Lorenzo’s Oil for Nervous Breakdowns

 

A Happy New Year to all our readers – and indeed to anyone who isn’t a member of that merry band!

What better way to start than with a salute to the miracles of modern science by talking about how the lives of a group of young boys have been saved by one such miracle.

However, as is almost always the way in science, this miraculous moment is merely the latest step in a long journey. In retracing those steps we first meet a wonderful Belgian – so, when ‘name a famous Belgian’ comes up in your next pub quiz, you can triumphantly produce him as a variant on dear old Eddy Merckx (of bicycle fame) and César Franck (albeit born before Belgium was invented). As it happened, our star was born in Thames Ditton (in 1917: his parents were among the one quarter of a million Belgians who fled to Britain at the beginning of the First World War) but he grew up in Antwerp and the start of World War II found him on the point of becoming qualified as a doctor at the Catholic University of Leuven. Nonetheless, he joined the Belgian Army, was captured by the Germans, escaped, helped by his language skills, and completed his medical degree.

Not entirely down to luck

This set him off on a long scientific career in which he worked in major institutes in both Europe and America. He began by studying insulin (he was the first to suggest that insulin lowered blood sugar levels by prompting the liver to take up glucose), which led him to the wider problems of how cells are organized to carry out the myriad tasks of molecular breaking and making that keep us alive.

The notion of the cell as a kind of sac with an outer membrane that protects the inside from the world dates from Robert Hooke’s efforts with a microscope in the 1660s. By the end of the nineteenth century it had become clear that there were cells-within-cells: sub-compartments, also enclosed by membranes, where special events took place. Notably these included the nucleus (containing DNA of course) and mitochondria (sites of cellular respiration where the final stages of nutrient breakdown occurs and the energy released is transformed into adenosine triphosphate (ATP) with the consumption of oxygen).

In the light of that history it might seem a bit surprising that two more sub-compartments (‘organelles’) remained hidden until the 1950s. However, if you’re thinking that such a delay could only be down to boffins taking massive coffee breaks and long vacations, you’ve never tried purifying cell components and getting them to work in test-tubes. It’s a process called ‘cell fractionation’ and, even with today’s methods, it’s a nightmare (sub-text: if you have to do it, give it to a Ph.D. student!).

By this point our famous Belgian had gathered a research group around him and they were trying to dissect how insulin worked in liver cells. To this end they (the Ph.D. students?!) were using cell fractionation and measuring the activity of an enzyme called acid phosphatase. Finding a very low level of activity one Friday afternoon, they stuck the samples in the fridge and went home. A few days later some dedicated soul pulled them out and re-measured the activity discovering, doubtless to their amazement, that it was now much higher!

In science you get odd results all the time – the thing is: can you repeat them? In this case they found the effect to be absolutely reproducible. Leave the samples a few days and you get more activity. Explanation: most of the enzyme they were measuring was contained within a membrane-like barrier that prevented the substrate (the chemical that the enzyme reacts with) getting to the enzyme. Over a few days the enzyme leaked through the barrier and, lo and behold, now when you measured activity there was more of it!

Thus was discovered the ‘lysosome’ – a cell-within-a cell that we now know is home to an array of some 40-odd enzymes that break down a range of biomolecules (proteinsnucleic acidssugars and lipids). Our self-effacing hero said it was down to ‘chance’ but in science, as in other fields of life, you make your own luck – often, as in this case, by spotting something abnormal, nailing it down and then coming up with an explanation.

In the last few years lysosomes have emerged as a major player in cancer because they help cells to escape death pathways. Furthermore, they can take up anti-cancer drugs, thereby reducing potency. For these reasons they are the focus of great interest as a therapeutic target.

Lysosomes in cells revealed by immunofluorescence.

Antibody molecules that stick to specific proteins are tagged with fluorescent labels. In these two cells protein filaments of F-actin that outline cell shape are labelled red. The green dots are lysosomes (picked out by an antibody that sticks to a lysosome protein, RAB9). Nuclei are blue (image: ThermoFisher Scientific).

Play it again Prof!

In something of a re-run of the lysosome story, the research team then found itself struggling with several other enzymes that also seemed to be shielded from the bulk of the cell – but the organelle these lived in wasn’t a lysosome – nor were they in mitochondria or anything else then known. Some 10 years after the lysosome the answer emerged as the ‘peroxisome’ – so called because some of their enzymes produce hydrogen peroxide. They’re also known as ‘microbodies’ – little sacs, present in virtually all cells, containing enzymatic goodies that break down molecules into smaller units. In short, they’re a variation on the lysosome theme and among their targets for catabolism are very long-chain fatty acids (for mitochondriacs the reaction is β-oxidation but by a different pathway to that in mitochondria).

Peroxisomes revealed by immunofluorescence.

As in the lysosome image, F-actin is red. The green spots here are from an antibody that binds to a peroxisome protein (PMP70). Nuclei are blue (image: Novus Biologicals)

Cell biology fans will by now have worked out that our first hero in this saga of heroes is Christian de Duve who shared the 1974 Nobel Prize in Physiology or Medicine with Albert Claude and George Palade.

A wonderful Belgian. Christian de Duve: physician and Nobel laureate.

Hooray!

Fascinating and important stuff – but nonetheless background to our main story which, as they used to say in The Goon Show, really starts here. It’s so exciting that, in 1992, they made a film about it! Who’d have believed it?! A movie about a fatty acid!! Cinema buffs may recall that in Lorenzo’s Oil Susan Sarandon and Nick Nolte played the parents of a little boy who’d been born with a desperate disease called adrenoleukodystrophy (ALD). There are several forms of ALD but in the childhood disease there is progression to a vegetative state and death occurs within 10 years. The severity of ALD arises from the destruction of myelin, the protective sheath that surrounds nerve fibres and is essential for transmission of messages between brain cells and the rest of the body. It occurs in about 1 in 20,000 people.

Electrical impulses (called action potentials) are transmitted along nerve and muscle fibres. Action potentials travel much faster (about 200 times) in myelinated nerve cells (right) than in (left) unmyelinated neurons (because of Saltatory conduction). Neurons (or nerve cells) transmit information using electrical and chemical signals.

The film traces the extraordinary effort and devotion of Lorenzo’s parents in seeking some form of treatment for their little boy and how, eventually, they lighted on a fatty acid found in lots of green plants – particularly in the oils from rapeseed and olives. It’s one of the dreaded omega mono-unsaturated fatty acids (if you’re interested, it can be denoted as 22:1ω9, meaning a chain of 22 carbon atoms with one double bond 9 carbons from the end – so it’s ‘unsaturated’). In a dietary combination with oleic acid  (another unsaturated fatty acid: 18:1ω9) it normalizes the accumulation of very long chain fatty acids in the brain and slows the progression of ALD. It did not reverse the neurological damage that had already been done to Lorenzo’s brain but, even so, he lived to the age of 30, some 22 years longer than predicted when he was diagnosed.

What’s going on?

It’s pretty obvious from the story of Lorenzo’s Oil that ALD is a genetic disease and you will have guessed that we wouldn’t have summarized the wonderful career of Christian de Duve had it not turned out that the fault lies in peroxisomes.

The culprit is a gene (called ABCD1) on the X chromosome (so ALD is an X-linked genetic disease). ABCD1 encodes part of the protein channel that carries very long chain fatty acids into peroxisomes. Mutations in ABCD1 (over 500 have been found) cause defective import of fatty acids, resulting in the accumulation of very long chain fatty acids in various tissues. This can lead to irreversible brain damage. In children the myelin sheath of neurons is damaged, causing neurological defects including impaired vision and speech disorders.

And the miracle?

It’s gene therapy of course and, helpfully, we’ve already seen it in action. Self Help – Part 2 described how novel genes can be inserted into the DNA of cells taken from a blood sample. The genetically modified cells (T lymphocytes) are grown in the laboratory and then infused into the patient – in that example the engineered cells carried an artificial T cell receptor that enabled them to target a leukemia.

In Gosh! Wonderful GOSH we saw how the folk at Great Ormond Street Hospital adapted that approach to treat a leukemia in a little girl.

Now David Williams, Florian Eichler, and colleagues from Harvard and many other centres around the world, including GOSH, have adapted these methods to tackle ALD. Again, from a blood sample they selected one type of cell (stem cells that give rise to all blood cell types) and then used genetic engineering to insert a complete, normal copy of the DNA that encodes ABCD1. These cells were then infused into patients. As in the earlier studies, they used a virus (or rather part of a viral genome) to get the new genetic material into cells. They choose a lentivirus for the job – these are a family of retroviruses (i.e. they have RNA genomes) that includes HIV. Specifically they used a commercial vector called Lenti-D. During the life cycle of RNA viruses their genomes are converted to DNA that becomes a permanent part of the host DNA. What’s more, lentiviruses can infect both non-dividing and actively dividing cells, so they’re ideal for the job.

In the first phase of this ongoing, multi-centre trial a total of 17 boys with ALD received Lenti-D gene therapy. After about 30 months, in results reported in October 2017, 15 of the 17 patients were alive and free of major functional disability, with minimal clinical symptoms. Two of the boys with advanced symptoms had died. The achievement of such high remission rates is a real triumph, albeit in a study that will continue for many years.

In tracing this extraordinary galaxy, one further hero merits special mention for he played a critical role in the story. In 1999 Jesse Gelsinger, a teenager, became the first person to receive viral gene therapy. This was for a metabolic defect and modified adenovirus was used as the gene carrier. Despite this method having been extensively tested in a range of animals (and the fact that most humans, without knowing it, are infected with some form of adenovirus), Gelsinger died after his body mounted a massive immune response to the viral vector that caused multiple organ failure and brain death.

This was, of course, a huge set-back for gene therapy. Despite this, the field has advanced significantly in the new century, both in methods of gene delivery (including over 400 adenovirus-based gene therapy trials) and in understanding how to deal with unexpected immune reactions. Even so, to this day the Jesse Gelsinger disaster weighs heavily with those involved in gene therapy for it reminds us all that the field is still in its infancy and that each new step is a venture into the unknown requiring skill, perseverance and bravery from all involved – scientists, doctors and patients. But what better encouragement could there be than the ALD story of young lives restored.

It’s taken us a while to piece together the main threads of this wonderful tale but it’s emerged as a brilliant example of how science proceeds: in tiny steps, usually with no sense of direction. And yet, despite setbacks, over much time, fragments of knowledge come together to find a place in the grand jigsaw of life.

In setting out to probe the recesses of metabolism, Christian de Duve cannot have had any inkling that he would build a foundation on which twenty-first century technology could devise a means of saving youngsters from a truly terrible fate but, my goodness, what a legacy!!!

References

Eichler, F. et al. (2017). Hematopoietic Stem-Cell Gene Therapy for Cerebral Adrenoleukodystrophy. The New England Journal of Medicine 377, 1630-1638.

 

Guess Who’s Coming to Dinner?

 

Question: when is a gene not a gene? Answer: when it’s a pseudogene.

Genes are familiar enough these days when the acronym DNA has become part of everyday speech “It is in Toyota’s DNA that mistakes made once will not be repeated”, as the CEO of Toyota rather sinisterly remarked. You could say that’s pseudo-scientific rubbish but, despite that kind of liberty-taking, most will know that a gene is a stretch of our genetic material (DNA) that carries the code to make a closely related RNA molecule that, in turn, may be used as a template to make a protein ­– it’s the molecular unit of heredity. Well known too is that the Greeks gave us ‘pseudo’ – but what’s a ‘lying’ or ‘false’ gene – and who cares?

No prizes for guessing that we should all be interested because it’s emerging that pseudogenes can be important players in cancer.

Player’s biography

Pseudogenes are somewhat disreputable because they are relatives of normal genes that along the evolutionary highway have become dysfunctional by losing the capacity to be ‘expressed’ – that is, their code can no longer be transformed into RNA and protein. You could think of them as an example of the shambolic way in which species have evolved by random happenstance so that they work in their own particular niches. And if you want the outstanding example of unintelligent design, look no further than yourself, as we did in Holiday Reading (2), Poking the Blancmange.

Just for background, although it doesn’t affect the main story, there are three ways in which our genome can acquire a pseudogene:

1. A normal gene becomes functionally extinct: odd mutational events disable the stretches of DNA that control its expression. The gene is like a siding on a railway that isn’t used for years and years until eventually the points  seize up (it would be a ‘switch’ on US railroads) and the cell machinery can no longer get at it – but when this does happen we get by without that gene.

2. During evolution genes quite often get duplicated – giving multiple copies: if one of these loses its regulatory bits the duplicate gene is switched off – it’s become a ghost.

3. We owe about 8% of our genome to viruses – mainly those with RNA genomes (retroviruses) whose life-cycle turns their RNA into DNA that has then been stuck into our genome. And that’s a lot (about 100,000 bits of retrovirus DNA) especially bearing in mind that only about 1% of our genome encodes proteins.

So our precious genome is littered with corpses and fragments thereof. In the past there’s been a regrettable tendency to label this material as ‘junk’ but increasingly we’re now discovering that there may be genetic life after death, so to speak. It’s not surprising if you think about it. If random events can inactivate a gene then they might do the reverse, even if that may be a much rarer event. And indeed it’s now clear that pseudogenes can be brought back to life through the random mutational events that characterise the rough and tumble of cellular life.

So not all pseudogenes are extinct then?

Correct. Obviously we wouldn’t be wittering on about them had not some bright sparks just shown that pseudogenes – or at least one in particular – can be re-awakened to play a part in cancer. The luminaries are Florian Karreth, Pier Paolo Pandolfi and friends from all over the place (USA, UK, Italy, Singapore) who found that a pseudogene called BRAFP1 (a relative of the normal BRAF gene) can help to drive cancer development. Some earlier studies had shown that BRAFP1 was expressed (i.e. RNA was made from DNA) in various human tumours but Karreth & Co extended this, detecting significant levels of the pseudogene RNA in lymphomas and thyroid tumours and also in cells from melanoma, prostate cancer and lung cancer, whilst it’s not switched on in the corresponding normal cells.

To show that this pseudogene can drive cancers they genetically engineered its over-expression in mice, whereupon the animals developed an aggressive malignancy akin to human lymphoma (specifically diffuse large B cell lymphoma). Short-circuiting an enormous amount of work, it emerged that the pseudogene up-regulated a signaling pathway involving its normal counterpart, BRAF, that drives proliferation.

106 pic

How a pseudogene (BRAFP1) might drive cancer. Top: The scheme illustrates the ‘central dogma’ of molecular biology: DNA makes RNA makes protein. In normal cells a family of micro RNAs (different coloured wiggles) regulate the level of BRAF RNA and hence of BRAF protein (above white line).  Bottom: When the pseudogene BRAFP1 is switched on its RNA competes for the negative regulators: the result is more BRAF RNA making more BRAF protein – making cancer (Karreth et al., 2015).

Interfering RNA

The pseudogene’s RNA manages to interfere with normal control by targeting another type of RNA – micro RNAs, so called because they’re very short (about 20 bases (units) long – so they’re encoded by tiny stretches of the over 3,000 million units that make up the genome). Small they may be but there are hundreds of them and it’s become clear over the last few years that they play critical roles in regulating how much protein is made from specific RNAs. Their method is simple: they recognize (i.e. bind to) stretches of RNA that encode proteins, thereby blocking translation into protein.

Karreth & Co showed that there are about 40 different micro RNAs that can stick to the RNAs encoding BRAF or BRAFP1. Normally when there’s no (or very little) BRAFP1 around they have only BRAF to act on – and their role is to control the proliferation signal it transmits – i.e. to keep that signal to what’s required for normal cell growth control. BUT, when the pseudogene RNA is made in significant amounts the attentions of the 40 micro RNAs are divided. Result: more BRAF RNA, more BRAF protein, higher cell proliferation.

It’s a bit like you’re just sitting down to a family dinner for four when there’s a knock on the door and in walks long lost Uncle Bert, complete with wife and two kids in tow. Of course you invite them to dine too – but now a meal for four has to stretch to eight. There is something for everybody – just not as much. Similarly for the regulators of BRAF: when BRAFP1 is present there’s half as much of the RNA regulators for each – and the result, bearing mind that they are negative regulators, is that the activity of BRAF goes up and the cells proliferate more avidly. The pseudogene is driving cancer.

First but not last

For decades pseudogenes were thought of as ‘junk’ DNA along with most of the rest of the genome that didn’t encode proteins – though I might say that was a concept I never promoted. Beware labeling anything in our genome as junk for it may rise, Kraken like, to remind us of our ignorance. And, now that one pseudogene has come in from the cold and been shown to drive some cancers, you can be confident that others will follow.

References

Karreth, F.A. et al. (2015). The BRAF Pseudogene Functions as a Competitive Endogenous RNA and Induces Lymphoma In Vivo. Cell 161, 319–332.