In Pursuit of Memory

by Joseph Jebelli

  PART IV

  Experimentation

  IN OCTOBER 2012, shortly after Abbas passed away, I attended the Society for Neuroscience conference in New Orleans, Louisiana, with almost 30,000 fellow neuroscientists. The conference has been a pilgrimage for researchers since its founding in 1969. It’s where the most dedicated academics gather to present data and share ideas. I had just finished the second year of my training and this was my first taste of international collaboration at the highest level. And it did not disappoint. For five glorious days I met and spoke with people conducting the most advanced experiments the world has ever seen. Each morning I was handed a hefty catalogue outlining the day’s presentations–you have to plan your day meticulously if you’re to see even a fraction of what’s on offer–and each morning I immediately flipped to the section on Alzheimer’s.

  My first feeling, as I wandered the endless corridors of lecture theatres and poster exhibits, was a hypnotic mix of awe, intrigue and frustration. So numerous were the breakthroughs and new theories, I wanted to clone myself and put a copy of me in every room. I wondered if my research using the brain’s immune cells was still cutting-edge. Like an octopus, the field appeared to have spread its arms into wholly uncharted territory.

  One evening that week, alone in my hotel room, I received a Skype call from my father. Like others in my family he wanted to know if I’d spotted anything that might have helped Abbas. I told him that the research here was experimental and probably wouldn’t have done much for Granddad. I could see the disappointment on his face. I think that after seeing Abbas’s decline he had grown fearful of dying in the same way. In the year preceding his death, the most visible change in Abbas’s personality was his inconsistency: snapshots from the past–a familiar piece of music, say, or an old photograph–prompted sudden sprees of mirth and coherence, while prolonged moments of introspection hurled his mind into a labyrinth of jittery, existential unknowns.

  I think my father would have tried anything at that point, and a part of me felt as if I’d failed him. I still had nothing to offer even with an army of neuroscientists by my side. It’s a reality every researcher confronts. Try as we might, there’s an infuriating spectral quality to research on the front line: a promising lead can emerge and then suddenly vanish.

  But despite that, I went on to tell my father, five areas of experimentation felt like a brief look into the near future. Neuroscientists were leaving no stone unturned. Granddad’s suffering would not be in vain.

  14

  Regeneration

  My dreams were all my own; I accounted for them to nobody; they were my refuge when annoyed–my dearest pleasure when free.

  Mary Shelley, Frankenstein

  THE YOUNG MAN peered down the microscope. The cells snapped into focus and he zoomed in. He’d spent the last ten years trying to get this experiment to work; he was getting impatient. What’s more, his funding was running out and his reputation hung perilously in the balance. Maybe his colleagues were right: maybe this was a fantasy. He was, after all, hoping to rewrite the laws of nature. He steeled himself for the moment of dejection.

  But it didn’t come. Still he didn’t know what he was looking at–but something was different. He walked away with one, all-consuming thought: could this be it?

  Shinya Yamanaka is a smartly dressed man of mild demeanour, keen intuition and playful humour. The son of a factory owner, he grew up in Osaka, Japan, in the 1960s, and spent his childhood playing with machines before deciding to study medicine. After suffering many broken bones from rugby and judo he became interested in sports injuries and qualified as an orthopaedic surgeon. For two years he worked as a resident at Osaka National Hospital–replacing bones, resurfacing joints, repairing torn ligaments. But he soon realised his real passion lay somewhere else entirely.

  In the mid-1980s biologists had started inserting genes into mouse embryos to create ‘transgenic’ mice. This allowed them to explore how single genes influence development and, since our genome is 99 per cent identical to that of a mouse, to explore how single genes influence human development. Yamanaka was captivated. As a surgeon the sheer number of untreatable conditions frustrated him. In this new world of molecular genetics, he realised, he could eradicate diseases by learning their underlying mechanisms. He looked for a doctoral programme that would teach him the basics of designing and executing experiments, and found one in pharmacology at Osaka City University.

  After a further three years’ training in America, Yamanaka returned to Japan and started generating his own mouse colony. Within six months he had 200 mice–after a year, nearly 1,000. He had to feed and clean every one himself, leaving him scant time for experiments. ‘I started to think, am I really a scientist, or just a mouse keeper?’1

  His goal was to study genes using mouse embryonic stem (ES) cells. There were two advantages to using ES cells over embryos: first, they grew and proliferated rapidly, providing a limitless supply of samples to test; second, they were pluripotent–that is, they had the potential to become any cell type in the body. By manipulating their genes Yamanaka could deduce which ones were necessary for them to become particular cell types.

  But the scientists working in his department didn’t see the point. ‘I was often told by my colleagues, “Shinya, working on those strange mouse cells may be interesting to you, but perhaps you should do something more related to medicine.”’ Depressed and exhausted, Yamanaka considered quitting science altogether. But then a major event rescued him.

  In 1998 a developmental biologist at the University of Wisconsin, Madison, named James Thomson, isolated the first human embryonic stem cells–the primordial material that creates an entire human being.2 With that, a new branch of medicine, regenerative medicine, was born. Suddenly, the treatment for virtually every human disease that involved cellular and tissue deterioration was reimagined. Researchers around the world became gripped by the idea of growing made-to-order cells in a petri dish for human transplants: cardiac myocytes for heart failure, motor neurons for spinal cord injury, islet cells for diabetes, photoreceptors for blindness, and cortical neurons for Alzheimer’s. And to achieve this, knowledge of how stem cells chose their cellular fate was paramount, making Yamanaka’s work indispensable. He made it his mission to keep going, and was given his very own lab the following year.

  As an associate professor at the Nara Institute of Science and Technology, Ikoma, Yamanaka could now begin to forge a career in science. The problem with ES cells, he soon realised, was that the patient’s immune system would recognise them as foreign and mount a deadly biological defence to remove them from the body. In addition, ES cells were rapidly provoking fierce controversy due to the moral dilemma of destroying human embryos to harvest them. But the more tangible problem for Yamanaka was that he desperately needed other scientists to help kick-start his lab.

  Every April, 100 students at the Nara had to select one of its twenty research labs to work for–which often left some labs with none. Students were drawn to the old, established professors, who published career-defining papers in prestigious journals like Nature and Science. At thirty-six years old, with no such papers, how, Yamanaka wondered, could he attract them? The answer came to him in a moment of breathtaking innovation.

  Yamanaka wanted to test if it was possible to turn adult human cells back into stem cells–to take skin, for instance, from an individual’s arm, and reprogramme it into an embryonic-like state. From this, an unlimited source of cloned human tissue could then be established. Better yet, obtained from the very person whom it was intended to help. These cells wouldn’t just evade the barrier of immune rejection, they would carry the person’s unique genetic signature, making them powerful tools for seeing how a patient’s particular form of disease manifests. And they would be derived from adults, thereby bypassing the ethical impasse of harvesting cells from the embryo.

  Not surprisingly, most scientists thought Yamanaka was dreaming. Yamanaka himself was daunted by the
challenge. ‘I knew how difficult it would be. It would take twenty years, thirty years, or more. But I didn’t tell the students that. I just told them how wonderful it would be!’ His inspired idea persuaded three students to join the lab.

  They set to work. Research had already shown that stem cells required twenty-four genes to remain in a stem-like state. By artificially inserting these twenty-four genes into adult cells, Yamanaka reasoned, they might reprogramme the cells into stem cells. But using all twenty-four genes didn’t work. So he tried using fewer. Again, no luck. He spent years testing various combinations of genes to see which ones were essential. ‘It was like swinging a bat in pitch darkness,’ he recalled. Only one student, Kazutoshi Takahashi, continued to work in the lab upon graduating. To keep Takahashi motivated, Yamanaka promised him a job for as long as he lived–so long as he kept trying to ‘swing the bat’.

  It worked. Just four genes, it turned out, were enough. In 2006 they demonstrated that adult cells could be reprogrammed in mice,3 and by 2007 showed it was possible in humans as well.4 Yamanaka called the cells ‘induced pluripotent stem (iPS) cells’. He received a Nobel Prize in 2012.

  In recent years, a series of landmark publications have shown that iPS cells can be turned into a host of human tissue–including liver and gut, heart, pancreas, eye and brain–paving the way for humankind to grow its own biological spare parts, and helping scientists simulate complex human diseases in a petri dish. Hence-forth, the technique was aptly nicknamed ‘disease-in-a-dish’.

  Alzheimer’s researchers had good reason to be excited about iPS cells. It represented the first step towards a wholly new kind of cure for dementia. Few organs are as inaccessible as the brain but now, for the first time, personalised neuronal cell lines from Alzheimer’s patients could be developed and scrutinised. On the table were gene-editing techniques to study the effects of inserting or removing single genes; fluorescent probes to track the early signs of plaques and tangles; and even, perhaps, a cellular road map to boost cognition beyond what is natural.

  As for therapeutics, their greatest strength is undoubtedly their human origin. Besides the shortcomings of mice to mirror dementia, studies also found that screening drugs in mice was far from ideal. In 2010 it was estimated that 90 per cent of drugs based on mouse models fail in clinical trials.5 The reason: unlike mice found in the wild, lab mice are inbred, and therefore don’t capture the huge genetic variation seen in people. And although their genome is very similar to ours, the way they use it–how they switch genes on and off–is very different. As the Harvard physician H. Shaw Warren put it, if one ‘tried to understand a station wagon by studying a motorcycle, one would learn something about wheels and spark plugs but have no idea about steering wheels, airbags, and sunroofs, and the larger picture would be substantially missed’.6 Indeed, one study found that only 12 per cent of the genetic changes seen in mouse models of inflammatory disorders mimicked those seen in humans7–providing ‘a sobering reminder’, wrote an editor for Nature Methods, ‘of what most thoughtful biologists already know: your biological conclusions are really only as good as the methods that get you there’.8

  But iPS cell modelling, too, had some troubling caveats. The reprogramming process often gave the cells strange and unpredictable characteristics. Some retained a ‘memory’ of their adult origins (in the form of chemical marks on their DNA) and wouldn’t conform to a single population of clones. Scientists were also wary about trusting data from stem-cell-derived neurons considering neurons in Alzheimer’s patients are so much older. For transplantation the concerns were more pressing: the cells might multiply uncontrollably and spawn tumours. They were also therapeutically impractical because it took roughly five months to generate them, creating the additional drawback of being extremely costly. And because the number of neurons that die in Alzheimer’s is so large, scientists questioned how much iPS cell transplants could actually do; what good were they if they couldn’t replace enough neurons in the time it took to harvest them? Was it even possible to grow enough? It was one thing to promise breakthroughs in the lab and quite another to translate those breakthroughs to the real world. Thankfully, many scientists believed there was little point dwelling on the obstacles, and a surge of research groups started asking patients to donate their skin.

  ‘Yeah! Bring it on! Reimplant me!’ Victoria Huntley exclaimed with a howling laugh that could probably fill a small concert hall. A professional caregiver and mother of two, she was thirty-five when she first learned of the early-onset Alzheimer’s in her family. A year later, she decided to have the test. It was positive.

  Victoria was one of four children born into a low-income family in Walthamstow, east London, in 1968. Growing up, she knew something wasn’t right with her mother, Susan, who’d become confused, forgetful and incapable of doing simple things at home, like making a cup of tea. Before long, Susan stopped work as a hairdresser. She just couldn’t do it any more: she made too many mistakes, such as giving her customers the wrong hair colour. But Susan was still in her thirties. Susan’s father had died of something brain-related–he’d spent the better part of his adult life in an asylum, and Susan seemed to be heading for a similar fate. Not wanting to upset her children, she tried to hide her symptoms and carry on as normal. ‘I can understand why she did that,’ Victoria conceded, ‘because we really were too young.’

  Susan died in April 2006, when she was just fifty-six years old. In the years preceding her death, Susan’s doctor’s found that Susan had a rare genetic mutation in a gene named Presenilin 1 (PSEN1). Discovered in a French-Canadian family by scientists at the University of Toronto, Canada, in 1995–four years after the APP mutation was detected–PSEN1 carriers can develop Alzheimer’s as early as thirty years of age. Exactly how the gene causes such an early form of Alzheimer’s remains unclear, but good evidence suggests it does so by indirectly creating plaques. That’s because unlike APP (the gene for amyloid itself), PSEN1 codes for an enzyme that normally chops amyloid up into small pieces. A mutated PSEN1 therefore makes abnormally large pieces of beta-amyloid, which then clump together in the brain as plaques. And if John Hardy’s amyloid cascade hypothesis is correct, that’s all it takes.

  In 2005, however, those kinds of particulars were the last thing on Victoria’s mind. She just wanted to know if she had the PSEN1 mutation. ‘Why did I want to find out?’ she repeated when I put the question to her. ‘Because I thought about it and thought about it, and to keep on thinking about it,’ she declared, squinting her eyes in fervent concentration, ‘drove me mad.’ Now, although mildly symptomatic, Victoria is still able to recount how the result made her feel. ‘It was the best thing I’ve ever done,’ she told me, releasing another burst of effervescent laughter. We were chatting in her living room on a wintry November evening in 2015, her husband Martin sitting nearby, helping her remember events from the past. On Victoria’s right shoulder is a tattoo of Scrabble pieces that reads: AL[…]HEIMER’S CAN KISS MY ARSE, the Z deliberately left blank, emphasising that something is ‘missing’.

  ‘I used to be quite a shy, sensitive person,’ she continued, ‘but once I found out part of me thought, You know what, fuck it now!’ Elaborating, Martin described some of the couple’s devil-may-care responses to the news since Victoria discovered what little time her mind had left. They travelled extensively–to America, Africa and the Mediterranean. They hosted big, blowout birthday parties–each year now utterly sacrosanct. And they got married; there was no point waiting any more.

  When I asked what year their wedding was, Victoria couldn’t remember and turned to Martin for help. He stared at the ceiling in silence for a moment too long. ‘Now now!’ Victoria reprimanded. ‘Come on, son! Don’t you slack now!’

  ‘It was 2007,’ he finally said.

  ‘Yeah! Well done!’ Victoria said. ‘Well done.’

  Despite their new-found insouciance, it wasn’t long before the couple did start thinking about the future. After all, Victoria had two yo
ung children to consider, both with a 50 per cent chance of a strikingly abbreviated life. So when neurologists at the London Institute of Neurology asked her if she’d like to donate skin for stem cell research, she said yes without a moment’s hesitation.

  The Institute of Neurology occupies a twelve-storey, nondescript building not far from the British Library and the crowded underground labyrinth of King’s Cross Tube station. Over 500 people work there–including Selina Wray. Growing up in the coal-mining town of Barnsley, South Yorkshire, Wray became the first in her family to go to university, and her interest in Alzheimer’s stems from the inspirational mentors she had there. After her bachelor’s degree in biochemistry she studied for a doctorate in the biology of Alzheimer’s, and in March 2009 began work at the institute, where she now makes iPS cells from Alzheimer’s patients. On 11 November 2015 I found Wray in her lab, a small windowless room containing glass beakers and pipettes, white shelves filled with chemicals and reagents, and a tall grey incubator silently inhaling carbon dioxide. A sponsorship sticker on the door reads: THIS LAB IS DEFEATING DEMENTIA.

  The entire procedure is a prolonged and complicated process, involving a series of unstable, transient culture steps: an initial culture of human skin cells known as fibroblasts, made by dicing a small chunk of skin no bigger than a pea and leaving it in nutrient broth (a liquid containing essential nutrients and amino acids) for around six weeks; another fibroblast culture in which the four reprogramming genes, called Oct4, Sox2, c-Myc and Klf4–collectively known as Yamanaka factors–are subsequently added; a three-month-long pluripotent stem cell culture, where individual fibroblasts begin to form colonies of stem cells; a neuronal precursor cell culture, composed of infant neurons which develop once the stem cells have been submerged in a special, neuron-promoting nutrient broth; and a 100-day-long period of neuronal maturation known as corticogenesis, where the neurons make synapses, fire electrical impulses, and release neurotransmitter–in this case glutamate–to finally assume a culture of adult, human, cortical glutamatergic neurons. If you’re persistent enough to perform all that on a regular basis, knowing that many of your attempts will likely fail, you have a vocation for biomedical research.