John J. Medina, PhD

By the time you read this, most of your patients will have made and broken their New Year’s resolutions many times over, and the reason is well known, mostly because of its subject: weight loss. More than 85% vow to make lifestyle changes, many of these involving a commitment to lose weight. Even though creating a resolution has been shown to be the most potent strategy for losing weight, many still fail. “If they could only make a fat pill!” is a cry heard around the New Year from many overweight Americans-estimated to be two-thirds of the current population.

They are not about to get their pharmacological wish, unfortunately, for we are nowhere near making a medication guaranteed to melt off the pounds. Yet there is every reason to be optimistic that one might eventually become available.

Here I discuss a promising finding: the discovery of a mechanism that makes mammals resistant to weight gain even in the face of a high-fat diet. To understand this progress, we will unsurprisingly review some basic biology of the hypothalamus and discuss the interactions of 2 very interesting molecules-a protein called synaptotagmin 4 (Syt4) and perhaps the world’s most famous nonapeptide, oxytocin. Feel free to skip to the section “The data” if the paraventricular hypothalamus and calcium-independent presynaptic vesicle fusion are working parts of your vocabulary.

A review: the hypothalamus and 2 molecules

As you know, the hypothalamus is involved in the regulation of body weight. Specific collections of neurons within this multitalented region are host to a bewildering number of metabolic feedback loops, mediating their effects on our weight maintenance through the release of specific neurotransmitters. These appetite-specific regions include the paraventricular hypothalamus and the supraoptic nucleus, both of which secrete oxytocin.

Oxytocin has received a lot of press over the years, garnering popular attention because of its involvement in social cognition. It has been shown to mediate feelings of trust in mammals ranging from voles to newlyweds. But oxytocin is also involved in uterine contractions for birthing mothers (the drug pitocin is a synthetic form of oxytocin) and in the let-down reflex for nursing ones. This famous molecule triggered the 1955 Nobel prize in chemistry for Vincent du Vigneaud.

In recent years, this versatile polypeptide has been implicated in obesity, at least in mouse models. The key finding is that a reduction in expression of oxytocin leads to fat animals. The mechanism involves a feedback projection, mostly from the paraventricular hypothalamus back to the hindbrain. This is a big deal because this neural highway is involved in meal size regulation. Even more recently, the neurons that secrete oxytocin have also been shown to express an enzyme made from a variant of a gene called 

. First isolated by a group of researchers interested in type 2 diabetes mellitus, this variant has been positively correlated with human obesity. People with 2 copies of this allele have almost a 2-fold higher rate of obesity than those who do not.

Another molecule we need to review is Syt4. To describe the function of this protein in obesity, we need to briefly review the secretory mechanisms that lead to neurotransmitter release, a process in which the synaptotagmin family of molecules is deeply enmeshed.

As you may know, neurotransmitters are stored in grease-lined vesicles, localized to 2 places in presynaptic neurons. Some are in the so-called active zones, close to the cell membrane, where release will eventually occur. Many are stored immediately behind these zones, held in place by a protein scaffolding whose components are collectively called vesicle-associated membrane proteins.

Neurotransmitter release occurs when the vesicles in the active zone fuse to the presynaptic membrane and dump their contents into the synapse. This fusion, triggered by an influx of calcium, is mediated via a group of proteins collectively called the fusion complex. An important member of this complex is synaptotagmin, whose job is to sense calcium and stimulate the final few steps of neurotransmitter release. Without synaptotagmin, there would be no neurotransmitter discharge.

Syt4 is one member of a family of synaptotagmin proteins, but it is the black sheep of the clan. Syt4 can-not sense calcium and, as a result, down-regulates synaptic release of neurotransmitters. This inhibitory function will prove to be an important component in our obesity story (more on that in a minute).

To understand the obesity data, we need to review not only the molecules but also how they were used in the lab. The first has to do with an old and well-established genetic engineering protocol known as “knock-out” technology. Without getting bogged down in the details, it is possible to create laboratory animals (mice) that are perfectly normal in every way except one: a specific gene has been knocked-out of their chromosomal complement. Assuming the animals can survive without the gene, the DNA’s function may be inferred simply by examining any anomaly the animal presents. It is possible to create animals with either one or both genes missing. In our story, both copies of Syt4 have been knocked out, creating an animal termed “syt4−/syt4−.”

The second bit of technology involves another review of your basic molecular biology coursework. You recall that most genes have an on/off switch. This switch, just a specialized patch of DNA (usually at the head of the gene) is called a promoter. If you want to express oxytocin, you will need to turn on the oxytocin promoter, and if you want to express Syt4, you will need to turn on Syt4s. If you want to turn on Syt4 using signals that normally incite oxyto-cin expression, you will need to do some swapping. Simply stitching the promoter of oxytocin onto the structural gene of Syt4, then introducing this modified gene into whatever cell that interests you, will do the trick. The insertion is often done by loading up the hybrid construct onto a virus, then allowing the virus to infect the cell. The signals that nor­mally drive oxytocin expression may also drive Syt4. (See the 

With these background comments in mind, we are ready to discuss the findings. There are 5 observations that make up the bulk of the work.

. This story starts on an associative hint: researchers noticed that specific hypothalamic neurons were chock full of Syt4 proteins in wild-type animals, and they had some pretty elite special company. Syt4 was colocalized exclusively with neurons that express oxytocin. This expression profile was so specific (eg, Syt4 was not expressed in neighboring neurons expressing oxytocin-associated molecules such as vasopressin) and the role of oxytocin in weight gain was so well established that it was tempting to investigate the role of Syt4 in obesity. Fortunately for our story, the researchers yielded to the temptation.

. But does this association mean anything functionally? The simplest experiment in obesity research is to feed mice a high-fat diet, then see what happens to their brains. That’s exactly what the researchers did. They looked for changes in Syt4 expression patterns in the presence of such caloric exposure in those oxytocin-hypothalamic cells-and hit pay dirt. Syt4 was indeed elevated in the mouse hypothalamus with the introduction of the enriched food.

. What if the mice did not have Syt4? Would that make them fat-resistant? The researchers next created syt4−/syt4−knockout animals. These animals were then fed the high-fat diet that makes most mice obese. The researchers hit pay dirt again, and with a negative result. These animals did not become obese when fed the high-fat diet. They were, in fact, completely protected! Indeed, it appeared that Syt4 played an important role in the ability of enriched food to make animals fat.

. What about that pesky oxytocin? Using the promoter-swap technology, the researchers made a Syt4 structural gene with an oxytocin promoter. This had the effect of rendering the gene sensitive to oxytocin signals, over-expressing Syt4. The investigators then used a virus to deliver the construct to oxytocin neurons in the knockout animals and unengineered controls. Regardless of the genetic background, over-expression of Syt4 led to body weight gain. The researchers could reverse the protective effects of the syt4−/syt4−animals. In fact, they could turn the effect on and off like a lightbulb.

. The above experiments could be done because of another observation regarding oxytocin: it had been previously noticed that the Syt4 knockout mice had elevated levels of oxytocin compared with controls. In fact, when the researchers gave these knockout mice a high-fat diet, the serum levels of oxytocin were almost tripled compared with controls.

Was the “normal” job of Syt4 to prevent oxytocin release? Given its natural (although unusual) inhibitory role, that would make a great deal of sense. Through another complex series of experiments, that is exactly what the researchers found. Syt4’s day job may be to prevent oxytocin release in the hypothalamus. Perhaps that is one reason why the 2 molecules are colocalized. To show this in a most elegant fashion, the researchers gave the knockout animals a drug that can block the action of oxytocin (a receptor antagonist). To the researchers’ delight, the protective effect vanished. These knockout animals gained weight when fed a high-fat diet.

The finding is quite clear: Syt4 is profoundly involved in the mechanisms that control high-fat–mediated obesity. It does so by regulating the release of oxytocin.

These data outline a previously undescribed mechanism for understanding the molecular relationships between the hypothalamus and high-fat diets. Do they also hint at the creation of a “fat pill”? If you could inhibit the expression of Syt4 pharmacologically, would you make people resistant to the obesogenic effects of a high-fat diet? Might they then, quite literally, have their cake and eat it too? They could consume all the fat they wanted and not get obese!

The consummation of that fantasy is clearly a long way off, given these data. The usual caveats and cautions with which readers of this column are familiar apply. They range from “there needs to be a lot more work in these animals” to “mice aren’t humans.” The point is simply to demonstrate that with a powerful com­bination of techniques, we are dramatically increasing our understanding of the molecular relationship between diet and obesity. I have no doubt that a “fat pill” will eventually be created, perhaps on the basis of this mechanism.

To be frank, however, I am not sure that everybody will need it. We already know how to keep people from being fat. They just have to keep their New Year’s resolutions.

 Zhang G, Bai H, Zhang H, et al. Neuropeptide exocytosis involving synaptotagmin-4 and oxytocin in hypothalamic programming of body weight and energy balance.

Articles

This article outline a previously undescribed mechanism for understanding the molecular relationships between the hypothalamus and high-fat diets. Do they also hint at the creation of a fat pill?

The Molecular Biology of Weight Loss: An Unexpected Linkage Between 2 Molecules

This column has always been about the world of molecular mental health research. 

, I discussed how adult stem cells were being used to aid in research on a neurological disorder (spinal muscular atrophy). I revisit the technology in this column, now aimed at one of molecular neuropsychiatry’s most intractable, frustrating lines of research: the molecular/cellular basis of schizophrenia.

I use the word “frustrating” because a biological explanation for the disease seems heartbreakingly just out of reach. Schizophrenia has a powerful genetic component (heritability percentage is in the low 80s), something I’ve known for years, something that could make it low-hanging research fruit. There is also a large clinical base on which to do studies: schizophrenia afflicts millions of people (the estimated prevalence rate is about 1% of the global population). Despite these seeming advantages, a molecular mechanism capable of describing all aspects of schizophrenia has almost completely eluded researchers.

There’s a simple reason for this. A deep understanding of schizophrenia at such an intimate level has been hampered by a single technical bottleneck: the lack of a robust 

That may all be about to change. The results from a study that used cells derived from a deceased patient’s skin tissue has recently been published.

 Findings from the study may provide just such a model. It is not yet full-fledged schizophrenia-in-a-dish, but the findings portend a powerful future for the field. I’d like to tell you what happened. I’ll start with a brief review of hiPSCs (human inducible pluripotent stem cells), then move to the data.

August 2006 is a landmark in the field of regenerative medicine. An article published that month from a group in Kyoto, Japan, described how to take garden-variety mouse skin cells and turn them into pluripotent stem cells.

 It was reported that stem cells have an ability to transform themselves into different cell types, depending on the tissue, essentially at the scientist’s whim.

Researchers had been trying to harness this power for a long time, and for good reason. Such technologies could be useful for screening the effects of drugs, valuable as replacement therapies for damaged tissues, and relevant to this article on creating disease models. The cells are formally termed “iPSCs.” I received a further shock when a year later, the same research group did the same trick with human cells.

Four separate gene sequences are loaded onto genetically reengineered retroviruses (members of the genus lentivirus, which includes HIV). These viruses are allowed to infect a host skin cell, and because they are retroviruses, they immediately integrate into the host cell’s DNA. For reasons not fully understood, this integrated gene combination goes right to work reprogramming the skin, transforming it into a batch of versatile iPSCs. One of the most interesting of the 4 retroviral-stitched sequences is called 

, which before this work was mostly known for causing cancer.

Once there was a reliable way to manufacture healthy hiPSCs, scientists could turn their attention toward their most important goal: learning how to create the cell type they wanted to study. It did not take long for researchers to discover how to turn hiPSCs into functional neurons. It took a slightly different combination of genes stitched onto their retroviral transports (

). iPSCs were infected with this combination and, voil, functional neurons began to grow in a dish.

With these technologies in mind, I now have the tools needed to understand how to create a dish-bound model of schizophrenia. It involves answering some simple questions: What if you took the skin cells from patients who had schizophrenia and turned them into neurons? Would they exhibit behaviors of typical, healthy cells? Or would they exhibit behaviors reminiscent of previously determined properties of neurons in patients with schizophrenia? If the latter were observed, would you have a robust cellular model of schizophrenia, the missing link in this line of work? A consortium of researchers from Pennsylvania, San Diego, and New York decided to pool their resources and find out.

Primary fibroblasts derived from postmortem samples of patients with a formal diagnosis of schizophrenia were obtained and cultured. These were infected with retroviruses using iPSC protocols such as the ones described above. Would skin cells from diseased patients make iPSCs that were similar to fibroblasts obtained from unaffected people? They did indeed. Once obtained, these iPSCs were even reprogrammable, and soon either neurons or NPCs (neural progenitor cells) from deceased schizophrenic individuals were growing in the dish.

The most interesting result came from what happened next. Even though the reprogrammed cells were clearly neural tissue, they did not behave like typically functioning neurons. Several observed differences were eerily similar to previous findings other researchers had seen in tissue samples from patients with schizophrenia.

Neurons derived from patients with previously diagnosed schizophrenia exhibit specific, aberrant properties. Postmortem examination of neural tissue reveals a significant reduction in dendritic arborization in many patients, which may result in aberrant neural connectivity and subsequent behavioral abnormalities.

This same reduction was observed in the reprogrammed cells. The cells showed a decrease in neurite numbers (a neurite refers to any projection from a neuron’s cell body). This resulted in a loss in neural connectivity in these same populations. It is interesting to note that the application of the antipsychotic loxapine to these cells significantly restored their neuronal connectivity. (Four other antipsychotics did not.)

It should be noted that these cells were all fully capable of spontaneous neural activity. In response to normal membrane depolarization events, for example, these cells displayed typical inward sodium currents (transient) and more sustained outward potassium currents.

A number of genes whose aberrant expression profiles are associated with the disease state in other individuals showed up in these reprogrammed cells. One of the most dramatic was an increase in 

 is part of a group of glycoproteins in the epidermal growth factor family. This elevation was not seen in the cultured fibroblasts or in the iPSCs (nor was it seen in the NPCs). Why is 

 important? Previous work has shown that mutations in this gene were highly associated with the prevalence of schizophrenia in Icelandic populations. The specific involvement of neuregulin in these cells thus represents an important finding.

There are techniques that allow researchers to assess the “gross domestic product” of gene expression profiles in given cell populations (essentially a global assessment of class 2 gene expression). Such analysis was performed, and findings were compared with those from nondiseased controls and with genes previously implicated in schizophrenia. It was found that 25% of the genes differentially expressed in the reprogrammed cells had been previously implicated in schizophrenia. These were not trivial genes. Further analysis (gene ontology examinations) demonstrated changes in glutamate signaling, as well as WNT and cyclic adenosine monophosphate signaling pathways.

The bottom line is that many of the major cellular and molecular aberrations previously demonstrated in diseased tissues were showing up in these reprogrammed cells. A robust living cell model, derived exclusively from postmortem samples from patients in whom schizophrenia had been diagnosed, seems to have been achieved.

Although this is truly a significant result, there are specific caveats. Some have to do with the fact that relatively “simple” tissues growing in a dish do not mimic the complex, real world of the living brain. Other objections have to do with the technology.

iPSCs are notorious for carrying mutations that may have very little to do with the real 

 world. Retroviruses insert at random DNA sites in the genome, which introduces the risk of gene disruption. Moreover, genes such as the aforementioned 

 greatly increase the efficiency of reprogramming cells, which means they are nice to have around if you are doing this type of research. However, 

is also an oncogene, which means it is a sequence capable of inducing cancer. Does that introduce any uncontrolled variables? At this point, no one knows.

None of these objections seriously undermine the great potential of this result, of course. Having a dish filled with cells that carry many characteristics of a human disease is a lot like having a flashlight in a dark cave. The greatest utility is in the ability to illuminate molecular mechanisms that might go undetected without such a model. It can go a long way toward relieving the frustration often associated with this line of work. Give it enough time and it might even-someday-illuminate a cure.

 Brennand KJ, Simone A, Jou J, et al. Modelling schizophrenia using human induced pluripotent skin cells. 

 Takahashi K, Yamanaka S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. 

 Takahashi K, Tanabe K, Ohnuki M, et al. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. 

 Sendtner M. Regenerative medicine: bespoke cells for the human brain. 

 This column has always been about the world of molecular mental health research. I revisit the technology in this column, now aimed at one of molecular neuropsychiatry’s most intractable, frustrating lines of research: the molecular/cellular basis of schizophrenia.

Modeling Schizophrenia: An In Vitro Model of a Tough Disease

I admit to having 2 mixed responses when I rented the 1992 movie 

 The first reaction didn’t concern the film at all, but something I had read about in a science magazine earlier that day. Eighteen-year-old 

 had just died, the first person to succumb during a human gene therapy trial. Gene therapy (sometimes called gene replacement therapy) attempts to ameliorate genetic-based disorders by introducing corrected genes into affected patients. Jesse suffered from ornithine transcarbamylase deficiency, an X-linked genetic disorder that results in the inability to metabolize ammonia. It is invariably fatal.

 gene had been injected into Jesse’s bloodstream, with hopes of restoring this metabolic activity. Unfortunately, he mounted a catastrophic immune response to the modified adenovirus and died of multiple organ failure. A flurry of investigations ensued, the trial was halted, and the family was eventually paid an undisclosed amount of money. Having worked with gene therapy technologies early in my career, I felt this disappointment acutely.

But back to the movie, and my second mixed reaction. Lorenzo’s Oil was based on the true story of 

, a boy suffering from a different X-linked genetic disease-adrenoleukodystrophy (ALD). ALD is a severe neurodegenerative disorder with no cure and, at the time, no successful treatment options. The movie concerned the efforts of Lorenzo’s parents to find an effective treatment for their son. They eventually hit upon a dietary supplement derived from olive oil-hence the title-a combination of 2 long-chain fatty acids. They had reason to believe it would help their son, and the film documents their struggle trying to convince skeptical scientists, friends, and clinicians.

The bad news was that the establishment initially resisted their attempts to obtain a therapeutic distillate of the oil for their child. The good news was that the parents eventually found a professional who would create the distillate. Astonishingly, the compound halted the progression of the disease (the actual Lorenzo died last year of aspiration pneumonia, having lived to an incredible age of 30 years). In this film, members of the scientific community were portrayed as churlish, narrow-minded, and subject to group-think. We can be all these things, of course-research is a very human enterprise-but coming on the same day as the report of Jesse’s death, it didn’t feel like a very good day to be a scientist.

Happily, gene therapy technologies have undergone a powerful revolution. Since Jesse’s failed trial, additional safety procedures and oversight activities have been put into play. Existing experimental protocols have been modified. Combined with technical advances in gene replacement strategies, the changes have resulted in a number of clinical success stories. One extraordinary achievement occurred this fall and involved ALD, Lorenzo’s disorder. The achievement brought back a reminder not only of the day I saw the movie but also of how far we’ve come since then. I want to tell you about what happened. I start with some background information about ALD, then move to an experiment that, at this writing, shows enormous promise.

ALD is a demyelinating disease of the CNS. The mutations responsible for the disorder occur in a gene called 

, which encodes an adenosine triphosphate–binding transporter protein. The protein normally hangs out in the membranes of peroxisomes of both oligodendrocytes and microglial cells. In its role as a peroxisomal transporter, it happily degrades very long-chain fatty acids in these cells. A loss of this function inhibits normal myelination maintenance by these cells.

Not surprisingly, active multifocal brain demyelination occurs in affected children, with an active degradation phase that manifests around age 6 years. Death usually occurs before puberty. It is a remarkable tribute to Lorenzo’s parents that their son lived so long.

Once there is onset of the lesion phase, it is impossible to stop the degradation, which is why therapeutic interventions are best initiated at the earliest ages possible. The only effective treatment to date is hematopoietic stem cell (HSC) therapy, classic stem cell transplantation therapy. It works by supplying replacement microglial cells derived from donor bone marrow myelomonocytic sources. There are limitations to this therapy, of course, ranging from the limited pool of compatible donors to its high fatality rate.

 gene with a healthier version in affected children would be the gold standard treatment option. Since you have a brain delivery system already available with HSCs, what if you could put a corrected 

 gene into those stem cells, then allow the cells to migrate via those normal pathways into the brain? What would be the first step?

Anyone contemplating gene therapy manipulations begins with the choice of molecular transport vehicles. These are normally genetically re-engineered viruses fashioned into benign vectors, each capable of delivering corrected genes to the patient.

Many laboratories have used mouse gammaretroviruses as the vectors of choice. The big problem is that they will only transduce (supply the foreign gene) dividing cells. Since Jesse’s death, researchers have genetically engineered HIV-1 viruses to do the shuttling (

). Although they have been made replicatively defective, these viruses readily transduce nondividing cells. This property makes them the vector of first resort for many laboratories interested in gene replacement studies. Modified HIV-1 viruses are remarkably efficient at infecting HSCs. Once inside these cells, a certain percentage will express whatever foreign gene is on board, setting up a long-term presence of gene product in HSC lineages.

The question, however, remains: Would this work for patients suffering from ALD?

Seemingly simple-the introduction of the foreign gene into HSCs-the initial leg of this journey is a cautious one. If integration is successful, the next step is to determine what happens after transplantation. Once the cells stabilize in the patient, then one would start looking for clinical effects. That’s exactly what teams of researchers-some from Europe, some from the United States-have undertaken to do.

They were given permission to treat 2 ALD-affected boys, aged 7 and 7.5 years. The children had no matched donor available or cord blood for a ready sources of HSCs, which made them ideal candidates for a gene therapy trial. In the laboratory, autologous HSCs were transduced with an HIV-1 vector genetically engineered to express healthy 

 genes. After myeloablative treatment, these HSCs were reintroduced into the patients.

The boys were followed up over the next 2 to 3 years. The researchers were looking not only to see whether the stem cells “took” but also to determine how many of them were expressing the corrected protein. To their excitement, the researchers found healthy 

 proteins expressed in a wide variety of HSCs, including monocytes, T and B cells, granulocytes, and bone marrow progenitors. It wasn’t a great percentage, hovering between 9% and 14%. Would it be enough to produce a positive therapeutic outcome? The answer was yes! More than a year later, progressive cerebral demyelination in both boys stopped.

This was quite an achievement. Despite its relatively low level of correction, the boys’ brains were getting better-or at least stopped getting worse. The results are similar to those in ALD patients who have had the good fortune to find a donor source and subsequent transplantation. In a single stroke, a positive effect had been produced in patients with no such advantage. It had been done with a technology previously under a cloud, with a disease whose research efforts had been couched as negative high drama.

Perhaps we have come a long way after all, I thought. After hearing about the breakthrough-and recalling Lorenzo-it turned out to be a remarkably good day to be a scientist.

 Gene therapy (sometimes called gene replacement therapy) attempts to ameliorate genetic-based disorders by introducing corrected genes into affected patients. 

Lorenzo’s Oil and the Rehabilitation of Gene Therapy

Elegant Knockdowns, DISC1, and Schizophrenia

It is ironic that an attempt to do a molecular end-run around a politically hot topic could result in an important breakthrough in the treatment of neurological disease with potentially strong implications for the psychiatric community. Ironic maybe, but true.

In this column, we explore how the judicious use of neural stem cells (NSCs) has led to a research Holy Grail: the creation of research-ready, patient-specific neurons. This technology did not use the famously controversial 

 stem cells. These custom-made NSCs were created from politically neutral 

 tissues (fibroblasts), which were originally isolated from an affected patient. With no embryo in sight, scientists genetically reprogrammed fibroblasts into stem cells, which were then induced to develop into NSCs. This is an extraordinary finding with many topics to be discussed here:

• The potential research utility for patient-specific neurons

• An explanation of how stem cells can be made from adult tissues

• A striking set of results that involve one of the most commonly inherited and lethal childhood neurological disorders: spinal muscular atrophy (SMA)

Of what possible utility could molecular investigations of a motor disorder have for the mental health community? Before getting into the specifics of the breakthrough, it might be useful to address a real-world psychiatric need, using depression and SSRIs as an example, to see where these data fit.

When we consider the molecular mechanisms of SSRI interactions, it is easy to resort to commonly taught ideas about interactions that involve a single synapse. Nothing could be further from the truth. The most comprehensive neurological view of SSRI interactions must take into account the participation of thousands of individual neurons strung together in coordinated, complex neural networks.

And not just serotonergic neurons. These cells are in contact with many other central nervous denizens, from adjacent glial cells to the extracellular matrix into which the cells are embedded. What do these circuits actually look like in patients who are vulnerable to depression? Is their architecture all that different from patients who do not exhibit this vulnerability? If there are differences, could they eventually predict drug efficacy? Could these differences only be detected by constructing parts of the circuit from scratch, or could they be observed at the level of a single cell?

The first step in answering these questions involves growing a custom-made batch of serotonergic neurons derived only from the affected patients, and then asking relevant structure/function questions. From attempting to understand molecular mechanisms of disease to testing the efficacy of potential medications, such patient-specific test beds would have a powerful research utility. Until recently, the creation of such tailor-made neural substrates had been an impossible goal.

While it will certainly be quite some time before we can grow entire parkinsonian dopaminergic pathways in a dish, it is now possible to create individual patient-specific neurons in culture. The technology comes from that end-run I mentioned earlier, through the use of a certain type of stem cell. It is to these interesting cellular substrates that we now turn.

To say that embryonic stem cell research has been subject to heated political debate is an understatement. The bugaboo has been the source materials from which the stem cells would be isolated-human embryos-many left over from embryos generated in in vitro fertilization laboratories.

In 2006, researchers found a way to create stem cells that bypassed the need for human embryos. The original technique involved the introduction of 4 specific gene products into mature mouse fibroblasts. Surprisingly, this cocktail was found to reprogram adult stem cells and reverse-engineer them into pluripotent stem cells. Like embryonic stem cells, the altered stem cells had the ability to differentiate into any cell type. Eventually, a protocol was developed that did the same thing in human tissues. The cells were called iPSCs, short for induced pluripotent stem cells.

This was quite a breakthrough. No longer would researchers need to harvest cells from extant human embryos to do stem cell research. Skin cells would do. Scientists were soon able to regenerate-and then correct-molecular dysfunction in a mouse model of sickle cell anemia using this technology.

Could any of this work apply to humans, specifically to human neural tissue? Another successful round of experiments (with amyotrophic lateral sclerosis neurons) prompted researchers to study motor disease, ie, SMA.

Of those hereditary neurological disorders capable of causing death in pediatric populations, SMA is easily the most common. The disease is unique to humans and associated with 2 genes, 

. For reasons that are not well understood, the absence of the survival motor neuron (SMN) protein results in an alteration of the function of spinal motor neurons. The primary feature is muscle weakness and atrophy. Death occurs at infancy in the most severe forms of the disease, with symptoms generally presenting several weeks after birth. There are many other, nonlethal forms of the disorder, however, with a wide spectrum of symptoms that range from trivial motor effects to catastrophic impairment.

Why this variation? Both genes express in unaffected individuals, but the biological heavy lifting belongs to the 

 gene. Because of structural constraints, the expression pattern of the 

 gene normally results in only 10% of its protein being processed as a full-length (and functional) polypeptide; 90% of its protein output is truncated (and nonfunctional). That is okay, as long as the 

 is mutated and silent, the disease condition results. Assuming there is a damaged 

, the severity of SMA varies according to the number of other 

 copies the infant may carry. The more copies of 

 gene, the greater the population of functional protein. This interaction explains in part why there can be so much varia-tion in the clinical presentation.

The great mystery is why SMN protein loss results in motor cell alterations that lead to the disease state in the first place. The protein is known to be essential for normal messenger RNA processing and is expressed throughout the body. Yet its absence most severely affects spinal motor neurons.

The most exacting way to attack this “black box” would be to isolate the motor neuron populations from the patient, then compare these populations with unaffected controls and look for differences, of which there are many. These include responses to various medications. It is well known that the application of valproic acid (an anticonvulsant and/or mood stabilizer) or tobramycin (an aminoglycoside) to cultured cells, for example, leads to changes in the expression patterns of both full-length SMN protein and truncated forms. What is the molecular basis of this unusual interaction? And could such differences be used as a “molecular flashlight” to ferret out other secrets regarding the SMN protein? Creating custom-made neurons-one population from an affected individual, another from an unaffected control-would certainly give a test bed capable of answering this question.

Studying these 2 populations is precisely what a group of investigators did. The researchers isolated fibroblasts from an affected child and also from the child’s healthy unaffected mother.

The next step was to generate custom-made neurons. Several steps would be required (

). First, using the iPSC protocols I mentioned, the researchers would attempt to create stem cells from both child and parent sources. If that worked, the researchers would then try to induce these patient-derived stem cells into motor neurons-ones that would carry the same biological mechanisms observed in both the diseased and the healthy populations. If successful, the researchers would have their custom-made test beds. They could begin characterization studies; reactions to valproic acid and tobramycin would make obvious first choices to try.

The first step worked. The researchers were able to generate custom-made stem cells from both child and parent. The researchers then tackled the hard part: manufacturing spinal motor neurons from these stem cell populations. They certainly generated promising cellular populations. But the iPSC technolo-gies are new enough that a visual inspection of the generated cells might be necessary-but certainly not sufficient-to show the presence of motor neurons.

There are ways to gain greater reassurance. One way to assay the success of the protocol is to look for bona fide molecular markers of developing spinal neurons. It is known, for example, that extant motor neurons express the protein SMI032 and choline acetyltransferase. Did these induced cells express such proteins? The answer turned out to be yes, both for the affected child and for the unaffected parent. Developing cells in these populations possess transcription factors such as HOXB4, ISLET1, HB9, and OLIG2 as well. Did the induced populations express these markers? They did indeed. While not completely conclusive, it appeared that the researchers had generated patient-specific motor neurons from known affected and unaffected sources.

The next characterization experiments also yielded fruit. They were able to find that the child and parent neurons reacted very differently to the normally stimulating effects of valproic acid and tobramycin. The child’s cells showed elevated levels of SMN protein, both of the truncated form and full-length version. In addition, SMN-containing nuclear structures were altered. No such elevation occurred in the unaffected maternal line of cells.

These differences were significant for 2 reasons. First, it gave the investigators a toehold in their attempts to characterize at a more intimate level the differences between affected and unaffected cells. Second, the differences were discovered as reactions to known medications. The hope is that similar approaches could be used to test the efficacy of various medications before committing to human trials.

These data, full of promising implications as they are, need to be treated with some caution. First, the experimental cells are pure populations derived from stem cells. This hardly reflects the physical in vivo situation. The cells and matrix components that normally surround such cells in nature, including skeletal muscle tissues and even other neurons, are not present in these studies.

Another objection concerns the fidelity of the conversion process itself. The differentiation pattern seen in various molecular markers hinted that the investigators generated real live spinal motor neurons; however, one cannot a priori say they have in every way created a motor neuron that precisely mimics the real-world situation. These cells may lack many subtle molecular processes-and a few extra, equally subtle interactions-that could easily escape detection, at least by current technologies. Because subtle differences can profoundly influence intracellular molecular interactions, especially when we think about reactions to medications, this is a true concern.

The most exciting aspect of these studies comes from what the future holds. A great deal of speculation has gone into thinking about how to tailor medications to individual patients. That certainly is a psychiatric issue . . . I need not talk to this audience about the variable effects of, say, fluoxetine on clinical outcomes. We have visited this topic in past columns. The ability to create patient-specific cellular test beds may go a long way toward solving some of these problems. Indeed, clinics of the future might routinely screen to decide what medications their patients should receive-and in what concentrations.

There is much work to do. To date, none has been applied to neurological systems relevant to mental health professionals. Even given the cautions mentioned above, there is no reason why it couldn’t. That’s not bad for having to do with an end-run around a hostile, politically charged issue such as stem cell debates. Would that all ethical issues could be decided so cleanly, or with so much fruit.

 In this column, we explore how the judicious use of neural stem cells (NSCs) has led to a research Holy Grail: the creation of research-ready, patient-specific neurons. 

Custom-Made Neural Stem Cells

One of the most interesting research efforts of the past few years seems to have taken a page from the preoccupations of the self-help magazine world: the cognitive decline of the brains of aging baby boomers and the obese nature of their grandchildren. These topics have been united by the well-established finding that restricting caloric intake leads to an increased life span in almost every animal tested. Because eating habits are formed early in life, the prediction is that a sensible diet will increase a person’s life span.

Is that true? The link between caloric restriction and life span is certainly solid, although the jury is still out on many of the claims made in the general media. Serious scientists have been studying the molecular biology behind this link for a number of years. One particular group of sequences seems to stand in the gap between aging and food, that of the sirtuin family of genes (also called silent information regulator [SIR] genes). One member of the family, the 

 gene, has been studied in particular detail. This article discusses age-related cognitive decline, caloric intake restriction, and the role 

It is the canonical experience of people older than 40 that senior moments become an increasingly familiar part of one’s thinking life. We forget names, we forget places, we forget where we put our car keys, and we wonder when-and why-our retrieval systems began to abandon us. Higher-order processing begins to change as well as memory, perhaps not as obviously, but apparently in a far more dramatic way. Although the types of cognitive decline clearly vary from one person to the next, no one escapes these behavioral changes completely or, perhaps, the panic that ensues for some individuals when they compare their previous talent with current abilities.

It is axiomatic that cognitive decline occurs because of physical changes that human brains undergo as they age. Yet demonstrating the specifics of the relationship has not always been easy. With the advent of more sophisticated technologies (and improvements in older technologies), that has begun to change. We now know that changes in the regulation of global gene expression patterns-mostly down-shifting-are observed in a broad swath of the CNS during aging. Gene products specifically involved in the physiological processing of inhibitory signals mediated by γ-aminobutyric acid) have shown particular vulnerability. These neuron-related changes are disproportionately large when compared with changes in other tissues (eg, kidney and muscle tissues show age-related up-regulation).

Alterations in gross neuroanatomical structure have also been observed for many years. Some of the most dramatic involve disruptions of the myelinated fibers that yoke disparate brain regions together to provide specific functions-particularly in the prefrontal cortex (PFC). The changes are generally not due to neuronal loss in the PFC, which is actually quite minimal in most aging brains, but to loss of functional connectivity. The accompanying disruptions of neural integration in these regions of the aging brain result in less organized activity than is found in the brains of youthful controls. Such alterations are thought to be associated with a measurable behavioral change in aging populations: disruption of executive function (a task involved in everything from impulse control to planning for the future). This is part of a general age-related decline in the brain’s higher-order functional abilities.

One of the challenges of studying cognitive decline in elderly populations is separating normal changes in cognition from abnormal pathological changes. Although these are not always easy to distinguish, examination of the aging hippocampus has provided valuable insights. The normal aging pattern of the hippocampus involves an inhibition of metabolic activities of the dentate gyrus and subiculum. That is not what you see in patients who have Alzheimer disease. At least initially, the inhibition primarily targets the entorhinal cortex. Neuronal death in these tissues, with a general volumetric shrinkage of the medial temporal lobe, has been shown to distinguish the disease state from typical aging processes.

Many of the data presented above appear to describe natural, typical processes. But are they inevitable? One of the first questions many people ask after going a few rounds with their senior moment brains is: can the decline be reversed? These are often the questions asked by people who want to increase their life span. The surprising answer to both questions, in a few cases described below, is yes.

One of the most remarkable discoveries in the field of life span alteration occurred in the past century and has to do with caloric restriction. (This does not mean caloric starvation; malnutrition does not provide the benefit and is a completely different issue.) A controlled decrease in the amount of calories consumed has changed the life span of a surprising variety of animals, including mammals. It truly does mean that if you eat less, you will live longer.

The benefits of caloric restriction have been shown to have brain-specific effects as well. Caloric restriction can alter the regulatory genetic down-shifting phenomenon discussed earlier. It has also been shown to change the age-related neuronal degradation in nonhuman primates. Most relevant to our story, caloric restriction can affect human cognitive functioning. In one remarkable study, a caloric restriction protocol that lasted 90 days dramatically improved the verbal memories of a healthy geriatric cohort. Caloric restriction has even been shown to inhibit amyloid-related plaque formation in transgenic mice models of Alzheimer disease.

Such robust findings work like scaffolding for researchers interested in the molecular biology behind the aging process and have certainly piqued the interest of people wanting to extend their life spans. What is the molecular mechanism behind the life span–extending properties of feeling hungry all the time? One of the earliest fruits of these research efforts was the isolation and characterization of the SIR genes.

The SIR genes were first isolated and characterized in yeast. They are a highly conserved family of sequences found in animals as diverse as roundworms and humans. One intensely studied member of this family is the 

 gene product functions as an NAD+-dependent deacetylase. In the presence of NAD+, 

 removes acetyl groups from proteins. Histone proteins are a favorite target of 

. As you may recall from your undergraduate days, histones are groups of proteins around which DNA molecules wrap themselves, somewhat like popcorn wrapped around string. Histones are deeply involved in regulating gene expression. Adding or subtracting subgroups to histones can profoundly influence the expression patterns of the genes in contact with the molecules.

 fit into the caloric restriction story? It was shown years ago that if you introduce 

 into yeast in such a fashion that you overdrive its expression, you can increase the yeast’s life span (measured as the number of times a cell completes a round of replication). If you severely restrict the caloric intake of an unmanipulated yeast, you can do the same thing. If you look for levels of 

 protein, you find that starvation has elevated its activity. The association appears to be strong, both by correlation and by direct intervention. The link between 

 protein and caloric restriction was first found in insects and then in mammals.

Subsequent research has muddied the waters of this otherwise seemingly tight story, however. Other researchers failed to replicate the results of the initial findings. Questions about various technical aspects that provided the original findings have been raised as well. There appear to be differences between genetic backgrounds in the primary test vehicles, from yeast to mice. These issues have yet to be fully resolved.

Such controversies are the nature of a good research project that, while hardly finished, has reached a certain maturity. These controversies are not deal killers regarding the association between the aging process and what goes in your mouth. The devil, as they say, is in the details. The fact that these issues can be raised at all demonstrates the enormous strides that researchers are making regarding the association between aging and eating-two of the most socially important issues of our time. That the arguments can revolve around deciding how the subtraction of acetyl groups changes the cell cycle simply shows how intimate, and how sophisticated, the progress has become.

Andrews-Hanna JR, Snyder AZ, Vincent JL, et al. Disruption of large-scale brain systems in advanced aging. 

Bishop NA, Lu T, Yankner BA. Neural mechanisms of ageing and cognitive decline. 

Haigis, MC, Guarente LP. Mammalian sirtuins-emerging roles in physiology, aging and calorie restriction. 

 One of the most remarkable discoveries in the field of life span alteration occurred in the past century and has to do with caloric restriction. 

To Sirtuin With Love: Caloric Restrictions and the Genes of the Aging Brain

Perhaps the seminal component of any clinician’s behavioral repertoire is the ability to understand the conscious motivations and intentions of their clients. This article addresses the work of conscious motivations at the neuroanatomical level.

I seldom address the notion of consciousness-let alone motivations-in this column for a very good reason. Nobody really knows what they are or even if there is a “they.” The literature is confusing, but it hasn’t stopped researchers from speculating on possible neuroanatomical and biochemical substrates that undergird the phenomena. Without a broad consensus about what is being studied, there can be no neurons, let alone molecules, for active experimental consideration. After all these years, researchers have yet to isolate an area of the brain solely devoted to the experience of consciousness. There may be none.

Given the importance of these issues to the mental health professions, I revisit the concept of motivations from time to time-but only when the data are conservatively presented, with sober, modest conclusions. The findings described here originate from experiments that have attempted to determine how we voluntarily choose to perform a motor task (action planning). This work requires reviewing background information on association cortices and the neural substrates behind a decision to initiate voluntary action.

Functionally, the cortical regions of the brain and their myriad interlocking circuits can be divided into 3 modules. These consist of front-, back-, and middle-end domains.

• Front-end functional domains are sensory information processing centers. The brain receives input from the eyes, ears, and other sensory systems. It sends the input off to various places for further processing.

• Back-end functional domains involve motor control systems. These systems essentially respond to whatever command the sensory cortices give to it (eg, execute a decision to move).

• The middle-end suite involves nearly everything other than front-end and back-end functional domains. These association cortices generally entail higher processing features and are some of the least understood and the most mysterious parts of the brain.

One such cortex, located in the inferior posterior parietal cortex, is a sensorimotor association region that links sensory stimuli to motor movement. It may even be involved in sensory prediction, which calculates the consequences of a given action through the simultaneous evaluation of input from both sensory (front-end) and motor (back-end) functional domains.

Many of the actions humans initiate on a day-to-day basis seem to depend on a kind of internal free will. This sequence of events (also known as volitional motor movement) gives humans a sense of control: we act because we want to act. That is why researchers use volitional motor movements in their research designs. Researchers interested in volitional behavior study neural prime movers behind decision making.

Exactly what does it mean to want to do something? We do not really know. The events that initiate movement occur in a fairly straightforward sequence (although it depends on the source of the signal). For example, a central processing area with directives for voluntary motor movements pass through a final staging area before the execution of an action. This region is the primary motor cortex.

Research on laboratory animals demonstrates that this cortex decides on a course of action that depends on the source of signals it receives before the execution of that action. One source originates in the premotor cortex. Signals in this area initi-ate movements in response to a specific external trigger, such as a visual cue.

The second source arises in the presupplementary motor area, which is stimulated when laboratory animals make the same movements mentioned above, but they do not originate from responses to an external source. The movement instead arises spontaneously; a thought is internally generated through intentional actions. There is an observed rapid rise in electrical signals that build up just before the brain executes these actions. This has led to the notion that the presupplementary motor area harbors some kind of readiness potential, a useful function in generating movement (

In terms of human behavior, complex human brains have many more research issues to solve than standard laboratory animal research can address. One potential confounder is conceptual. With research of this type, scientists often tell subjects to choose (or not to choose) from a variety of options. Is that voluntary? Hardly. This is like saying, “Okay, it’s time to have some voluntary volitional behavior now,” or like runners at a race who respond to the starting gun. Do volitional actions disappear in these experiments with human subjects? Are these subjects simply reacting to commands to respond, not to respond, or to respond however they want? To test volition, researchers should not control the input. Nevertheless the experimenter must, almost by definition.

Another complexity involves engineering. How does conscious intent to move an arm relate to the actual movement of the arm? This could be partially resolved with electrical stimulation mapping in which surgeons create a map of the brain on conscious patients to understand what tissues need to be avoided during certain manipulations (such as resection). No pain neurons exist in the brain. The patient, immobilized in a stereotactic frame, can be consciously interrogated while the surgery takes place. The surgeon applies a gentle electrical current to the open tissue, talks to the patient about what he or she is experiencing, and makes a map that discerns what areas to avoid during cutting. Working primarily with epileptic patients, the legendary Canadian physician Wilder Penfield first performed these techniques.

This technique has proved to be of great value in understanding volitional components of motor movement. It was discovered almost 2 decades ago that if one stimulates a specific area of the human presupplemental area, the patient will experience a conscious urge to move.

 This gets around the runner’s starting gun problem mentioned previously. An external electrical stimulator supplies a specific quantity of electricity-and a desire to do something is suddenly generated!

As important and well-characterized as these data are, they hardly explain what causes the presupplemental area to generate the signal in subjects not undergoing surgery. Some research findings answer this question and have led to some intriguing results.

When the inferior posterior parietal cortex was stimulated, the patient experienced an urge to move specific body parts. Stimulating one area caused patients to want to move their arms. Another region, the lips. Another region, the chest. This is similar to what one observes in frontal lobes, except that you are nowhere near the frontal lobes. Recall that this is the associative cortex region (a sensorimotor associative area at that), quite distinct from anything observed in the well-characterized general motor areas of the frontal lobes. Was this simply a remote stimulation?

This result showed that the answer would be no. The parietal cortex urges were qualitatively different from those obtained by stimulating parts of the presupplementary cortex. It is well known that if the presupplementary cortex is stimulated at a low current, the urge to act is acquired. However, if the same region is stimulated at high current, actual movement occurs. That’s not what happened in the parietal cortex. The urge was stimulated at low intensities, but movement was never generated at higher ones. Instead, subjects felt that they had already performed some movement.

This is important. The desire to move did not result from subtle motor contractions that may have been generated by motor regions (an alternative idea that has been put forth as a rational explanation for the results in previous experiments). Parietal stimulation never produces muscle activity, regardless of the intensity. The stimulation of the premotor cortex itself produces large-limb movements in subjects, but never the desire to move the limbs. They usually remain unaware that movement has occurred when these regions are stimulated.

These results suggest the presence of 2 specific aspects of conscious intention (however one defines it). One might be the conscious correlation of preparatory motor commands in the presupplemental cortex region, as is clearly observed in laboratory studies of animals. The other might involve sensory prediction of the consequences of those commands, under the domain of the association cortex region. A portion of conscious intent seems to be a specific class of experiences housed within the parietal lobe.

It appears that the parietal lobe contributes to the conscious experience of intention, at least in regard to motor movement. These results cement 1 more brick onto the great construction project that seeks to define intention. But they hardly hint at the overall building.

Pushing the edge of our understanding into the murky world of association cortex only means that future experiments will be trickier to interpret. Electrical stimulation mapping, as good as it is, is necessarily a blunt instrument that stimulates thousands of neurons simultaneously. Not isolated modules, these regions connect to each other in complex, little-understood ways. That the regions produce different behaviors is an important finding but not a defining one.

How do the frontal and motor aspects of volitional experience differ from the parietal, sensory versions? What factors stimulate the parietal lobes in the first place? What about remote effects?

Questions such as these remain to be answered and are just a few of the many that researchers will face as they attempt to define intentional and conscious experiences.

Penfield W, Erickson TC. Epilepsy and cerebral localization: a study of the mechanism, treatment and prevention of epileptic seizures (Review). 

 Fried I, Katz A, McCarthy G, et al. Functional organization of human supplementary motor cortex studied by electrical stimulation. 

 Haggard P. Human volition: towards a neuroscience of will. 

 Custers R, Aarts H. The unconscious will: how the pursuit of goals operates outside of conscious awareness. 

 Pushing the edge of our understanding into the murky world of association cortex only means that future experiments will be trickier to interpret.  

The Neurobiology of Conscious Intent

The neuroanatomical linkage that emerges from a normal part of business experience-the reaction to success and also to failure (especially if that failure happens to someone else)-is the focus of this column. I am sometimes asked to speak to groups of business executives, mostly to discuss a possible connection between neuroscience and business practices. These meetings are always challenging for me, because I don’t think brain science has much to do with the world of business. My own opinion is that the field of neuroscience is simply not mature enough to tell business executives how to manage their subordinates or how to lure customers into buying their products. “I have nothing real to say to you,” I usually start, “We don’t even understand how humans know how to put their socks on in the morning.”

There are usually some murmurs in the crowd at this point, but since I still have 45 to 60 minutes to burn, I continue, “My perspective isn’t hopeless, though. In fact, almost all of the brain’s neural circuitry can be easily explained-especially if you are looking at people’s interior motivations.” Then I continue with what turns into a Darwinian lecture: “People will do whatever they think will ultimately benefit them. And people will do whatever they can to avoid pain. Almost everything we know about how the brain generates behavior can be couched as combinatorial activations of these 2 broad sets of purpose-driven circuits-seeking pleasure, avoiding pain.”

The human brain as a mass of biological tissue is most clearly understood as a survival organ-the world’s most sophisticated. Given this performance envelope, a great deal of theoretical common ground exists between what we know about the brain and the needs of business. Even though not much of the brain has been mapped, my corporate audiences and I usually end up with lots to say to each other.

This month’s column is all about mapping a specific parcel of this common ground between pleasure and pain and gives a suggestion for a specific investigative direction. We will explore how a subset of these circuits supports the social experience of pleasure and pain.

There is a powerful bridge between pleasure and pain and their social equivalents; indeed, to the brain, they are nearly identical. Recent findings confirm that the same reward circuits are activated during sex and also while delighting in someone else’s misfortune (

 Similarly, both physical pain and envy over another person’s success activate these circuits.

We start with a basic review of canonical circuits normally associated with pleasure and pain, and then discuss interesting data from a collaboration of scientists in Japan and the United Kingdom. Much of the brain’s pleasure circuitry has been studied through the lens of reward reception and the establishment of addictive behavior. Invariably, this involves the neurotransmitter dopamine and a number of neural circuits that have been isolated and characterized in surprising detail.

Three networks are briefly reviewed. The first circuit involves the interaction of dopamine in neurons within the ventral tegmental area, especially in response to external rewards (eg, sexual activity, drugs, food) (Figure). Associated with these circuits, the second network comprises neurons embedded in the nucleus accumbens, within the ventral striatum. The nucleus accumbens has been shown to play a vital role in the learning of reward and the regulation of pleasurable states. The third circuit involves the ventromedial prefrontal cortex in association with the amygdala. These 3 networks are also vital parts of the dopaminergic system and are thought to mediate reward processing and the emotional responses involved in the ex-perience of pleasure.

Association never means causation. If you could somehow temporarily  deactivate the ventral striatum, would schadenfreude suddenly disappear?

The various circuits associated with mediating the experience of pain are collectively termed the “cortical pain network” (

The neuroanatomical linkage that emerges from a normal part of business experience-the reaction to success and also to failure (especially if that failure happens to someone else)-is the focus of this column.

Author | John J. Medina, PhD