroth did because of the opposite. That’s all. This is not about squabbles about whose work is more elegant, who presented his work as a scientific discovery or a technical report or whose title is more catchy, whose language is more boisterous or native English-speaker or luck or anything like that. It is about bias and, why not?, let’s call a spade a spade, discrimination. Nature and Journal of Neuroscience are not caught doing this for the first time. Not by a long shot. The problem is that they are still doing it, that is: discriminating against scientific work presented to them based on the name of the authors and their institutions.

Personally, so I don’t get comments along the lines of the fox and the grapes, I have worked at both high profile and low profile institutions. And I have seen the difference not in the work, but in the reception.

That’s my piece for today.

Source:  STAT, Scientific American.

References:

1) Bi A, Cui J, Ma YP, Olshevskaya E, Pu M, Dizhoor AM, & Pan ZH (6 April 2006). Ectopic expression of a microbial-type rhodopsin restores visual responses in mice with photoreceptor degeneration. Neuron, 50(1): 23-33. PMID: 16600853. PMCID: PMC1459045. DOI: 10.1016/j.neuron.2006.02.026. ARTICLE | FREE FULLTEXT PDF

2) Boyden ES, Zhang F, Bamberg E, Nagel G, & Deisseroth K. (Sep 2005, Epub 2005 Aug 14). Millisecond-timescale, genetically targeted optical control of neural activity. Nature Neuroscience, 8(9):1263-1268. PMID: 16116447. DOI: 10.1038/nn1525. doi:10.1038/nn1525. ARTICLE 

By Neuronicus, 11 September 2016

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Another puzzle piece in the autism mystery

Just like in the case of schizophrenia, hundreds of genes have been associated with autistic spectrum disorders (ASDs). Here is another candidate.

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Féron et al. (2016) reasoned that most of the info we have about the genes that are behaving badly in ASDs comes from studies that used adult cells. Because ASDs are present before or very shortly after birth, they figured that looking for genetic abnormalities in cells that are at the very early stage of ontogenesis might prove to be enlightening. Those cells are stem cells. Of the pluripotent kind. FYI, based on what they can become (a.k.a how potent they are), the stem cells are divided into omipotent, pluripotent, multipotent, oligopotent, and unipotent. So the pluripotents are very ‘potent’ indeed, having the potential of producing a perfect person.

Tongue-twisters aside, the authors’ approach is sensible, albeit non-hypothesis driven. Which means they hadn’t had anything specific in mind when they had started looking for differences in gene expression between the olfactory nasal cells obtained from 11 adult ASDs sufferers and 11 age-matched normal controls. Luckily for them, as transcriptome studies have a tendency to be difficult to replicate, they found the anomalies in the expression of genes that have been already associated with ASD. But, they also found a new one, the MOCOS (MOlybdenum COfactor Sulfurase) gene, which was poorly expressed in ASDs (downregulated, in genetic speak). The enzyme is MOCOS (am I the only one who thinks that MOCOS isolated from nasal cells is too similar to mucus? is the acronym actually a backronym?).

The enzyme is not known to play any role in the nervous system. Therefore, the researchers looked to see where the gene is expressed. Its enzyme could be found all over the brain of both mouse and human. Also, in the intestine, kidneys, and liver. So not much help there.

Next, the authors deleted this gene in a worm, Caenorhabditis elegans, and they found out that the worm’s cells have issues in dealing with oxidative stress (e.g. the toxic effects of free radicals). In addition, their neurons had abnormal synaptic transmission due to problems with vesicular packaging.

Then they managed – with great difficulty – to produce human induced pluripotent cells (iPSCs) in a Petri dish in which the gene MOCOS was partially knocked down. ‘Partially’, because the ‘totally’ did not survive. Which tells us that MOCOS is necessary for survival of iPSCs. The mutant cells had less synaptic buttons than the normal cells, meaning they formed less synapses.

The study, besides identifying a new candidate for diagnosis and treatment, offers some potential explanations for some beguiling data that other studies have brought forth, like the fact that all sorts of neurotransmitter systems seem to be impaired in ADSs, all sorts of brain regions, making very hard to grab the tiger by the tail if the tiger is sprouting a new tail when you look at it, just like the Hydra’s heads. But, discovering a molecule that is involved in an ubiquitous process like synapse formation may provide a way to leave the tiger’s tail(s) alone and focus on the teeth. In the authors’ words:

“As a molecule involved in the formation of dense core vesicles and, further down, neurotransmitter secretion, MOCOS seems to act on the container rather than the content, on the vehicle rather than one of the transported components” (p. 1123).

The knowledge uncovered by this paper makes a very good piece of the ASDs puzzle. Maybe not a corner, but a good edge. Alright, even if it’s not an edge, at least it’s a crucial piece full of details, not one of those sky pieces.

Reference: Féron F, Gepner B, Lacassagne E, Stephan D, Mesnage B, Blanchard MP, Boulanger N, Tardif C, Devèze A, Rousseau S, Suzuki K, Izpisua Belmonte JC, Khrestchatisky M, Nivet E, & Erard-Garcia M (Sep 2016, Epub 4 Aug 2016). Olfactory stem cells reveal MOCOS as a new player in autism spectrum disorders. Molecular Psychiatry, 21(9):1215-1224. PMID: 26239292, DOI: 10.1038/mp.2015.106. ARTICLE | FREE FULLTEXT PDF

By Neuronicus, 31 August 2016

Painful Pain Paper

There has been much hype over the new paper published in the latest Nature issue which claims to have discovered an opioid analgesic that doesn’t have most of the side effects of morphine. If the claim holds, the authors may have found the Holy Grail of pain research chased by too many for too long (besides being worth billions of dollars to its discoverers).

The drug, called PZM21, was discovered using structure-based drug design. This means that instead of taking a drug that works, say morphine, and then tweaking its molecular structure in various ways and see if the resultant drugs work, you take the target of the drug, say mu-opioid receptors, and design a drug that fits in that slot. The search and design are done initially with sophisticated software and there are many millions of virtual candidates. So it takes a lot of work and ingenuity to select but a few drugs that will be synthesized and tested in live animals.

Manglik et al. (2016) did just that and they came up with PZM21 which, compared to morphine, is:

1) selective for the mu-opioid receptors (i.e. it doesn’t bind to anything else)
2) produces no respiratory depression (maybe a touch on the opposite side)
3) doesn’t affect locomotion
4) produces less constipation
5) produces long-lasting affective analgesia
6) and has less addictive liability

The Holy Grail, right? Weeell, I have some serious issues with number 5 and, to some extent, number 6 on this list.

Normally, I wouldn’t dissect a paper so thoroughly because, if there is one thing I learned by the end of GradSchool and PostDoc, is that there is no perfect paper out there. Consequently, anyone with scientific training can find issues with absolutely anything published. I once challenged someone to bring me any loved and cherished paper and I would tear it apart; it’s much easier to criticize than to come up with solutions. Probably that’s why everybody hates Reviewer No. 2…

But, for extraordinary claims, you need extraordinary evidence. And the evidence simply does not support the 5 and maybe 6 above.

Let’s start with pain. The authors used 3 tests: hotplate (drop a mouse on a hot plate for 10 sec and see what it does), tail-flick (give an electric shock to the tail and see how fast the mouse flicks its tail) and formalin (inject an inflammatory painful substance in the mouse paw and see what the animal does). They used 3 doses of PZM21 in the hotplate test (10, 20, and 40 mg/Kg), 2 doses in the tail-flick test (10 and 20), and 1 dose in the formalin test (20). Why? If you start with a dose-response in a test and want to convince me it works in the other tests, then do a dose-response for those too, so I have something to compare. These tests have been extensively used in pain research and the standard drug used is morphine. Therefore, the literature is clear on how different doses of morphine work in these tests. I need your dose-responses for your new drug to be able to see how it measures up, since you claim it is “more efficacious than morphine”. If you don’t want to convince me there is a dose-response effect, that’s fine too, I’ll frown a little, but it’s your choice. However, then choose a dose and stick with it! Otherwise I cannot compare the behaviors across tests, rendering one or the other test meaningless. If you’re wondering, they used only one dose of morphine in all the tests, except the hotplate, where they used two.

Another thing also related to doses. The authors found something really odd: PZM21 works (meaning produces analgesia) in the hotplate, but not the tail-flick tests. This is truly amazing because no opiate I know of can make such a clear-cut distinction between those two tests. Buuuuut, and here is a big ‘BUT” they did not test their highest dose (40mg/kg) in the tail-flick test! Why? I’ll tell you how, because I am oh sooo familiar with this argument. It goes like this:

Reviewer: Why didn’t you use the same doses in all your 3 pain tests?

Author: The middle and highest doses have similar effects in the hotplate test, ok? So it doesn’t matter which one of these doses I’ll use in the tail-flick test.

Reviewer: Yeah, right, but, you have no proof that the effects of the two doses are indistinguishable because you don’t report any stats on them! Besides, even so, that argument applies only when a) you have ceiling effects (not the case here, your morphine hit it, at any rate) and b) the drug has the expected effects on both tests and thus you have some logical rationale behind it. Which is not the case here, again: your point is that the drug DOESN’T produce analgesia in the tail-flick test and yet you don’t wanna try its HIGHEST dose… REJECT AND RESUBMIT! Awesome drug discovery, by the way!

So how come the paper passed the reviewers?! Perhaps the fact that two of the reviewers are long term publishing co-authors from the same University had something to do with it, you know, same views predisposes them to the same biases and so on… But can you do that? I mean, have reviewers for Nature from the same department for the same paper?

Alrighty then… let’s move on to the stats. Or rather not. Because there aren’t any for the hotplate or tail-flick! Now, I know all about the “freedom from the tyranny of p” movement (that is: report only the means, standard errors of mean, and confidence intervals and let the reader judge the data) and about the fact that the average scientist today needs to know 100-fold more stats that his predecessors 20 years ago (although some biologists and chemists seem to be excused from this, things either turn color or not, either are there or not etc.) or about the fact that you cannot get away with only one experiment published these days, but you need a lot of them so you have to do a lot of corrections to your stats so you don’t fall into the Type 1 error. I know all about that, but just like the case with the doses, choose one way or another and stick to it. Because there are ANOVAs ran for the formalin test, the respiration, constipation, locomotion, and conditioned place preference tests, but none for the hotplate or tailflick! I am also aware that to be published in Science or Nature you have to strip your work and wordings to the bare minimum because the insane wordcount limits, but you have free rein in the Supplementals. And I combed through those and there are no stats there either. Nor are there any power analyses… So, what’s going on here? Remember, the authors didn’t test the highest dose on the tail-flick test because – presumably – the highest and intermediary doses have indistinguishable effects, but where is the stats to prove it?

And now the thing that really really bothered me: the claim that PZM21 takes away the affective dimension of pain but not the sensory. Pain is a complex experience that, depending on your favourite pain researcher, has at least 2 dimensions: the sensory (also called ‘reflexive’ because it is the immediate response to the noxious stimulation that makes you retract by reflex the limb from whatever produces the tissue damage) and the affective (also called ‘motivational’ because it makes the pain unpleasant and motivates you to get away from whatever caused it and seek alleviation and recovery). The first aspect of pain, the sensory, is relatively easy to measure, since you look at the limb withdrawal (or tail, in the case of animals with prolonged spinal column). By contrast, the affective aspect is very hard to measure. In humans, you can ask them how unpleasant it is (and even those reports are unreliable), but how do you do it with animals? Well, you go back to humans and see what they do. Humans scream “Ouch!” or swear when they get hurt (so you can measure vocalizations in animals) or humans avoid places in which they got hurt because they remember the unpleasant pain (so you do a test called Conditioned Place Avoidance for animals, although if you got a drug that shows positive results in this test, like morphine, you don’t know if you blocked the memory of unpleasantness or the feeling of unpleasantness itself, but that’s a different can of worms). The authors did not use any of these tests, yet they claim that PZM21 takes away the unpleasantness of pain, i.e. is an affective analgesic!

What they did was this: they looked at the behaviors the animal did on the hotplate and divided them in two categories: reflexive (the lifting of the paw) and affective (the licking of the paw and the jumping). Now, there are several issues with this dichotomy, I’m not even going to go there; I’ll just say that there are prominent pain researchers that will scream from the top of their lungs that the so-called affective behaviors from the hotplate test cannot be indexes of pain affect, because the pain affect requires forebrain structures and yet these behaviors persist in the decerebrated rodent, including the jumping. Anyway, leaving the theoretical debate about what those behaviors they measured really mean aside, there still is the problem of the jumpers: namely, the authors excluded from the analysis the mice who tried to jump out of the hotplate test in the evaluation of the potency of PZM21, but then they left them in when comparing the two types of analgesia because it’s a sign of escaping, an emotionally-valenced behavior! Isn’t this the same test?! Seriously? Why are you using two different groups of mice and leaving the impression that is only one? And oh, yeah, they used only the middle dose for the affective evaluation, when they used all three doses for potency…. And I’m not even gonna ask why they used the highest dose in the formalin test… but only for the normal mice, the knockouts in the same test got the middle dose! So we’re back comparing pears with apples again!

Next (and last, I promise, this rant is way too long already), the non-addictive claim. The authors used the Conditioned Place Paradigm, an old and reliable method to test drug likeability. The idea is that you have a box with 2 chambers, X and Y. Give the animal saline in chamber X and let it stay there for some time. Next day, you give the animal the drug and confine it in chamber Y. Do this a few times and on the test day you let the animal explore both chambers. If it stays more in chamber Y then it liked the drug, much like humans behave by seeking a place in which they felt good and avoiding places in which they felt bad. All well and good, only that is standard practice in this test to counter-balance the days and the chambers! I don’t know about the chambers, because they don’t say, but the days were not counterbalanced. I know, it’s a petty little thing for me to bring that up, but remember the saying about extraordinary claims… so I expect flawless methods. I would have also liked to see a way more convincing test for addictive liability like self-administration, but that will be done later, if the drug holds, I hope. Thankfully, unlike the affective analgesia claims, the authors have been more restrained in their verbiage about addiction, much to their credit (and I have a nasty suspicion as to why).

I do sincerely think the drug shows decent promise as a painkiller. Kudos for discovering it! But, seriously, fellows, the behavioral portion of the paper could use some improvements.

Ok, rant over.

EDIT (Aug 25, 2016): I forgot to mention something, and that is the competing financial interests declared for this paper: some of its authors already filed a provisional patent for PZM21 or are already founders or consultants for Epiodyne (a company that that wants to develop novel analgesics). Normally, that wouldn’t worry me unduly, people are allowed to make a buck from their discoveries (although is billions in this case and we can get into that capitalism-old debate whether is moral to make billions on the suffering of other people, but that’s a different story). Anyway, combine the financial interests with the poor behavioral tests and you get a very shoddy thing indeed.

Reference: Manglik A, Lin H, Aryal DK, McCorvy JD, Dengler D, Corder G, Levit A, Kling RC, Bernat V, Hübner H, Huang XP, Sassano MF, Giguère PM, Löber S, Da Duan, Scherrer G, Kobilka BK, Gmeiner P, Roth BL, & Shoichet BK (Epub 17 Aug 2016). Structure-based discovery of opioid analgesics with reduced side effects. Nature, 1-6. PMID: 27533032, DOI: 10.1038/nature19112. ARTICLE 

By Neuronicus, 21 August 2016