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Murrays ProblemBy Mark Johnson

M urray Blackmore stood at the lectern and tried to takein the dark conference room, the men and women inwheelchairs waiting for him to wrest a little hopefrom science. But in his preoccupied state, the room was a blurand hope a struggle. The 39-year-old researcher took a deepbreath.

An assistant professor at Marquette University, Blackmorehad looked forward to addressing the symposium on spinalcord research in Boston. Work filled his daylight hours; inter-rupted his dreams at night. Often he would wake at 2 or 3 in themorning, pitched from sleep into the scientific puzzles of a bro-ken spinal cord. Ideas in the midnight hours seldom bore fruit,but his mind churned through them just the same.

He felt a responsibility. The National Institutes of Healthhad awarded him a $1.6 million grant. He ran a lab outfittedwith cutting edge equipment. He pursued the newest ideas inthe field.

But in the fall of 2013, the researcher with the short, blackhair and slim cyclists build was facing a year that would testthe balance in his scientific life.

On one side of the scale weighed his deeply personal desireto cure spinal cord injuries; on the other, his professions de-mand for detachment. He would need to balance the two if hewanted to produce strong results in the lab, an important pa-per, a groundbreaking technique, something promising.

As Blackmore addressed the audience in Boston, a shoulderdrooped. His voice slowed.

Ive been thinking a lot about speed lately.A small sigh escaped and a slide appeared on a large screen

up front showing several photos of an older woman. In one, the

woman was laughing beneath an umbrella as her canoecruised downriver past gray cliffs. Another showed her sittingin an office; only a close look revealed she was in a wheelchair.

As some of you know, this is my mom. She was injured in1987 and lived for 26 years as a C4 quadriplegic. She lived well.. . . She never stopped going on adventures.

She passed away recently, and to me its really broughtsomething into focus.

Blackmore scratched the back of his neck and blinked.Twenty-six years. Thats a long time. And every day that

she was living with spinal cord injury, there were scientistsout there trying to solve spinal cord injury, and in the last sev-eral years thats included me and my lab.

And when you get right down to it, we didnt get it done. Notyet.

He clenched his teeth.So I guess what Im saying is, if theres anyone in this room

thats feeling frustrated that we havent finished this yet, be-lieve me, I understand how you feel.

And Id like to have a conversation, a continuing conversa-tion about how do I, as a researcher, move this faster.

For a moment, it was as if the scientist had stepped downfrom the stage and joined the 273,000 Americans living withspinal cord injuries and wondering if an answer will come intheir lifetimes.

The grief was still fresh. Blackmores mother had died twoweeks earlier. Devastated, was how his wife, Dawn, de-scribed him.

The next slide appeared. He gathered himself and began: Ithink everyone here is familiar with the basic problem of axonregeneration . . .

RICK WOOD / [email protected]

Murray Blackmore, an assistant professor at Marquette University, shows his disappointment after discovering cells had been contaminat-ed. He leads a lab that has received a $1.6 million grant to study spinal cord injury treatments research he was inspired to undertake by

MURRAYS PROBLEM | A JOURNAL SENTINEL SPECIAL REPORT

Quest for cure personalMothers injury inspires scientist as he works to balance his desire totreat spinal cord injuries with his professions demand for detachment

By MARK [email protected]

DECEMBER 14, 2014

He might have chosen an easier path to contentment, thishusband and father of three, a man who enjoys hiking andcamping, fish fries and music in the park.

But in the pursuit that dominates his life, the quest to curespinal cord injuries, experiments fail. Under the microscope, atreated mouse shows no improvement. In the cage, it cannotgrip a food pellet.

And later, at dinner with his family, Blackmore is quiet anddistant. He sleeps fitfully. Then he rises at daybreak and re-turns to the lab.

One winter morning, several months after the conference,Blackmore sat at his desk explaining how he came to work inan area of medical research that seems so high in desperation,so low in hope.

What Im passionate about is solving a problem, he said.A spinal cord injury is a wiring problem.The wiring works like this: When you decide to change chan-

nels on the TV, a neuron in the brain sends an electrical signaldown a long, wire-like structure called an axon.

The signal reaches the spinal cord and passes to a messengercalled a motor neuron. The motor neuron dispatches its ownaxon carrying the message to the finger. Then the finger press-es the remote.

Almost every movement from bending a knee to movingthe diaphragm in order to breathe depends upon messagestraveling along axons.

Its the interruption of that signal thats the heart of theproblem, Blackmore said. No axon, no signal. No signal, nomovement. Thats paralysis.

Its such a simple principle. Its the exact same problem thephone company has when a tree falls on one of their cables.

In theory, repair ought to be simple, too.Before were born, in the developing embryo, broken axons

can repair themselves. When the axons mature, however, theylose that power. (Scientists know about this change from ani-mal tests, but they believe it holds true in humans.)

This leaves one overriding mystery: What makes embryonicand mature axons behave so differently?

Figure that out and maybe you can reverse the process. May-be old wires can be made new again.

Thats the essence of the problem facing Blackmores team but nowhere near its full dimensions. More than 1,000 genesdiffer between an embryonic and a mature axon. Some becomemore active as the axon develops, others are dimmed down.

Each axon is composed of thousands and thousands of pro-teins interacting in complex ways.


Murray Blackmore and postdoctoral researcher Zimei Wang examine cells under the microscope. The National Institutes of Health awardedBlackmore a $1.6 million grant to study spinal cord injuries.

FAMILY PHOTO

Bonnie Blackmore visits the White Cliffs along the Missouri River inMontana. She lived for 26 years as a quadriplegic.

FAMILY PHOTO

Bonnie Blackmore on a canoe trip in 1968, years before her spinalcord injury. Blackmore was injured in a car crash in 1987. Her paral-ysis inspired her son Murrays research.

Imagine a jet engine thats not working, Blackmore said.And it comes from outer space. We dont understand all thecomponents. Were trying to figure out which are working andwhich are not working.

Blackmore takes apart the human engine, testing compo-nents. He looks for genes that make axons grow, then insertsthose genes into mice with spinal cord injuries. He searchesfor one gene, or maybe a combination, that will help the mousefunction better.

But growing axons isnt enough. Injuries to the spinal cordcause scars and those scars block new axons, preventing themfrom establishing a connection. So Blackmore also tests en-zymes, hoping to find one that breaks down scar tissue.

Scientists have grown axons in a dish and even in animals,but despite sporadic success, the injured animals are seldommuch better. And success in humans has been rarer still.

If before the injury, the human spinal cord could score 1,000touchdowns, Blackmore said, so far in animals we can moveone football only 1 yard.

As he turns the problem over in his mind, Blackmore con-structs a visual image: the communication between brain andspinal cord as a train carrying cargo over tracks. The cargo issignals to and from the brain. There is a break in the track, theinjury. Maybe the signals are requests for repair supplies.Maybe the axons failure to regrow is a communication prob-lem, a muddled message between brain and spinal cord.

He freezes the picture. What signals are in the cargo?This is how he thinks.These are his tools.A $90,000, state-of-the-art microscope allows Blackmore and

his team to test 20 individual genes in a single hour and mea-sure how much axon growth each one triggers. Previously ittook weeks just to test a single gene.

Recently, he began applying a new technique called optoge-netics, activating groups of brain cells by shining light onthem. This lets the lab compare the behavior of cells that havereceived a gene treatment with cells that have not.

Dr. Blackmore is doing exciting, cutting edge research, said Lyn Jakeman, who oversees grants on spinal cord inju-ries for the National Institutes of Health.

Across the United States, 250 scientists work on spinal cordresearch. The NIH spent about $95 million last year on re-search studying spinal cord injuries, a little less than it spenton cervical cancer, a little more than it spent on traumaticbrain injury.

Competition for the grants is fierce. Labs burn 80-hourweeks preparing applications only to get rejected; only about15% receive NIH money. Success requires focusing researcharound the right question, a skill for which Blackmore hasshown a knack.

Hes really, really logical and very reductionist. He canbreak things down into small problems, explained VanceLemmon, who worked with Blackmore at the Miami Project toCure Paralysis. Hes one of the most talented people at doingthis that Ive ever met.

When Blackmore turned his mind to the big problem theinjury that changed his mothers life he saw a group ofsmaller problems, each one now the focus of a project in hislab.

One involves hunting genes that make axons grow, narrow-ing long lists of candidates and testing the best on mouse braincells in a lab dish.

Another: the study of astrocytes, star-shaped cells that showpromise in helping axons to pierce the scar tissue barrier.

Yet another: a groundbreaking attempt to determine wheth-er optogenetics can be used to assess spinal cord treatments.

For months, Blackmore and the half-dozen students and twopostdoctoral researchers in his lab have chipped away at theseproblems, conducting experiments, then discussing the re-sults at weekly lab meetings. Theyve tested, learned and ret-ested, questioning what they saw, advancing cautiously, a lit-tle like a baseball team stringing together singles.

Then, at a meeting in early April, Blackmore decided toswing away.

This is a crazy, unfunded project, he told his students andresearchers.

Their eyes followed him as he explained where his recentthinking about how to grow axons had led.

Cancer. A disease of uncontrolled growth.The idea of trying to use genes that make cancer grow to do

the same for axons had been proposed before. But scientistslacked the technology for a systematic examination. Now,armed with the powerful screening microscope, Blackmorewanted to revisit the idea, to make it the labs new line of in-quiry.

Through online searches, he identified 1,400 genes that ei-ther trigger cancer or block it by suppressing tumors. Toomany. He narrowed the list, focusing on genes that turn other

genes on or off, master regulators. That dropped the number to219. Then he looked at genes linked to axon growth.

So far, scientists have identified a dozen axon-growing genes all but one either activate or suppress cancer.

The axon growth field is already studying cancer genes,they just dont know it. Blackmore told his team. So I wrote agrant to do that and it got laughed out of the room. And I said,Screw it. Were going to do it anyway.

The inquiry into cancer genes had begun as a side project.Now, he said, it is going to be one of the labs focal points.

As the students returned to their experiments, Blackmorecould tell hed sparked their interest. He was both excited andworried. The lab had several promising lines of research and apressing need to publish a paper soon.

The situation made Blackmore think of a professor hedstudied with in Hawaii, a man who had an odd way of stayingfit. He chased wild goats.

Chasing goats was not an easy business. Run at the pack andthey scatter. The only way to succeed, the professor hadlearned, was to lock in on a single goat and follow it withoutgetting distracted by the others.

Blackmore wondered if he ought to be applying the sameprinciple to the different projects his lab had been pursuing.

He remembers the precise moment he decided what to dowith his life.

It was in the fall of 1997, while bicycling on the Pan-Amer-ican Highway through the Andes. He was 23 years old. He and afriend were in the midst of a 6,000-mile ride from St. Paul,Minn., to Santiago, Chile.

Theyd slept under the stars. Theyd been robbed at machete-point. Theyd made it across flooded-out stretches of road.Now, they were riding in the mountains of Ecuador.

Blackmore had an undergraduate degree in environmentalscience from Stanford. But he no longer believed he could offerthe field what it needed most. When it came to the environ-mental challenge of our time, climate change, Blackmore feltscientists had firmly established the predicament and the so-lutions. Missing was the will to respond.

The bike trip would be one last adventure before he settledinto a career, marriage and parenthood. And maybe, some-where in those miles, he would figure out what to do.

I knew that I wanted to do something good in the world,something worthwhile, he said. But I didnt know what thatlooked like.

So, there he was, pedaling at 10,000 feet above sea level, be-neath a brilliant blue sky. To his left rose a steep cliff. To hisright, the landscape fell away through a field of boulders.

The Pan-American Highway is one of the main truck routesthrough South America. The shoulders are narrow. Whitecrosses dot the landscape, marking death on the highway.

Here, he found clarity.The right problem had been in front of him all along, the leg-

acy of another day, a decade earlier, on a distant highway.

Thanksgiving weekend, 1987.Theyd been driving all night in the family station wagon,

heading home to St. Paul, Minn., from Montana after visitinggrandparents.

It was cold. In the early morning, black ice painted patchesof Interstate 94, as the Blackmores approached the small west-ern Minnesota town of Fergus Falls.

There were five in the car: Blackmores older sister, Jenny,


Matt Simpson and Denise Coley prepare culture samples to studythe effects of genes on axons. The lab is trying to find genes thatwill trigger axon growth to help repair spinal cord injuries. Almostevery movement we make depends on messages traveling alongaxons.

the driver; their mother, Bonnie, in the passenger seat; Black-more behind them, and next to him, his other sister, Leah; andin the very back, sleeping on top of the luggage, their brother,Cameron.

Blackmore was reading Agatha Christies Murder on theOrient Express. He had just taken off his seat belt to get morecomfortable.

Suddenly, the car was spinning. Outside the window, a con-crete overpass rushed in, then receded.

Now, the car was rolling backward. The car was crossing themedian. The car was in oncoming traffic. A semi was bearingdown. White headlights flooded the station wagon.

Then nothing.Sprawled on the ground. Face down. His eyes opened. The

first thing he saw in front of him: Murder on the Orient Ex-press. Shards of glass were embedded in the cover. He was 100yards from the car with no idea how hed gotten there.

Cameron was dead; Leah badly injured. The roof of the carhad buckled on top of their mother. Her spine had beencrushed at the C4-C5 level, a little below the chin. She could notmove her legs or hands. She could only control her shouldermuscles enough to make the limp mass of her arms swing rightor left, backward or forward. She was a quadriplegic.

The goal posts changed radically, Blackmore would say ofher life.

His mother was 45. She had just divorced his father. She wasfiguring out what she wanted to do. She loved the outdoors. Shehad a keen interest in psychology. She was taking lessons intennis and guitar.

For the next two years, Bonnie Blackmore spent much of hertime either in a hospital or a rehabilitation center. She camehome in a motorized wheelchair. She had a splint that allowedher to hold a spoon and feed herself. For the other tasks of ordi-nary life, she had personal care attendants who lived in thehouse.

It took months before she could use a mouth stick to type andoperate the phone.

Try to envision what it must have been like to be BonnieBlackmore.

Maybe once, for a disability awareness program, a teacherhad you sit in a wheelchair and imagine how different your lifewould be. The wheelchair was supposed to represent the har-dest-to-bear of all losses from a spinal cord injury: walking.

Only that isnt the case.A 2004 paper in the Journal of Neurotrauma surveyed qua-

driplegics (those with partial or complete loss of movement inall four limbs and torso) and paraplegics (loss of movement inthe legs and torso, but not the arms). They were asked to rankthe abilities they would most want to regain.

Walking did not finish first with either group. Or second.Not even third.

Quadriplegics said they wished most of all to have arm andhand function restored, then sexual function, then trunk sta-

bility (the ability to keep oneself upright), then bladder andbowel function. Walking ranked fifth.

Among paraplegics, the order was different because they re-tained arm and hand function. They wanted most to have sex-ual function back; then bladder and bowel; then trunk stabil-ity; then walking.

If these priorities seem odd, imagine you cannot hold a pen,open a door, scratch an itch, stroke your daughters hair. Youcannot swipe a bee away from your face. Youre helpless to stopa charging dog from jumping on you.

Now, imagine that you must depend on someone else in themost private moment every time you go to the bathroom.

Blackmore didnt have to imagine. He had only to look in hismothers eyes.

An incident early on lodged in his memory. It was the sum-mer after the accident, and his mother sat at the dining roomtable with the rest of the family. A splint clamped her handaround a spoon so that she could feed herself.

Unfortunately, she could not attach the splint herself, noteven close. Someone had to put on the device and arrange herfood perfectly, or it wouldnt work. After the meal, Blackmorewatched as his mother tried to remove the splint. She was us-ing her teeth. The intensity burned in her eyes. Hed neverseen her so determined.

In the end she couldnt do it, Blackmore recalled. I dontthink I have ever seen a person so defeated.

FAMILY PHOTO

Murray Blackmore bikes along the Pan-American Highway through the Andes in 1997 during a 6,000-mile ride from St. Paul, Minn., to San-tiago, Chile. It was during that trip that he realized he wanted to devote his life to studying spinal cord injuries.

ABOUT THE STORYMarquette University professor Murray Blackmore allowed

Milwaukee Journal Sentinel reporter Mark Johnson and photo-journalist Richard Wood to sit in on virtually all weekly labmeetings, watch experiments and witness discussions ofresults. Johnson interviewed Blackmore and lab membersextensively, as well as experts in the field and scientists whoworked with Blackmore earlier in his career. He also read keyscientific papers by Blackmore and others and, with Wood,traveled to the 2014 Working 2 Walk symposium on spinal cordresearch in Seattle, where Blackmore was a presenter.

Many of the scenes in the story were witnessed firsthand bythe reporter or photographer. Scenes that took place before2014 were described through interviews with those present,except for the opening scene, which was drawn from videotapeof Blackmores 2013 talk at a conference in Boston. The de-scription of the 1987 accident was drawn from Blackmoresaccount and confirmed through news coverage from the time.The state of Minnesota no longer has the original accidentreport.

In a few instances a characters thoughts are described. Inall such cases, people described their thoughts to the reporter.

Thats when he understood.

On a chilly spring morning in late April, a few weeks afterhed announced the cancer gene investigation to his team,Blackmore arrived at his office feeling nervous. Hed sleptpoorly.

Results were coming back on experiments involving some ofthese genes. As he saw it, genes that cause tumors to startgrowing or stop were like on-off switches that might be tai-lored to make axons grow.

Matt Simpson, a 20-year-old sophomore in the lab, had car-ried out the experiments. The data would appear on bar charts,and Blackmore knew the result he wanted. Heck, he coulddraw the chart he hoped to see. And this worried him.

There are two things I think about, he said. Its not thebest idea for a scientist to be so invested in an outcome, eventhough we totally are. The other thing is about leadership.Even if it totally crashes, I cant show that. I have to find someway to keep Matt completely invested in the project.

The professor entered the lab and found Simpson sitting byhis computer. The student called up the chart for the first of thetwo genes theyd tested. Blackmore stared at it hard.

Interesting, he said. Were so close to a slam dunk.They were looking at a gene called HHEX, which shuts down

growth. In the lab, theyd used a trick to alter the gene, hopingto turn the growth switch from off to on. The bars on thechart showed they had succeeded but the growth was mod-est.

Not bad. Not bad, Blackmore said, pointing a little higheron the chart. We would have opened the champagne if the barswere up here.

Results from the other gene ended any talk of champagne.The gene called ETV5 appears in the brain and Blackmore

hoped it would function like a close relative. The relative stopsgrowth. Blackmore saw another off switch that might be turn-ed on.

Only now that he looked at the charts, nothing made sense.ETV5 failed to stop growth. So did its relative, a baffling result.Either there was an error in the experiment, or the informa-tion theyd begun with was wrong.

Thats as bad as it gets, Blackmore said, staring at thechart. Are you kidding me?

The student and professor went over the experiment andtried to guess what had happened. They would have to run theexperiment again. From Day 1, Blackmore had warned stu-dents in his lab about confusion; it is the way 90% of experi-ments end, hed said.

After Simpson left the lab for class, Blackmore admitted hehad gambled. Believing ETV5 would behave like its relative,hed jumped the gun. Hed already modified the gene to make aversion that turned on growth. The safest approach wouldhave been to test a normal version of the gene on brain cellsbefore going to the cost and trouble of modifying it.

But following that procedure felt too methodical, and worse,too slow. Now, he worried he had wasted lab resources.

Its part of a bigger problem for the lab, Blackmore said.Its my problem.

When a gene shows promise in growing axons, he wants sobadly to bypass the confirmation tests and the tests to under-stand how the gene is triggering this growth.

His heart and mind leap to the bottom-line experiment: Willit help an injured animal?

Thats where I want to go, dammit, he said. The problemis that the answer is almost always, Nope, it doesnt work.

And if the answer is no, then you cant publish and youhavent learned very much.

This was the tension between his two selves.The skeptical scientist who approached experiments with

detachment.The idealistic son who wanted to cure his mother.

Dendrites

What happens in a spinal cord injury?

Currently, the best that treatment can offer is to:

1. Maximize function of spinal circuits and any spared fibers: The spinal cord is a vast bundle of fibers. If not all the fibers are severed, then doctors will seek more function from the remaining connections.

2. Improve area of injury: The bodys natural response is to produce scar tissue around the injury. Axons cannot grow through scar tissue. Some treatments include removal of scar tissue combined with nerve grafts to connect parts of the spine above and below the injury.

This can produce limited axon growth, but neurons that control hand function and movement do not respond well to this treatment. Therefore scars still present a barrier to axon regrowth.

Can axons regrow?Nature has plenty of examples of nervous system regrowth. Some animals, embryonic mammals and our own peripheral nervous system have the ability.

But in adult neurons this function has been turned off. One goal of Blackmores research is discovering how to turn this switch back on.

Neurons have two types of appendages:

1. Dendrites, which receive information from

other neurons 2. Axons, which send

information to other neurons.

Neurons are classified into three main types:

Unipolar: Neuron has one axon connected to

the cell but the axon has two branches and dendrites

branch from the axon.

This type is found mainly in the sensory nerves of the peripheral nervous system.

Bipolar: These have one axon and one dendrite connected to the

cell body. The dendrite and axon each convey a process.

Bipolars are the standard neuron design and are found mostly in embryos. Theyre only found in specialized

sensory areas of the eyes, nose and ears.

Multipolar: Has many processes with only one axon and several dendrites attached to the cell body.

Most of the neurons of the brain and spinal cord are multipolar.

Billions of neurons reside in the brain, and millions more can be found in the spinal cord. These specialized cells include a nucleus but they cant reproduce.

Their specialty is communication. They use cable-like appendages called axons to carry signals through the spinal cord and out to the rest of the body.

The brain communicates with every part of the body via the spinal cord. When the spinal cord is injured, a communication problem results. Injury severity is based on where the damage occurs along the spinal cord. Generally, the cord above the injury site is intact, but it cant regenerate. Below the injury, the cord is capable of responding but can no longer receive signals. Re-establishing this damaged connection is the hope of many patients and drives researcher Murray Blackmore of Marquette University.

1

2

2.infor

Synapse

Neuron

Axon

Astrocytes are found in the central nervous system. Some, called scarring astrocytes, form the scar tissue barrier at the site of a spinal cord injury. However, others called bridging astrocytes have the ability to pierce the scar tissue. Blackmore's lab has been trying to use these bridging astrocytes to penetrate the scar so that axons can get through and make connections below the injury.

Source: The Human Body Book; Murray Blackmore, assistant professor, department of biomedical sciences, Marquette University

Graphic: Lou Saldivar/[email protected]

How neurons communicate

Neuron

Axon Dendrite

C4-C5 level of spine(This is where Bonnie Blackmore was injured in a 1987 accident that left her a quadriplegic.)

Multipolar neurons

O ne afternoon in April, a razor-thin beam of blue lightzipped like a dart into the exposed brain of an anesthe-tized mouse at Marquette University. The creatures ti-ny limbs jerked instantly. All four.

Naveen Jayaprakash, a 30-year-old postdoctoral researcherin Murray Blackmores lab, watched the mouse, then rusheddown the hall to Blackmores office.

I have good news, Jayaprakash told his boss, and bad news.At 39, Blackmore has spent more than one-third of his life on

a quest to find a treatment for spinal cord injuries. In the lab,hed embraced some of the newest techniques, including theone Jayaprakash had just tested: optogenetics, the use of laserlight to stimulate brain cells.

It happens in tiny fractions of a second. The blue laser lightis absorbed by channels along the surface of a brain cell. Thechannels open, and a burst of positively charged ions rushesinside the negatively charged cell. This is whats meant by aneuron firing.

Optogenetics is new to the study of spinal cord injuries, sonew that Blackmore believed his lab was the first in the nationto use it.

Now, as his researcher reported on the test, Blackmoreleaned back in his chair, listening intently.

The animal is moving, Jayaprakash explained. The badnews is all the limbs are moving.

Only one was supposed to.They walked back to the lab and Jayaprakash ran the experi-

ment again. Blackmore watched.Jayaprakash triggered the laser and the needle of blue light

ran straight into the unconscious animals exposed brain.Again, all four limbs moved. The experiment had not gone ex-actly as planned. And yet, it had succeeded in one crucialsense: Light had clearly activated the mouses brain cells.

Jayaprakash, who was born and raised in the southern Indi-an state of Tamil Nadu, felt a wave of happiness; it lasted nomore than a minute or two.

For Blackmore, the feeling was relief. A burden lifted, hesaid, the constant fear that its all going to fall apart and thiswill be the stumbling block that brings the whole thing down.

The relief lasted maybe 15 seconds. Then came a question:What really caused the mouse to move? Maybe it was startled.Maybe the eyes, not the brain, had reacted to the light.

The experiment would have to be repeated, then repeatedagain. That is because of a fundamental conflict at the core ofscience.

You try to disprove what you hope is true, Blackmore said.You try to kill your own baby and yet you want it so badly tolive.

The accident that paralyzed Blackmores mother in 1987changed his life long before he became a scientist and took upthe search for a cure.

His mother had taught him to cook and do the laundry. Butthere was more his mother needed.

Bonnie Blackmore would not ask the children to help withmost of her personal cares dressing, bathing, going to thebathroom. Those jobs fell to the attendants who lived in theirhouse in St. Paul, Minn.

Still, from time to time, Blackmore emptied urine from hercatheter bag. He felt guilt for not helping more, and the guilttriggered resentment. He hated that his mother needed help inthe bathroom; and he hated himself for feeling this way.

Their relationship changed in other ways. After the acci-dent, she could not be the same attentive mother she had beenbefore; and that meant he had to be a different son.

Late one night 4 a.m. to be exact he drove home from afriends house. He was 16. As he pulled into the driveway, herealized no one knew where hed been. No one would ask. His


Naveen Jayaprakash, a postdoctoral researcher in Murray Blackmores lab, triggers a laser sending a shaft of blue light into a mousesexposed brain. He is trying to determine whether this technique, optogenetics, can be used to assess treatments for spinal cord injuries.


Lab prepares for challengesMarquette researcher finds some of the biggest obstacles he faces in finding cure for spinal cord injuries are tricks of the Natural WorldBy MARK [email protected]

DECEMBER 15, 2014

mother was not tracking his activities. She couldnt.Unlike almost every one of his high school classmates, he

returned to a home where there were no expectations, no rules.No one could structure his life. No one could prevent him fromdrifting. Only one person could push him to succeed.

In that moment, Blackmore resolved to structure his ownlife. He would focus on school.

Blackmore discovered discipline, and it became his pass-port to Stanford University.

There, he poured everything into his classes and got all Asand A+s. He spent his summers doing environmental field-work in Hawaii. He even achieved a rare distinction for an un-dergraduate: first author on a study in a professional ecologyjournal.

He was also miserable. His roommates freshman year werean Olympic-level gymnast and a first violinist. In other words,at Stanford everyone stood out, which really meant that no onedid.

He made no friends, not a single person he would stay intouch with after graduation. He visited nearby San Franciscojust once in four years. Mostly he studied by himself in the li-brarys subbasement.

Those years taught him he needed balance. He couldnt livein the library.

Yet even when he chose to unwind, he favored solitary pur-suits. He liked to bow hunt, perched in a tree for hours, motion-less, alone, thinking.

After graduation, he had set out on the long bicycle trip toChile with a friend, but eventually the friend went home.Blackmore rode into Santiago alone.

A few weeks after he watched the blue laser light enter themouses brain, Blackmore introduced three new undergradu-ates to the lab and to the science of the spinal cord.

In the life cycle of a university lab, new students arrive insummer, filling the gaps left by graduating seniors. Black-more, the senior investigator, remains the one constant. Hepicks the projects, applies for grants, buys equipment. He setsthe scientific course.

The students run the different projects, perform the experi-ments and update the others at the weekly meetings. Thisweek, however, Blackmore addressed the new and old mem-bers of his team. Hed rehearsed the speech in his head as helay in bed the previous night.

As we do science, he told the students, I would like you tobe innovative, constantly innovative. I dont mean in some ab-stract, lofty sense. I mean in a very concrete way.

As your hands are moving, I want your brain to be asking,


Laura Thiel, an incoming senior, measures the length of axons as she works on a study to determine whether genes that trigger or blockthe growth of cancer can be engineered to make axons sprout.


Axons, the strands that communicate information along the spinalcord, are measured in the lab.

FAMILY PHOTO

Murray Blackmore with his father, Ronald Blackmore, his mother,Bonnie, and his sister Jenny on the day of his graduation from highschool in 1992.

Is this the best way to do things? The new students had never worked in a professional sci-

ence lab. He needed to excite them, but also to steel them for thehard, humbling work. The disappointments.

One thing youre going to learn in lab is that what we knowis a really small subset of the knowledge in the universe, hesaid. And a lot of what we think we know is wrong. . . . Wethought that we knew that this particular enzyme was not ac-tive in the adult spinal cord and it turns out it is. And thereforethe whole rationale for our project fell apart. Right there.Boom . . .

As we go through this summer, mistakes will be made.Youre going to do experiments wrong. Youre going to forget acontrol. Youre going to use equipment wrong and its going tobreak. This is inevitable. Its not only inevitable, its desirable.Its something I demand of everyone in the room. I insist thatyou make mistakes, and then I insist that you take them to thegroup, we talk about the mistakes and we learn about them as agroup.

Blackmore sought to prepare them, too, for the world out-side the lab: the gladiatorial arena of peer review. You sendyour grants to a group of people who are competing for the ex-act same pool of money, he said. You send your papers andthey are evaluated by people who are competing for the exactsame slot in those high-profile journals. So they have a vestedinterest in tearing you down, and they will.

In this harsh world, he explained, they must help themselvesand each other by being skeptical of their own ideas and bychallenging those of the other lab members.

Part of the reason we do that for our friends and part of thereason we do that for ourselves is because its a rough worldout there and weve got to toughen each other up before we goout there and try to survive.

The other thing thats even harsher than all of the otherscientists out there evaluating our ideas is the Natural Worlditself. It is a cruel, cruel mistress. It will trick you. It will leadyou on. It will crush your hopes and dreams again and againand again.

Blackmore smiled as he said this.Spinal cord researchers had seen once-exciting ideas fall

short of initial promise. In the late 1980s and 1990s many fo-cused on molecules called neurotrophins, discovering thatthey help the crucial wiring called axons grow a little, but notnearly enough to cure an injury. In the late 1990s and 2000s,excitement built around the idea that axons were beingblocked by molecules in the myelin, the insulation around thewiring. So far, neutralizing the molecules has only resulted inslight improvements.

The way to protect yourself, Blackmore said, and the wayyou protect your friends is to try to spot the cruel trick thatshes going to play before she plays it.

A week later, in early June, one of the new students, LauraThiel, sat in the lab staring at neurons under the microscope.

What she saw looked like an image from outer space a vastblack background broken up by small white globes, each withlong, curling lines extending out from the surface. The globeswere neurons, the bright lines were the axons.

Thiel, an incoming senior, was working on the labs newestproject, an unusual study to determine whether genes thattrigger or block the growth of cancer can be engineered tomake axons sprout.

She traced each axon on the microscope with a red cursorrecording the length. The cells in the culture dish had beentreated with a version of the HHEX gene. Although HHEXshuts off growth, Blackmore had engineered this version to dojust the opposite to make axons flourish.

So far, the gene had shown promise. The professors hopeswere high.

He looked over Thiels shoulder. The axons looked long.Whats more, neurons usually have one axon, perhaps two, onrare occasions three. This neuron had four axons. So did thenext one and the next one.

If this is true, Blackmore said, its really interesting. . . .Weve only looked at 13 neurons so far. You have to be so cau-tious, because weird things happen in culture.

Before he could allow himself to show excitement, Black-more would have to see 200 neurons or more. They would haveto be spread out over three separate experiments. Each experi-ment would have to be carried out on a different day.

This is exciting, Thiel said.


Murray Blackmore works on an optogenetics experiment. Optoge-netics is new to the study of spinal cord injuries.


Blackmore discusses culture preparation protocol in the lab withpostdoctoral researcher Ishwariya Venkatesh.

Blackmore paused, then told her, We need to blind you.By blinding, he meant that she should not know which ax-

ons had been treated with the gene and which had not. Black-more would know, but he would not be in a position to affect theoutcome. The professor explained the reason experiments areoften carried out this way so that the experimenter cannottell if a result supports or contradicts a particular theory.

Thats the only defense against the human craving for pat-terns, he said, patterns that support our ideas.

After his South American bike trip, Blackmore returned toMinnesota and began the transition from environmental sci-entist to spinal cord researcher. He did not share the plan withhis mother.

Bonnie Blackmores first inkling was the envelope that ar-rived at her home in St. Paul with her sons test results forgraduate school. The test was in biology.

Whats this about? she asked.Im going to grad school, he told her. Im going to study neu-

roscience and work on spinal cord injuries.She gave a little smile and said something like, Youre a fun-

ny guy, a phrase she used to mean strange rather than hu-morous.

Beyond that, Blackmores mother offered no opinion; itwasnt her style. The children were old enough to make theirown choices.

The lack of discussion helped mother and son avoid whatwas an awkward subtext.

I could never say, Im going to study this and cure you,Mom, I could never make that promise, Blackmore said.

And she never made me feel like she expected me to actual-ly cure it.

In late June, Kristen Winsor stood at the SmartBoard, up-dating lab members on her project as she had every week forthe last several months.

Winsor, 21, was going into her senior year. Shed grown up innearby Waukesha and could not ever remember wanting to doanything but science.

A year and a half earlier, Winsor had been scheduling meet-ings with different labs, trying to decide where to work. Whenshe met Blackmore, she canceled her last appointment.

His approach fit her view of science.Its not just about gaining knowledge and advancing your-

self, she said. Its about advancing somebody else. My knowl-edge is going to be used to improve somebody elses health.

Blackmore had not mentioned at that first meeting andWinsor would not learn until months later that his motherhad a spinal cord injury. Since coming to work for him, shednoticed how much time he spent in the lab, how he celebratedgrants and good news, and how patient he was when experi-ments did not work or results did not hold up.

Now she stood at the front speeding through the slides thatexplained her project. She was testing whether cells called as-trocytes help axons to grow through the stubborn scar tissue atthe site of a spinal cord injury.

Results in the spring appeared promising. Astrocytes werehelping axons to break through the scar tissue barrier. Black-more and Winsor were so encouraged that they planned to pre-sent their findings at a major neuroscience conference in No-vember.

Now, something had gone wrong. The cells that form thescar tissue were supposed to stop axons from growing. Theywerent. As long as the scar tissue refused to behave like scartissue, the experiment was useless. There was no barrier tobreak through.

Winsor had repeated the experiment placing the cells in

both glass and plastic dishes to see if she could get the scar tis-sue to do its job. When she finally succeeded, the conclusionwas discouraging. The astrocytes did not promote any axongrowth, Winsor told the group. None.

Somethings gone off, Blackmore said quietly, almost tohimself.

Now, Winsor continued, Im working on something com-pletely different.

As she explained her new project, the end of the old one ho-vered strangely in the air.

How do we explain the results we got all through thespring? Blackmore wondered aloud. Thats rough.

Based on the spring results, they had already submitted anabstract for the fall neuroscience meeting, a brief descriptionof the talk that would be included in the conference schedule.

Negative findings showing that something doesnt work seldom impress journal editors, conference organizers orfunders. The professor still had a hunch the promising earlyfindings might have been right. But do you ask a student tokeep going when theres no progress and the results make nosense?

For days afterward, Blackmore agonized. He hated having ahypothesis proved wrong, but this was worse. To perform anexperiment over and over and come up with wildly differentresults, this says that for this project at least, we are doingscience wrong, he said.

Disappointment had clouded his mind. He knew that. Hewould set the project aside until he could view it without emo-tion.

In graduate school, Blackmore surprised his professors.Even before hed started at the University of Minnesota, hecontacted neuroscience professor Paul Letourneau about join-ing his lab. He told Letourneau he wanted to dive into the ques-tion of how to make axons regenerate.

It was a good question, but not the one Letourneau and hislab were asking. Letourneau wanted to know how the spinalcord gets wired in early development. Besides, his lab wasstudying chickens; to learn about spinal cord regeneration,Blackmore would want to study mammals.

Letourneau explained all this. He did not expect to hear backfrom Blackmore.

But the student showed up at his lab anyway. Blackmorecame with a prestigious Howard Hughes Medical Institute fel-lowship, a five-year scholarship that paid for all of his gradu-ate studies. He quickly proved that working in Letourneauslab was not such a strange choice.

Knowing how the spinal cord is wired gave him a good startin considering how to repair it. Chickens also offered usefulcomparisons for spinal cord damage. Like mammals, chickensstart in the embryo with the ability to repair a severed spinalcord; then they lose the power.

Blackmore found he could open a chicken egg, cut the em-bryos spinal cord, then seal the egg back up. The chickenwould live. If he cut the spinal cord before the 10th day of in-cubation, the axons would regenerate and the spinal cordwould heal. By the 14th day, the axons would not regrow.

Murray designed new experimental paradigms, Letour-neau recalled. He thought very deeply about his experiments.He would think about all of the possible outcomes before hestarted, so that he could really understand what he saw.

Blackmore used his observations of the chicken embryo toconstruct a clever experiment that would take him two yearsand ask a fundamental question for the field:

To repair communication between the brain and spinalcord, to fix the downed wire, which end should researchers fo-cus on?

Was it the brain, or the spinal cord that needed more help?

Optogenetics, a tool for testing treatmentsUntil recently, researchers monitoring the nervous system and neuronal activity were limited to general regions of the brain. Optogenetics allows single cells to be activated, giving researchers a highly focused tool to map brain activity.

Heres how it works:

Some species of algae have light sensitive proteins called channelrhodopsins that respond to blue light by opening an ion channel in a cells membrane. This creates an electro-chemical signal that can be turned off and on with light.

There are generally two parts to a gene; one encodes for a protein and the other regulates, or defines how much protein is made. In Murray Blackmores optogenetics experiments, channelrhodopsin from algae is combined with the regulatory section from selected mouse neurons.

The combination is inserted into a virus and injected into the mouse. The virus, used as a delivery system, enters targeted neurons.

The selected neurons now contain a light-activated switch allowing researchers to measure the effects of individual treatments.

m,

fects of ments.

Algae

Gene

Channelrhodopsin protein

Virus

Neuron

Gene for channelrhodopsin protein

Graphic: Lou Saldivar/[email protected]: wiringthebrain.com

A bad week got worse. At the end of June, Marquette Uni-versity researcher Murray Blackmore was forced to puton hold one of his labs spinal cord research projects. Early experiments had suggested cells called astrocytes

might help axons, the wiring so crucial to healing a spinal cordinjury. Then results turned baffling and inconsistent.

Other projects ran into problems. Experimental controls,the baseline comparisons that tell a scientist a test is valid,were failing. Tests had to be redone. Plates of cells were con-taminated. A $30,000 microscope wasnt working, and Black-more worried hed broken it.

That weekend he took a break from contaminated cells andfailing controls and went camping with his wife and three chil-dren.

The following Monday morning, Blackmore stopped on hisway to the weekly lab meeting and bought a bag of bagels andmuffins for his crew. His mind was busy drafting the pep talkhe would give them.

He left the bakery feeling determined. As he approached thedoors to his building at Marquette, a gull passed overhead andscored a direct hit on the bag. The food escaped unscathed.

So, last week was a tough week, Blackmore began.The students sat around the long table snacking on the ba-

gels and muffins.The first thing I want to say is that good things in the lab

tend to come in waves. Bad things in lab also come in waves.Ive seen it before.

Blackmore explained that sometimes problems are a signthat projects are overreaching. Inevitably, you overreach andthings crash around you. Its a teaching moment. What do youdo when everything is going wrong? You just keep doing sci-ence. You do the fundamentals.

He reminded them of all theyd accomplished. Last summertheyd learned and refined techniques for delivering genes toneurons. Now, it was routine. Their optogenetics experiments,a completely new approach for the field of spinal cord re-

search, showed great promise; they could shine light into amouses brain and watch limbs move in response.

They just needed to take a step back perform a few simplerexperiments, get procedures working again and rebuild confi-dence.

Its very easy when things are going well to say, You guysare great. Youre my crew, Blackmore said. Well, you guysare really good. Youre amazing and talk is cheap. So thatswhy I bought you treats.

He was a student once like them. A decade before he becamea professor, Blackmore was a graduate student at the Universi-


Murray Blackmore, a researcher at Marquette University, examines contaminated cells with Kristen Winsor, a senior who works in Blackmo-res lab.


Research hits setbacksScientist, students must overcome challenges in spinal cord experimentsBy MARK [email protected]


Axons grow in long strands. Blackmores lab is looking for ways toencourage axon growth, an effort that has grown to include tests ofcertain cancer genes. He turns the genes into on-off switches forgrowth.

DECEMBER 16, 2014

ty of Minnesota working in someone elses lab, but cutting hisown path.

He was certainly a unique individual, said Paul Letour-neau, the neuroscience professor who ran the lab. He was oneof those people who just decide how theyre going to live theirlives and they do it. And thats how he did science.

Throughout the brutal Minnesota winters, Blackmore rodehis bicycle to campus every day. He arrived at the lab wearinga Gore-Tex suit splattered with snow and dirt. Classmates inthe lab learned his quirks; he would not eat meat unless hedkilled the animal himself.

As for the science, Blackmore knew exactly how he wantedto attack the problem of a spinal cord injury.

He drilled deeper into the question of why a chicken embryocan repair broken axons at 9 days of age, but not at 14 days. Hebegan by observing the change in a lab dish.

Blackmore placed two slices from the embryo beside eachother in the dish, one from the brain, the other from the spinalcord. If the embryo was 9 days old or younger, axons wouldgrow from the brain to the spinal cord. If the embryo was just5 days older, nothing, no growth.

Scientists wanted to understand the change because a simi-lar transition is believed to occur in human embryos; brokenaxons repair themselves early on, then lose the ability. Mostresearchers had been seeking the explanation in the spinalcord. Blackmore wondered if that was the best place to look.

So he devised a clever way to figure out where the criticalchanges were taking place. He started mixing the ages of theslices. A 9-day-old brain slice next to a 14-day-old spinal cordslice. Then the reverse: older brain, younger spinal cord. Hetried many different combinations.

It was a unique approach. It was not something people haddone, Letourneau said. He completely figured it out on hisown. And it worked.

If the prevailing wisdom was correct, and the spinal cordwas the center of this crucial change, the best results shouldhave come from young spinal cord slices. The age of the brainslices shouldnt have mattered so much.

But it was just the opposite. Blackmore saw almost nogrowth when the brain was 14 days old and the spinal cord was9 days old. Growth improved considerably when he usedyounger brain slices.

During those five days, as the embryo lost its ability to repairthe broken wiring from a spinal cord injury, the most criticalchanges were occurring in the brain.

It took him two years of experiments, but Blackmore

emerged with an important paper in the journal Developmen-tal Neurobiology and a new direction.

I came out of that with my marching orders, he said, tofigure out what was happening in the brain.

In August, a month after the bagels and muffins, the profes-sor returned to the lab following a family vacation in the BlackHills of South Dakota. In his absence, key experiments had tak-en place. Now, Blackmore would learn the results.

We ready to do this? he asked Matt Simpson, an incomingjunior.

Simpson nodded. He had been performing experiments tosee whether certain cancer genes help axons grow.

Blackmore was on guard for bad news.Im not liking the big sigh, he told Simpson, who had not

actually uttered much of a sound.Its nothing bad, Simpson said.Although some of the experiments had run into problems

with controls, one had produced encouraging news about thegene the lab had been especially interested in: HHEX. Normal-ly the gene stops growth, but theyd engineered a version to dothe opposite, and it appeared to have helped axons sprout.

Simpson called up charts on his computer that compared thelengths of axons on two types of brain cells: one type treatedwith HHEX, the other a control group treated with a geneknown to have no effect on axon growth.

Yes! Blackmore said, raising his arms in triumph. HHEXhad outperformed the control and by a convincing margin.

The most important results were still to come. They had test-ed the modified version of HHEX directly on injured mice, in-jecting the gene into the brains cerebral cortex. This was a keystep toward someday testing the treatment in humans.

Inside three black boxes of microscope slides rested all thehope the lab had invested in HHEX. The slides contained thinslices from the spinal cords of mice, some with the HHEX gene,others without.

The professors face looked calm, but it was a mask.I feel like Im going to puke, he said, placing a slide under

the microscope.If the gene worked, Blackmore would see ghostly white

threads. He began with the control group, scanning to get a feelfor what normal looked like.

Normal, however, turned out to be something wholly unex-pected. He could see that at least some of the broken axonswere regenerating on their own. This odd result raised the bar


Matt Simpson, an incoming junior, presents his research on axon growth. The half-dozen students and two postdoctoral researchers inBlackmores lab discuss their research as a group each week.

for HHEX. It would not be enough for axons to grow when treat-ed with the gene; they would have to outgrow those sproutingin the control cells.

OK, Matt, here we go, said Blackmore. Heres HHEX.The image from the microscope appeared onscreen: White

strands, long ones.Thats substantial, Blackmore said, trying to restrain his

excitement.But the slides that followed delivered no consistent message.

Some showed impressive growth, others very little. He wentthrough them methodically, his mood rising, falling, risingagain. He circled back to the controls, trying to size up thelengths of axons with HHEX and those without.

Lacking precise measurements, it was impossible to tellwhether the gene made a difference. Knowing the excitementtheyd all felt about HHEX, Blackmore had tried to trick hisbrain into neutrality.

I pretended to myself that the purpose of the experiment isto show that HHEX has no effect, he explained later. HHEXwould have to have a huge and consistent effect before Id letmyself believe it.

Its very hard to figure out whats actually true when youwant something to be true.

By 2005, almost two decades had passed since the auto acci-dent that left his mother, Bonnie Blackmore, a quadriplegic.

She still lived in St. Paul, Minn., sharing her house with areliable team of personal care attendants who helped herdress, bathe, go to the bathroom and move back and forth be-tween her wheelchair and bed.

She had adjusted to the loss of privacy, though she referredwistfully to the time before the accident, back when I was my-self. Despite her injuries, she continued going on adventures,flying to Scotland, traveling the mountains of the BitterrootValley in western Montana and floating down the MissouriRiver in a canoe.

All things considered, Blackmore said, she was in a goodplace.

He was, too. Hed spent six years at the University of Minne-sota. Hed married his wife, Dawn, who taught Spanish and En-glish as a second language, mostly to elementary students.

He could see the next step on the scientific ladder, a post-doctoral research job. The move would bring him closer to oneday running his own lab, but it would take him many milesaway from his mother.

Sometimes he wondered why she never talked to him abouthis research. She was plenty smart. She had a masters degreein journalism. Many times he had watched her ask dinnerguests about their jobs.

When it came to his work, she was chiefly interested ingrants and lab politics. She wanted him to be happy and suc-

cessful. Yet she showed no interest in the experiments he car-ried out and the treatments he pursued.

Her injury had inspired his science. But his science left herin a conversational cul de sac. When he talked about his work,she would say, Uh huh, uh huh, uh huh in that way people dowhen theyre impatient. Then shed mumble, Its all beyondme, and change the subject.

He wondered if maybe his mother avoided the subject to en-sure that he never felt pressured to cure her. Or perhaps, it wasan act of self-protection; she harbored no hope of ever beingcured and preferred not to be reminded that her injury waspermanent.

In 2005, he was recruited to work for The Miami Project toCure Paralysis. Founded in 1985, the University of Miami pro-ject was one of the national leaders in spinal cord research.

Minnesota was home, but Blackmore knew it was time tomove.

Having decided to focus his research on the brain, he wasnow pondering a new question: What genes tell axons in thebrain to grow?

The question had led Blackmore to the curious gene HHEX.For objectivitys sake, the professor forced himself to be

skeptical, so skeptical that he was now convinced that the genewas little help.

Two weeks after first peering through a microscope at thetwo sets of mouse spinal cord cells, the professor was in a lowplace. Dawn Blackmore recognized the look in her husbandseyes and knew what it meant: his body home with the family,his mind far away.


Postdoctoral researcher Ishwariya Venkatesh and Murray Black-more enjoy a lighter moment in the lab.

Blackmore canoes to work down the Milwaukee River. The unique commute helps him relieve tension during stressful periods in his re-search.


A really cool effect in the culture dish so often just doesntcarry through to a living animal, she said. He takes it verypersonally.

The professor fought through his disappointment. For sev-eral days, he retreated to the basement, where hed put up apunching bag Dawn gave him for Christmas one year. He hitthe bag as hard as he could until his arms felt rubbery.

When he stopped punching, he focused on thinking, the kindthat informed those pep talks he gave to his students. Otherprojects showed promise. What about the groundbreaking useof optogenetics, light that stimulates brain cells?

Blackmore reminded himself the HHEX experiments werenot simply pass-fail. Sure, there was personal disappointment,but there was also a professional responsibility not to show it.As head of the lab, he must keep students enthusiastic and en-gaged in projects.

And there was still cause for enthusiasm. What about theworks value to basic science? Leaving aside the question ofwhether HHEX can repair a spinal cord injury, Blackmoresteam had established that the gene plays a role in the biology ofaxons. In a culture dish, the gene could be turned into an on/offswitch for axon growth. They had shown, too, that the gene isin the brain; nature must have had a purpose in putting itthere.

Still, he could not truly set aside the question of whetherHHEX would help a spinal cord patient. It was the question, theonly one that would matter to all the men and women in wheel-chairs.

The professor couldnt help feeling that HHEX had failed theeasiest test. Theyd only asked the gene to make axons grow inan injured mouse. They hadnt asked the axons to work.

When I think like a scientist, Blackmore said, HHEX isexciting. Its brand new. Who knows what it might teach usabout axon growth . . .

When I think like a very impatient bioengineer who wantsthis problem done, I wish (the modified gene) had knocked itout of the park with the first softball we threw its way.

Blackmore spent the years 2005 to 2011 at The Miami Projectto Cure Paralysis, testing genes, trying to pin down the differ-ences between the young neurons that could repair axons andthe old neurons that couldnt. He made important progress,helping to identify a new family of genes called KLF, that madeaxons grow.

The discovery led to a paper in one of the elite journals, Sci-ence. Blackmore was one of two lead authors.

He moved up in rank from postdoctoral fellow to researchassistant professor, a step closer to receiving his own labspace. For now, he worked in a corner of someone elses lab.

The Miami Project immersed young researchers completelyin the problem they sought to solve.

One of the great things about working where I do is that itsa building where every person works on spinal cord injuries,explained Vance Lemmon, a professor of neurological surgerywho worked closely with Blackmore at the Miami Project.There are people in wheelchairs that we see every day. Itsconstantly in our faces in a good way.

Blackmore impressed Lemmon with his rigor. He is obsessed with trying to get things right, Lemmon

said. Repeating an experiment three times is the norm. WithMurray, I dont think he believes anything until hes done it 10times.

Where the intensity came from was a mystery, at first. Lem-mon said it was two years before Blackmore mentioned hismothers injury. The subject came up only because he was re-turning to Minnesota to visit her.

In Miami, Blackmore and his wife had three children. Butthey realized the city was a poor fit for them. They did not wel-come the prospect of sending the children to the citys schools.

Blackmore had loved the woods and wide open spaces ofMinnesota; Miami offered little in the way of green space.

Most of all, however, the 1,800 miles between himself and hismother proved too great. The distance had not been a problemwhen she had reliable attendants caring for her. Gradually,though, trusted aides had moved on and her care became a run-ning crisis.

She simply could not live alone and there were no relativesnearby to take charge of her care. One of Blackmores sisterslived far from Minnesota; the other had been badly brain dam-

aged in the same auto accident that injured their mother. Thatleft Blackmore desperately trying to hire health aides long dis-tance. The problem drove home the need to find a job closer toMinnesota, a lab of his own this time.

Schools were still mired in the recession, but he received of-fers from Temple, Ohio State, Kentucky and Marquette. OhioState was particularly tempting. The school had a strong spi-nal cord injury center, and its offer was substantial.

But Marquette impressed Blackmore with the quality of itsresearch. The school offered a few advantages over larger in-stitutions, including lower lab expenses and a less bureaucrat-ic finance system. When the Blackmores visited Milwaukee onSt. Patricks Day 2011, they fell for the city in a big way.

Seven months later, in October, the new assistant professorwalked down a fourth floor hallway of Marquettes SchroederComplex into a vacant room. His new lab. There were nochairs. Furniture was one of his new responsibilities.

How high should chairs be? he wondered. Should they be ad-justable? He realized he needed to learn about chairs.

Its like starting your own business, and all the fears that goalong with that, he said. Failure is a real possibility.

Naveen Jayaprakash, a 30-year-old postdoctoral researcherin Blackmores lab, kept adjusting the power of the laser 1milliwatt, 2 milliwatts, 3 but to his dismay, the blue lightbeam had no effect on the cells in the mouses exposed brain.The machine graphing the brain waves showed no peaks orvalleys. A flat line, nothing.

It was August 2014, about the same time the HHEX gene wasstarting to lose some of its luster. Jayaprakash led the labsgroundbreaking effort to use optogenetics stimulation ofbrain cells with light to test spinal cord treatments.

This was at the heart of Blackmores $1.6 million grant fromthe National Institutes of Health. But the success that had ex-cited Jayaprakash and Blackmore in April seemed to have de-serted them. In that dark closet of a room, Jayaprakash boost-ed the laser to 4 milliwatts and still saw no reaction from thebrain cells. Five milliwatts nothing.

The grant application had been a calculated gamble onBlackmores part. Two years earlier, while gathering data forthe application, he had run into the same problem. The lightworked initially, causing neurons to fire.

Then we had failure after failure after failure, Blackmoresaid.

He gave up temporarily. But he submitted the grant anyway.Its this Catch-22, Blackmore explained. It takes large

amounts of time and research to make things work. The onlyway to be sure something is possible is to do it, and to do it, youhave to have money.

Now, over a period of several days, failure dogged the experi-ment. Jayaprakash tried dialing the laser way up to 40 milli-watts. The brain brightened, blue light glowing back at him.Still, the neurons refused to fire. He turned the laser backdown to 5. Nothing.

Why isnt it working?Jayaprakash was certain it would. Certain. Maybe the high

laser settings had overstimulated the cells, in effect silencingthem. He decided to give the cells a break. He went to the bath-room, got some coffee, grabbed a few snacks.

When the process works, a special protein injected into theneurons makes them respond to the blue laser light. The lightis absorbed by channels along the cell surface, causing thosechannels to open, and ions to rush inside. The channels thenclose when the light switches off.

After 15 minutes, Jayaprakash returned and put the laser atthe lowest setting, one-tenth of a milliwatt. This time, he saw asmall response. With growing excitement, he nudged up thepower. The response peaked when the laser stood at 1 milli-watt.

He was skeptical, but tried again, then a third time. As longas the cells werent overstimulated, 1 milliwatt did the trick.

Jayaprakash told Blackmore what hed discovered.Can we document it? Blackmore asked.In the laser room, the professor checked the results himself.

He was delighted. The problem that had stumped him twoyears ago had now been solved by his researcher.

There is a role for faith in science, Blackmore said. Thereason Naveen got it to work was that he had faith that itwould.

Manipulating the DNA on-off switch

Chromosome

Transcription molecule

Cell

Nucleus

Beyond individual genes, researchers look at gene systems how collections of genes function to produce a particular characteristic, function or trait.

This study is called epigenetics.

In human development, genes can be activated or repressed. For example, in embryos, the genes to trigger axon growth are busy building a nervous system. When finished, those genes remain, but are turned off.

Each gene has two parts. The first describes a protein. The second regulates the protein whether its off or on and, if on, by how much.

Murray Blackmores lab is testing ways to turn those switches on and off in order to get axons to grow for spinal cord repair.

This requires:

A. Identifying which transcription factors turn on genes to promote axon growth.

B. Identifying which neurons in the brain control specific functions, such as hand movement.

C. Locating neurons in the spine connected to those functions.

D. Stimulating axon growth from brain neurons, past the

injury site to corresponding neurons

in the spine.

A

C

A. Identifyingtranscriptionon genes to pgrowth.

B. Identifyingneurons in tcontrol specifsuch as hand

C. Locating nespine connecefunctions.

D. Stimulatinfrom brain ne

injury site tocorrespo

in t

B

C

D

1. Transcription factors unzip the

intertwined DNA strands

where the target gene

resides.

2. The transcription factor reads the

sequence and produces a copy.

The copy contains a genetic recipe

for that protein.

Source: Sciencemag.org Graphic: Lou Saldivar/[email protected]

O ne afternoon in September, Marquette University spi-nal cord researcher Murray Blackmore peered into aglass enclosure as the first brown mouse saunteredalong a few rungs of a horizontal ladder. Less than halfwayacross, the mouse stopped, sat up, and began grooming its face.

Come on, keep going.Blackmore stared. So did two members of his lab: postdocto-

ral researcher Zimei Wang and undergraduate Matt Simpson.As intently as the scientists gazed, they could not will the in-jured animal to finish the two-foot journey across the ladder.

It was not as if the mouses injury was severe; its spinal cordhad been damaged in such a way that only the right front pawwas impaired. The experiment required 28 mice with the sameinjury to make the trek all the way across the ladder fourtimes each. At this rate, it would take days.

Dont go down there, Blackmore cried out, as the mousetried to squeeze between the rungs to reach the bottom of theenclosure.

He tried Mouse No. 2, but it ventured only a short distance,then turned around.

The mice now meandering indifferently along the ladderwere from the control group.

But Blackmore had only the faintest hope that the micetreated with the once-promising HHEX gene might walk anybetter. Under the microscope, HHEX had failed to impress.Mice treated with the gene did not grow significantly more ax-ons the wiring essential to repairing a spinal cord injury than those without the gene.

Despite the long odds of success, Blackmore and his teamhad to try the live-animal experiment. Theres no way to mea-sure axons in mice without sacrificing them. And if, by somemiracle, scientists found that axons had sprouted, they wouldneed to know whether the mice had walked any better.

Mouse No. 3, another control, wandered a short way downthe ladder, turned and headed back toward the bright light thatwas supposed to deter wrong-way travelers.

Blackmore had seen quite enough. Rungs had been removedfrom the ladder to make the task more difficult; the professortold Simpson to put them back.

Blackmore thought about what mice like. They like dark-ness and their home nest. So he took the home nest and placedit at the end of the ladder. Then he took apart a box and made anenclosure to place over the nest.

For the moment, success had acquired a new definition. Al-though the scientist wanted to test the effect of the gene on aninjured mouse, first he would have to persuade the mouse tocross a ladder.

Heres your home cage, your favorite place in the world tobe, Blackmore told the mice. And lets sweeten the deal withthis high-tech light shield.

The next mouse hustled across the ladder.

After arriving at Marquette in the fall of 2011, Blackmore satalone in an empty lab, writing grant proposals, ordering equip-ment, filling out regulatory forms on biosafety and animalcare.

Months passed before the room filled with other voices. Firstcame Wang, a collaborator of Blackmores from his previousjob at The Miami Project to Cure Paralysis. Then a few under-graduates volunteered to work in the lab.

Although he had start-up money from Marquette and the re-mainder of a grant hed received in Miami, Blackmore neededmore. He needed the kind of grant that pays for years of re-search and defines success at a university. He applied to theNational Institutes of Health.

In February 2012, news came. His application had been re-jected.


Murray Blackmore, a researcher at Marquette University, listens to students and postdoctoral researchers present data during a weekly labmeeting. The lab is searching for ways to treat spinal cord injuries, something Blackmore was inspired to do because his mother sufferedsuch an injury.


Following natures leadScientist, students see exciting potential in research of genes that might hold secret to repairing downed wires of spinal cord injuriesBy MARK [email protected]

DECEMBER 17, 2014

house.In St. Paul, his mother was hospitalized, struggling to

breathe.That summer he agonized over her care. For 26 years, she

had lived at home and traveled and done all she could to keepfrom being defined by her injury. But her latest caregiverswere leaving soon. She could not stay in her own home anylonger.

Near the end of the summer, he found a house in a St. Paulsuburb where a few patients received full-time nursing care.They could take his mother.

In the new place, it soon became clear that she had not exag-gerated how badly she wanted to remain at home.

She didnt have a will to live anymore, Blackmore said.She just wanted out.

A month after the move, he visited. It was a weekend in earlySeptember and she wanted to see her old house. Blackmorehelped her into the wheelchair, then into the van, and theydrove across town.

As she rolled inside the front door, he sensed conflictingemotions. Shock at seeing her house empty, a lifetime of pos-sessions gone, the message clear: She would not be comingback. But a burst of energy, too; she was home.

Later, they drove by the yard of a favorite attendant to checkon his garden. They stopped at a vegetable stand and boughtpeaches. Then, back to the new place, tired but content.

Bonnie Blackmore never again left her bed.Just over a week later, after returning from a scientific con-

ference in London, the researcher got a call that his motherwas very sick. It was Sept. 12.

Before starting the drive to Minnesota, he spoke to her onthe phone. She was too weak to talk. He told her she could stopfighting. He would be OK.

I lied, he said.

Two weeks after his mothers death, Blackmore spoke at thespinal cord symposium in Boston. He made no effort to camou-flage his heavy eyes and melancholy voice. Scientists, himselfincluded, didnt get it done, not in time for his mother and

Dawn Blackmore knew her husbands moods. He needed tobe by himself.

In graduate school, using paper route money that hed savedand invested in a stock fund, Blackmore had bought a small,spartan cabin near Spooner in northwestern Wisconsin. Now,his wife suggested he go. Actually, she insisted.

On the drive up, the bleak, wintry landscape mirrored hismood. Finally, he arrived at the one-room A-frame on the edgeof a small bluff overlooking a river.

Surrounded by woods. No electricity. No running water.Miles to the nearest house. It was quiet and beautiful.

That weekend, Blackmore chopped wood. He made a fire andread by it. He cooked and ate.

Being alone wasnt hard. It was a relief. No demandsweighed on him.

On the drive home, a crazy idea came to him, a new kind ofvirus that he could use in experiments. As he drove and toyedwith the idea, he couldnt wait to get back in the lab.

Blackmore applied again to NIH. This time, he learned thatthe new application had received a higher score, putting himon the borderline between approval and rejection. He felt thefamiliar disappointment, assuming hed come close only tomiss once more.

Then, early in the spring of 2013, he saw the email from NIHin his inbox. The grant had been approved: $1.6 million overfive years.

Each dollar is a vote of confidence, he recalled telling thestaff at a celebratory dinner. Weve gotten a lot of votes of con-fidence. Now I expect you to turn it into real results, real pro-gress.

Its time to walk the walk.

The professors mother had lived more than two decades as aquadriplegic. In the fall of 2011 she was 69 and still in her ownhouse in St. Paul, Minn. Home attendants watched her aroundthe clock, but finding reliable help had become a problem.

When it came to hiring, Bonnie Blackmore had a soft spot forpeople whod led hard lives. This time her empathy got her introuble. She wound up with abusive caregivers who refused toleave the house. Murray Blackmore had to call police to re-move them.

His mother suffered from pressure sores and bouts of infec-tion. She struggled to cough, as she had since her injury asmost quadriplegics do. Muscles needed to cough had been de-stroyed. Murray, please cough me, she would say, and hedlift her using the Heimlich maneuver to force the air out.

Yet she remained determined to stay in her house.She always said, If I go to an institution, exit stage left,

Blackmore recalled. That was her code for throwing in thetowel.

Shed become very particular about her care. There was on-ly one service station in town she trusted to supply the gaso-line for her specially equipped van; it was miles from home.When her son or others cooked, the carrots had to be cut a cer-tain way, a specific angle and thickness for each slice.

As hed done in the past, Blackmore mostly kept his moth-ers injury to himself at Marquette. He told his students onlywhen her health worsened in 2013 and he began making the350-mile trip from Milwaukee to St. Paul more frequently.Whenever he drove to one city, something demanded his atten-tion in the other.

In Milwaukee, his family was packing to move to a new


A mouse with an impaired right front paw has trouble pulling itself forward on a ladder. The mouse was given a slight injury so researcherscould test whether treatment with a certain gene would help axons grow after a spinal cord injury.


A mouse with an impaired front paw tries to grab a food pellet butsends the pellets flying away.

many others, he told the audience. They must move faster.Back in Milwaukee he found it hard to move very fast. Weeks

passed. He felt listless in the lab. Some days he sat in his office,just staring. Nothing felt important. He kept asking: What isthe reason to do anything?

Im not proud of it, but Ive never had such a profound lackof motivation, he would later say. On some level, my pointhas always been to earn my moms approval. And suddenlythis whole context is gone. The trophy is gone.

He puzzled over conflicting observations about their rela-tionship. He read her journals and found his name mentionedjust once in 12 years, as a participant on a trip. And yet, herfriends approached him at the memorial service to say howproud of him she was. They surprised him by knowing somany of the details of his career.

Still he wondered: What is the reason to do anything?The only answer he came up with was: Other people need me.It didnt seem like much, but it was enough. Slowly, his na-

ture began to reassert itself. What Im passionate about, hed said, is solving a prob-

lem.The trophy was gone; the problem remained.

Now that he could make brain cells fire using laser light,postdoctoral fellow Naveen Jayaprakash was asking the nextquestion: When the brain cells talk, are their messages beingreceived in an injured spinal cord?

The new experiment required a more elaborate setup. He an-esthetized an injured mouse and stretched it out so that its ex-posed brain rested beneath the laser. Carefully, he positioned atiny electrode in the animals spinal cord, which was also ex-posed.

Then, in the dark, cramped room, he triggered the laser, hiseyes shielded by goggles from the fierce, blue light.

On a side table, a laptop recorded the electrical activity inthe spinal cord. Methodically he moved the electrode to differ-ent positions up and down the spinal cord so that he could tellwhether messages were being received both above and belowthe injury site.

Day after day, Jayaprakash sat in that room for up to 12hours, so long that his head ached and his eyes squinted whenhe walked out into the hallways fluorescent glow. It took a fullday to examine a single mouse.

There was more at stake than simply testing the new tech-nique. Some of the mice had been treated with a gene calledSOX11. In previous experiments, the gene had helped axonsgrow after a spinal cord injury. Until now, no one knew for surewhether the new axons actually worked.

In early October, after days of experiments, Jayaprakashtold Blackmore what hed discovered. When he fired the laserinto the brains of three animals treated with SOX11, all showedelectrical activity below the injury site. The three control ani-mals showed none.

The data still needed to be analyzed, but Blackmore did nothold back.

Thats huge, he said.He and Jayaprakash were already assembling graphs and

photos to illustrate the paper they would write. It would pre-sent the first proof that a reborn axon can carry messages fromthe brain to the spinal cord, Blackmore said.

At least a few of the downed wires could be repaired.

The professor had told his team that bad things in the labcome in waves, but so do good things.

HHEX, so promising in a dish, so disappointing in a mouse,experienced a resurrection of sorts. The lab had confirmed thatthe gene was present in adult brain cells. Then the scientistsdiscovered something odd. When they looked for the gene inearly embryonic neurons it wasnt there, at least the test failedto detect it.

Could this explain why an axon in the embryo repairs itselfearly on, then loses that ability?

The question rose in importance when the scientists learned


Laura Thiel (from left), Ishwariya Venkatesh, Murray Blackmore and Denise Coley work in the lab at Marquette.


Murray Blackmore talks about axon regeneration at the Working 2Walk Conference in Seattle in October. The conference has beenBlackmores favorite over the years because spinal cord injuryresearchers mingle with the people they are trying to cure.

FAMILY PHOTO

Blackmore andhis mother, Bon-nie, stop for aphoto in thebackyard gardenon Bonnies lastvisit to her for-mer home in St.Paul, Minn.

that HHEX switched off another gene, a growth promotercalled MYC. They had not known this when they tested HHEXon injured mice. Maybe thats why it had not worked.

Only one experiment could put to rest the questions aboutHHEX. They would have to knock out the gene in a mouse andsee if the animal regained the ability to repair broken axons.

Its a completely novel gene, Blackmore told his lab mem-bers. Were presenting something brand new to the world.

Hope tended to swing like the needle on a bathroom scale, asthe saga of HHEX showed. When researchers tried out a theo-ry, the needle jumped from one extreme to the other: success tofailure. Only after more experiments would the needle settleinto the middle ground of what is true.

The swings could be demoralizing. Kristen Winsor, a senior,had learned this lesson with a series of experiments using cellscalled astrocytes in an attempt to pierce the scar tissue from aspinal cord injury. After riding a wave of good early results,shed endured problem after problem, until one day she toldlab members that the technique had failed and shed be work-ing on something else.

That something else turned out to be an enzyme calledCHASE. The enzyme cuts through one of the structural fea-tures of the scar tissue. Blackmore had engineered his ownversion.

In the summer, when everything was going wrong, Winsorkept reminding herself why she walks through the lab dooreach day. Her mother has diabetes; research into insulin is thereason her mother is still alive.

Even if there is no cure, Winsor said, we dont acceptthat.

By fall, her CHASE experiments were working consistently.She and Blackmore even discussed the possibility of teamingCHASE with the gene HHEX to grow axons able to breakthrough scar tissue.

Recently another student in the lab had begun screening thenext batch of promising genes. The search would continue.

Blackmore entered the ballroom at Seattles Hilton Hotel ona Friday morning in October and was quickly met by Chet Mo-ritz, a young scientist from the University of Washington.

Moritz attended last years symposium on spinal cord inju-ries in Boston. He remembered Blackmores talk. The Mar-quette researcher had seemed downcast, not himself. WhenBlackmore shared the news of his mothers death with the au-dience, the sadness made sense. He looked better now, Moritzsaid.

Blackmore settled into a chair near the front. The annualWorking 2 Walk Conference has remained his favorite over theyears because researchers mingle with the people they are try-ing to cure.

When youre at this conference, its a place to be honest,said Marilyn Smith, executive director of Unite 2 Fight Paraly-sis, one of the events organizers. This is the place to ask hardquestions.

Over the next two days, there were X-rays of spinal cords andreports on clinical trials; videos showing injured rats walkingand injured humans controlling assistive devices with theirthoughts. There was also film of a young man in a wheelchairdiscussing the loss of sex, perhaps the most powerful humansensation erased by a spinal cord injury.

In the ballroom, the word wheelchair seemed vague giventhe diversity of injuries and chairs. Some of the attendees pow-ered their chairs with muscular arms, others with electric tog-gle switches. Still others, the most severely injured, controlledtheir chairs by breathing into a tube.

In the ballroom, it was OK to laugh at the absurd indignitiesof paralysis. A man named Barry Long, the master of ceremo-nies for the conference, described getting a ticket for parkingin a handicapped spot, then rolling his wheelchair into CityHall for an explanation.

Your disability expired in March, the clerk told him.My wife is going to be so happy, he replied.

Blackmore waited nervously, wishing he were already onthe third slide.

On good days, that third slide of the PowerPoint is when hesettles down and the presentat

murray's problem needs letter

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