Rett Syndrome, Long-Term Memory and Neuren Pharmaceuticals' NNZ-2566

Over the last couple months I keep getting sucked back into the question of how Rett affects long-term memory. First I saw this write-up talking about how adult women with Rett seem to have better memory for events and objects from their early childhood. Then a few weeks later I was looking up long-term memory for an unrelated project and came across mentions of our good friend BDNF. I dug in a little then, but it's coming up again now as I'm working with Becca on learning her letters, and I wanted to better understand her learning process.

BDNF

So! Here's the somewhat limited (but still long) results of my foray into the relationship between Rett Syndrome and long-term memory.

Quick refresher from my previous post on BDNF, brain-derived neurotrophic factor is a protein and gene that have various effects, mostly on the brain. I believe it is also the first identified protein that is "downstream" of MECP2 (Rett). When you compensate for a BDNF deficiency in a mouse with Rett, their heart begins to function properly, their energy and strength improve -- many of the main symptoms of Rett are lessened significantly.

Then what about BDNF and long-term memory? Long-term memory is (I believe) defined as anything that's remembered for more than around eight hours. That includes childhood memories, lessons from school last year, and what you learned yesterday in class. The process of long-term-memorization seems to be getting something into short-term memory, then transitioning it to long-term memory and strengthening its placement in long-term memory over time. BDNF does a lot for the brain and is deficient in Rett Syndrome, so that's a great starting point for research.

 The first article I found was a winner, "BDNF is essential to promote persistence of long-term memory storage". The study shows two things, 1: BDNF is necessary for long-term memory persistence as part of a process that happens at some point after the original learning experience, and 2: even when other components of the long-term persistence process are missing ("hippocampal protein synthesis" is apparently the other part of the process), BDNF alone is enough to persist the long-term memory -- "in an ERK-dependent manner".

Cue the vocab lesson :-). Gathering these terms was no small feat, the neuroscientists of the world do not like to share their language with us peons.

Protein synthesis - the creation of proteins by cells. Hippocampal protein synthesis is the creation of proteins by brain cells, and is necessary for the brain to create connections. Apparently even if you suppress protein synthesis in the brain, a sufficient supply of BDNF is enough to allow for long-term memory. BDNF pretty much rocks.

Kinase - a kinase is an enzyme that allows for energy transfer. If I understand this correctly, a kinase is essentially a pathway through which actions can occur.

ERK - extra signal-related kinase. This is a specific kinase that is "activated" or used by, among other things, BDNF and growth factors (IGF-1, anyone?). This study was focused on spine growth, but showed that BDNF most definitely leverages the ERK "path" for its purposes.

CREB - CREB is a transcription factor (a protein) that helps regulate the transcription of certain DNA pieces. In other words, when a cell uses its DNA to send a message via RNA to create certain proteins,  needs transcription factors like CREB in order to send the correct message and have the protein created correctly. CREB regulates the transcription of a number of genes including BDNF.

Thanks for sticking with me, here's a picture.
CREB != crab.

PKMζ - PKMζ is another kinase. It appears that this kinase is necessary for the maintenance of long-term memory. Without it existing long-term memories seem to break down.

Akt/mTOR - Akt and mTOR are two kinases that I see lumped together a lot in the papers I'm looking at. The Neuren study implies they are a joint pathway that is inhibited by Rett Syndrome (more on that in a minute). 

IL-6 - this is a protein that is somewhat similar to BDNF and IGF-1. It does a bunch of cool stuff. I honestly haven't dug into it much, but the Neuren study suggests that their new drug may help via the IL-6 route in addition to the IGF-1 route. The only other paper I could find mentioning Rett and IL-6 wasn't directly related (but is wicked interesting and I'm going to dig into it later for sure).

Sorry, that was rough, but hopefully it'll help if you decide to dig in to any of the linked papers. I can proudly say I understand at least 30% of the Neuren presentation now :-). Anyway, back to our research. 

BDNF can facilitate long-term memory via ERK. In fact, another study said BDNF is a requirement for ERK activation (and also that CREB is necessary for correct ERK activation). There also appears to be a time element involved. In yet another study Alonso and company found that injecting rats with BDNF blockers prevented learned fear reactions -- but only some of the time. When BDNF was blocked 15 minutes before, or 1 or 4 hours after the training then the rats didn't developer the learned reaction, but when blocked at training time or 6 hours after, it had no effect. There were a few other studies that talked about time-critical moments in the long-term memory process. If there were a way to temporarily increase BDNF it seems like it could be done at a strategic time in order to improve retention when teaching new topics.

So is there anything to be done? Not sure. The whole "BDNF is necessary and also sufficient" thing is kind of a downer. I found some articles on how dopamine (2) and PKMζ increases can enhance long-term memory, but if BDNF is a must-have then I don't see how useful that is for Becca. Maybe it would help some even if it doesn't remove the main barrier, I don't know. I did also find some articles on memory strategies, the most promising of which is to try to encourage connections between existing long-term memories when introducing a new idea ("B" is for "bus", you ride the school bus every day to school), which can essentially make it more likely the idea will be pulled along into long-term memory (though somewhat paradoxically, novelty encourages attentiveness which improves retention as well). I'm trying to offer more variety while I teach Becca, pulling in both familiar and novel concepts, to hopefully do what I can to help her with retention.

Obviously long-term memory isn't completely nonexistent since Becca recognizes people from the past, she remembers things she doesn't like (her car seat) and things she does like (Wall-E, chocolate). She seems to recognize places she's been before. But when I work with her on school topics, it feels like she has a harder time making things stick between sessions. That's what this study says, too, that LTP exists in a weakened way in individuals with Rett Syndrome.

Oh right, LTP is another vocab word. Long-term potentiation is the biological process that most likely equates to long-term memory. They strongly correlate, anyway. As I look at my old notes, I remembered there were Rett studies mentioning plasticity, so I did a quick check on the relationship between plasticity and LTP. Apparently LTP is one type of plasticity, so if your plasticity is messed up then your long-term memory is probably messed up too. Like with Rett Syndrome.

Ok, so we're basically hosed when it comes to long-term memory, we do what we can but it's a very uphill battle. Lots of studies showing poor plasticity caused by Rett Syndrome. But remember, the IGF-1-as-a-replacement-for-low-BDNF studies on mice with Rett showed significantly improved plasticity (which should also mean improved long-term memory).

In fact, that's one of the main areas of study for Neuren Pharmaceutical's research drug, NNZ-2566. NNZ-2566, they claim, is an "analogue" of IGF-1 that can be taken orally but can still cross the blood-brain barrier, which is what needs to happen if it's going to have a positive impact on brain function.

Neuren has performed studies on mice where they "knocked out" or disabled FMR1, which is the equivalent of giving someone Fragile X. Fragile X is obviously not the same thing as Rett Syndrome (they may even be on opposite ends of the spectrum), but there are both genetic disorders with neurologic impact and do have some similar characteristics. Not sure I'm reading things right, but it seems like Fragile X *might* have the same problem of too little BDNF in the brain... The relationship between Fragile X and BDNF (search results) doesn't seem as cut-and-dry to me as with Rett, but I'm having a harder time understanding those results so don't read too much into that.

As far as the Neuren study goes, the Fragile X mice showed poor long- and short-term memory in different mazes when compared to wild-type mice. When placed in a simple maze 10 minutes and 24 hours after an initial exposure, the Fragile X mice were exploring the maze anew while the wild-type mice more quickly re-settled into their environment. In a different maze with essentially tall cliffs, the wild-type mice spent significantly less time on the anxiety-inducing cliffs than did the Fragile X mice. When given NNZ-2566 the Fragile X mice performed basically the same as the wild-type mice in both mazes.

Only one of these things has a head
full of fluff.  Also, stripes are cuter
than polka-dots.

So that's potentially very promising. It says to me that NNZ-2566 as an IGF-1 alternative may be a sufficient supplement for a BDNF deficiency as far as memory is concerned. We know from the other study I linked to before that IGF-1 positively affects brain weight and plasticity of Rett mice, so maybe that's enough of a correlation to be hopeful for Rett in addition to Fragile X. 

At any rate Neuren is working on Phase II of a clinical trial for NNZ-2566 as a treatment for Rett Syndrome. They are looking for adolescent and adult subjects (if I remember correctly, they're targeting older subjects because IGF-1 is a *growth* hormone and they don't want any confounding factors from younger subjects who are still growing), though if the drug is effective they will obviously work for approval for younger cases as well. They claim that in pre-clinical models of TBI, Fragile X and Rett there was a "normalization of Akt and ERK activation profiles" (you know what that means now, sort of!). Hopefully the clinical trials will back up their previous research.

What do the rest of us do in the mean time? Be patient is all I can suggest :-). It's been a tricky balance as I've been working with Becca on "school time" because she seems to remember enough to get annoyed if I'm too repetitive or spend too many days on the same subject, but it's also clear there are holes in her understanding that I need to fill before I can go too much farther. It does help to know there's the potential for learning, and that I'm not crazy in thinking she's getting it albeit sometimes at a slower pace. Even with all of her constraints she still surprises us quite often with what she knows or remembers, we just have to believe in her and keep remembering to ask.

Rett Syndrome Research and the Boston Studies

I've been doing a lot of research over the last month on papers published relating to Rett Syndrome. There's been plenty of talk about reversing or lessening the symptoms of Rett, mainly because of a drug trial going on at the Boston Children's Hospital. I heard there was a study published a couple years ago talking about some promising results in mice that were given Rett Syndrome that lead up to the clinical trial, but it wasn't clear how promising the research was. I thought I'd heard that the hormone they were using was happened upon by accident, and they weren't even sure what its effects would be, so I decided to dig in and do a bunch of research of my own. This post is a place for me to collect my thoughts and summarize the findings I came across. Maybe it will be useful for someone else as well.

I started with what I understood was the Very Important Paper. I found a link to it on Wikipedia, so I knew it was a big deal :-). Actually there were a bunch of links to it, some of them related to the Boston study.

The Very Important Paper is titled Partial reversal of Rett Syndrome-like symptoms in MeCP2 mutant mice and was written in 2008. It starts by talking about how they've had a hard time finding exactly what things are downstream effects of the MeCP2 mutation that causes Rett, one that they have found is BDNF (brain-derived neurotrophic factor) regulation.

Before I keep going on that paper I had to switch to another paper, The Ups and Downs of BDNF in Rett Syndrome, since unlike the authors I didn't know anything about BDNF or how exactly Rett affects it (researching protip: "etiology" means the study of causation or origination). As explained in the second paper (something of a summary paper), the BDNF connection was first discovered in 2003. In something of a surprise, BDNF levels are actually lower than normal (this is the important part), in spite of the fact that the MeCP2 mutation causes BDNF repression to fail. In other words, MeCP2 normally helps keep BDNF levels in check, so it seems strange at first blush that an MeCP2 mutation would causes lower levels of BDNF instead of higher levels. It turns out that BDNF levels actually start out high for Rett mice and drop to lower levels some time after that. The paper never actually concludes why the levels drop, and I'm still digging for more studies on that, but at least I had what I needed on the relationship between BDNF and Rett.

Back to The Very Important Paper.

The paper continues by referencing another paper, The disease progression of Mecp2 mutant mice is affected by the level of BDNF expression, that talks about how giving mice extra BDNF fixes a bunch of Rett symptoms including "locomotor activity levels". Unfortunately, BDNF doesn't cross the blood-brain barrier well, so it's hard to create a treatment using BDNF.

However. IGF-1 (Insulin-like Growth Factor 1, which incidentally is used by body builders) is a hormone that has a lot of the same results as BDNF, and is much better at crossing the blood-brain barrier. The point of this study was to try using IGF-1 as a treatment for mice with Rett.

The results were very positive. IGF-1-treated mice have a longer lifespan than untreated Rett mice, their physical activity is almost back in line with "normal" (they called them "wild-type") mice, and their breathing variability (Rett causes a lot of breathing irregularities, especially when sleeping) and heart rate (there's lots of weird heart problems related to Rett, e.g. Long QT Syndrome) are also improved.

Next came the really interesting stuff. Rett mice typically have less-heavy brains than normal mice, but when they were given IGF-1, brain weight also increased. That's very promising for something that is clearly a neurological disorder.

Rett is known to result in immature synapses in the brain. For a long time it was assumed that this was permanent, and that there would be no way to improve synapse development for girls with Rett should a cure ever be found. People assumed if a treatment were ever found, it would have to be given *before* Rett presented in order to do much good. (here are two sources for this statement -- though it's one I didn't find until later in my research)

However, as part of this study, the team tested "cortical plasticity" of MeCP2 mice. Plastic-what? Here's the deal: when you're young, your brain is pretty flexible and adjusts to changes very quickly. This is called "plasticity". As you get older and your brain is more developed, it can't adjust as quickly. So to study this, the researches recorded the eye dominance for a bunch of normal and MeCP2 mice. Then they stitched one of their eyes shut. Young mice should be able to adapt quickly to a change like that and shift eye dominance to the open eye, and old mice wouldn't adjust very well. Since Rett causes immature synaptic development, an old MeCP2 mouse should be more like a normal young mouse, and should be able to adjust quickly to the change. That's exactly what happened -- except for the mice that were given IGF-1. IGF-1-injected Rett mice responded like the normal old mice, which suggests that IGF-1 helps to "mature" brain synapses to more like what they're supposed to be.

Remember, that's a restoration. The immature synapses that are seen in Rett cases can actually be addressed, which means treatment can help existing Rett cases, not just brand new cases. This sounded like big news to me.

It was at this point that I started checking more sources, and came across more research. I thought the Very Important Paper was where they concluded that Rett symptoms could be reversed without permanent damage, but there were actually two prior studies that had already shown that: Reversal of neurological defects in a mouse model of Rett syndrome (full text not available) and Partial rescue of MeCP2 deficiency by postnatal activation of MeCP2. Those two studies focused on actually restoring the MeCP2 deficiency itself. But getting exactly the right amount of MeCP2 is very difficult and potentially dangerous, which is why work is being done on things like BDNF and IGF-1, for example.

Anyway, this all led me back to the Boston study, which I now felt like I understood the reasoning for. I'll summarize according to my understanding now.

This is the new Rett dude at Boston Children's

Boston Children's Hospital's Rett Team is running a multi-phased clinical study of the effects of IGF-1 on girls with Rett Syndrome. The first phase of the study, why has already concluded, was focused on making sure there were no major negative side effects that came from giving girls the IGF-1 hormone. It was originally a 4-week study with 12 girls that, after it went well, was extended an extra 20 weeks in which time all the girls (even the control group from the 4-week study) were given IGF-1. I missed the webinar where they summarized the results, but it sounded like there were no major concerns. The only thing probably of note is that the study was limited to girls 12 and under, since there are concerns with regularly prescribing growth hormones to more matured individuals (but apparently a separate study for a different disorder is already investigating this, so they're waiting on the results of that study... I'll see if I can find a link and post it here).

Phase two of the study hasn't started quite yet (I think it got delayed after the Rett dude at Boston Children's ended up leaving for family issues), but should start relatively soon. I just got an email update on it via the private Rett mailing list, RettNet. The age limits were restricted to between 5 and 10 to try to get a more homogenous set. The goal with phase two is to actually examine and measure the results of regular IGF-1 intake on Rett symptoms (behavioral changes, motor impairments, seizures, breathing abnormalities, hand stereotypies, etc.). Once that's done, if it looks like it actually helps, they're hoping for a phase three, which would be a major stepping stone toward getting FDA approval of the drug for treatment of Rett.

Anyway, that's what I got out of the research I've done so far. I have more digging I want to do for sure.