Does the Mother’s Diet Affect Her Offspring?


Can what a mother eats affect her baby? Claudia Buss of the Charité – Universitätsmedizin Berlin and the University of California, Irvine and her colleagues conducted a longitudinal study of mothers and their newborn babies, and discovered that increased production of the cytokine interleukin-6 (IL-6) in mothers can lead to alterations in the brain connectivity of her offspring.

Buss and coworkers took blood samples of pregnant women and measured levels of the cytokine IL-6 early in pregnancy, during the middle of the pregnancy, and near the end of their pregnancy. Shortly after the birth of their babies, Buss and others conducted MRI scans of the newborns. “This is the only way that we will be able to understand prenatal influences that are not confounded by post-natal influences,” Buss said at a November 17th press conference at the Society for Neuroscience (SfN) annual meeting in Washington, DC. In particular, Buss and her team looked for patterns of synchronized activity in the default mode network (DMN). The DMN is a network of brain regions that are active when the individual is not focused on the outside world and the brain is awake, but at rest. During goal-oriented activity, the DMN is deactivated and another network called the task-positive network (TPN) is activated. The DMN may correspond to task-independent introspection, or self-referential thought, while the TPN corresponds to action. Dysfunction of the DMN has been linked to psychiatric disorders.

The group found that the infant DMN “doesn’t look like adult network, but it’s emerging,” Buss said. “It’s there in an immature state.” More importantly, higher maternal gestational IL-6 concentration predicted reduced DFM connectivity. The infant brain was “less strongly connected under conditions of high maternal IL-6 concentrations,” Buss said.

In another study by neuroscientists at Duke University showed that the maternal diet of mice can cause inflammatory and behavior changes in offspring. Staci Bilbo of Duke University and her team found that a high-fat diet in the mother can lead to inflammation in the body’s fat tissue as well as immune changes in brain that may be linked to psychiatric disorders like anxiety and depression. The researchers fed mice either a low-fat diet or a high-fat diet, either enriched or not enriched for branched chain amino acids (BCAAs). Bilbo’s group examined the mothers’ brains midway through pregnancy and found increased expression of inflammatory cytokines in the hypothalamuses of mice fed a high-fat diet. These changes were also accompanied by postpartum increases in depressive-like behaviors in mice fed a BCAA-enriched diet and an increase in anxiety-like behaviors in mice fed a high-fat diet.

According to Bilbro, the offspring of these mothers showed “striking” differences in the expression of inflammatory cell types and in the behavior of the newborn pups. Infants born to moms fed a high-fat diet showed decreased expression of microglia markers and increased anxiety-like behaviors. However, mice born to moms on a high-fat, high-BCAA diet showed increased expression of a marker for astrocytes.

“Maternal diet does matter,” said Bilbo. “We believe [these changes] may be contributing to both metabolic changes as well as mood changes” in the moms and their offspring.

Society for Neuroscience Conference – More to Report


A very interesting poster at the SfN meeting described experiments with the antihypertensive medicine Telmisartan and its ability to protect brain cells from dying from an overdose of neurotransmitters.

During a stroke, dead or dying neurons tend to dump enormous quantities of neurotransmitters into their surrounding environment, and these excessive concentrations of neurotransmitters are deleterious for the surrounding neurons. This phenomenon is called “excitoxicity,” and it is an important killer of neurons in a stroke.

In this poster, a Chinese scientist used Telmisartan to pre-treat cultured neurons that were then given large quantities of the neurotransmitter glutamate. The drug protected the neurons from dying from the excessive concentrations of glutamate. Telmisartan also profected cells by binding to the AT[1A] receptor, and activating the PPAR[gamma] transcription factor. While these results may sound cryptic, PPAR[gamma] is a target for a group of anti-diabetic drugs called the triglitazones. By activating this transcription factor, telmisartan rescued these cultured neurons from certain death, and Dr. Wang (the poster presenter) suggested that Telmisartan could potentially be prescribed to delay the effects of stroke are even Alzheimer’s disease.

I also attended a series of short oral presentations at this meeting, and one symposium included modeling diseases with induced pluripotent stem cells. That was a fascinating symposium and I felt like a kid in a candy store. One Japanese researchers discussed his successes at using induced pluripotent stem cells (iPSCs) to make brain “organics.” These organoids contain multiple organ-specific cell types, recapitulate some function of the organ, and share at least some of the cellular organization of the organ. Brain organoids were made by deriving iPSCs from cells taken from human volunteers, which were ten grown in embryonic stem cell medium for one week to expand the cells. Then the cells were for about another week in Neural Induction Medium, and then shaken for four more weeks. The cells self-organized into minibrains that exhibited cortical organization with the layered structure of a brain that expressed many of the same genes as the layers of a developing brain. These minibrains also showed glutamate-induced calcium mobilization. Thus these minibrains qualify as a brain organoid.

Next, he used this same procedure to make minibrains from iPSCs derived from patients with fragile X syndrome, which, besides Down Syndrome, is the leading cause of mental retardation, globally speaking. Minibrains from these Fragile X Syndrome patients formed and looked normal. However, they showed abnormal connections between neurons. This tremendous model system can provide ways to study neurological diseases at very detailed levels.

The next talk was by Haruhisa Inoue from Kyoto University who examined the use of iPSCs as a way to treat neurological diseases. In particular, Dr. Inoue was interested in Amyotrophic Lateral Sclerosis or ALS. In the case of ALS, a cells called astrocytes are the problem. The astrocytes generate a foul environment that causes the neurons in the spinal cord to die off.

Dr. Inoue used iPSC technology to derive mature astrocytes from non-ALS and ALS patients. The two sets of astrocytes showed profound functional differences. When he transplanted normal astrocytes into the spinal cords of ALS mice, her also discovered that the mice showed rather significant functional improvements. Thus, Dr. Inoue thinks that transplantation of astrocytes made from iPSCs derived from the cells of healthy volunteers might provide an excellent way to delay or even reverse the effects of ALS.

Society for Neuroscience Conference 2014 Continued


Let me emphasize that the huge number of posters and talks at the SfN conference made it impossible to attend all of them, so my recollections here are some of the high points that I was able to take in. There is a lot of terrific science going on out there and these conferences are windows into it.

One poster described a feeding study in rats. One group of rats received a diet rich in omega-3 fatty acids, which are found in fish oils and soy. Another group was fed a standard laboratory diet that tends to skim on the omega-3 fatty acids. In the brains of the omega-3-fed rats, the expression off the gene that encodes Brain Derived Neurotropic Factor or BDNF increased significantly.

This is significant because BDNF promotes the survival of nerve cells (neurons) by playing a role in the growth, maturation (differentiation), and maintenance of these cells. In the brain, BDNF protein is active at the connections between nerve cells (synapses), where cell-to-cell communication occurs. The synapses can change and adapt over time in response to experience, a characteristic called synaptic plasticity, and BDNF regulates synaptic plasticity, which is important for learning and memory.

When these researchers examined why the BDNF gene was unregulated in rats fed the omega-3-rich diet, they discovered that the starting point of the gene, which is called the promoter was nice and clear. In the standard diet rats, the promoter of the BDNF gene was chemically modified with methyl (-CH3) groups. In the absence of the methyl groups, the transcription factor CTCF was able to bind and increase the rate of transcription. If the promoter was chemically modified with methyl groups, then a protein called MeCP2 bound to the promoter and prevented expression of BDNF.

This group looked further and discovered that the omega-3-rich diet seemed to influence the expression of BDNF by means of the balance of reduced and oxidized versions of electron carriers in cells, in particular, the ratio of NAD+ to NADH. NAD is a major electron carrier in cells and the ratio of NAD+, the oxidized version of this molecule, to the reduced version of this molecule, NADH, is a measure of the energy charge of the cell and how well-fed the individual is. More importantly, NAD is a substrate for another regulator of gene expression called Sirtuins.

Sirtuins are protein deacetylases, but they are unusual deacetylases since many of them they do not simply hydrolyze acetyl-lysine residues. Instead they couple lysine deacetylation to NAD hydrolysis. This hydrolysis produces O-acetyl-ADP-ribose, which is the deacetylated substrate and nicotinamide, which is an inhibitor of sirtuin activity. The dependence of sirtuins on NAD links their enzymatic activity directly to the energy status of the cell via the cellular NAD:NADH ratio.

The fact that a diet high in omega-3 fatty acids affects the NAD/NADH ratio is significant for Alzheimer’s disease because the sirtuin, SIRT1, deacetylates and coactivates the promoter for the gene that encodes the retinoic acid receptor beta gene, which subsequently upregulates the expression of alpha-secretase (ADAM10). Alpha-secretase is able to suppress beta-amyloid production. ADAM10 activation by SIRT1 also induces the Notch signaling pathway, which is known to repair neuronal damage in the brain. All of this begins with a dietary factor that actually protects the brain from Alzheimer’s disease by profound changes in gene expression.

Another poster from an Italian group used the 5XFAD mouse model of Alzheimer’s disease to test a growth factor called “painless Nerve Growth Factor” on mice with protein plaque formation in their brains. The growth factor was given by placing droplets of the growth factor in the noses of the mice while they were anesthetized. The results were stunning. Normally, 5XFAD mice get plaques quickly in their brains and lots of them. However, the growth factor was able to rescue the onset of behavioral deficits and reduces, although not eliminate, plaque formation. Other brain-specific pathologies found in these mice were reduced, such as astrocytosis. The wandering white cells in the brain known as microglia did a better job of gobbling up protein aggregates and clearing them from the brain, and the markers of inflammation were significantly reduced. I asked the investigator if there were plans to try to move this to clinical trials, and she said that she was unable to do so because of a lack of funding. Maybe someone will collaborate with this dear lady to make it so?

In another poster, the overexpression of an enzyme called heparanase in the brain decreased the burden of protein aggregates in the brains of mice with Alzheimer’s disease. I was not able to get into the details of this poster because of time.

In another poster, a very energetic young man told me about his very interesting work with a Parkinson’s disease model in rodents. If mice are administered a drug called MPTP (short for 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine), the dopamine-using neurons in the brain will specifically take up this drug in high concentrations and it will kill them. Therefore, this drug is an excellent model system to study Parkinson’s disease in mice.

Prokineticin-2 is a gene that is expressed in high quantities in the surviving dopamine-using neurons that came from the brains of Parkinson’s disease patients after their deaths. When Prokineticin-2 was overexpressed in cultured dopaminergic neurons, they unregulated a protein called Bcl-2. Bcl-2 is one of the group of proteins can protect cells from dying. Therefore, Prokineticin-2 is a prosurvival protein.

Next, this chap switched from a culture system to a “in a living animal” system or an in vivo system. By using genetically engineered viruses that overexpressed Prokineticin-2 in the brains of mice, he discovered that this viruses did not adversely affect the mice and he did in fact achieve high levels of Prokineticin-2 in the brains of mice with this recombinant viruses. The overexpression did not affect the mice in the least. When he did the same experiment with MPTP-treated mice – oh, just to be clear, he overexpressed Prokineticin-2 first and then administered the MPTP because it takes about 30 days for the viruses to properly upregulate Prokineticin-2 – he saw decreased inflammation in the brain, and increase in Bcl-2 and Pink1 expression in the brain (both of these genes are pro-survival genes), and the behavioral problems of the mice never emerged with the severity of the MPTP mice. When he examined TH – an enzyme that makes the neurotransmitter dopamine, he saw that levels of this enzyme were up too. This means that the dopamine-using neurons were surviving. Is this cool stuff or what?

That’s enough for now. More later.

Cardiac Stem Cells or their Exosomes Heal Heart Damage Caused by Duchenne Muscular Dystrophy


One of the research institutions that has been at the forefront of developing investigational stem cell treatments for heart attack patients is The Cedars-Sinai Heart Institute. Recently, a research team at Cedars-Sinai Heart Institute (CSHI) has injected cardiac stem cells into the hearts of laboratory mice afflicted with a rodent form of Duchenne muscular dystrophy. This disease can also adversely affect the heart, and these stem cell injections actually improved the heart function of these laboratory animals and resulted in greater survival rates for those mice. This work might provide the means to extend the lives and improve the quality of life of patients with this chronic muscle-wasting disease.

The CSHI team presented their results at the American Heart Association Scientific Sessions in Chicago. Their results clearly demonstrated that once laboratory mice with Duchenne muscular dystrophy were infused with cardiac stem cells, the animals showed progressive and significant improvements in heart function and increased exercise capacity.

Specifically, 78 lab mice that had been given laboratory-induced heart attacks were injected with their own cardiac stem cells, and over the next three months, these mice demonstrated improved pumping ability and exercise capacity in addition to a reduction in heart-specific inflammation. The CSHI team also discovered that the stem cells work indirectly, by secreting tiny vesicles called exosomes that are filled with molecules that induce tissue healing. When these exosomes were purified and administered alone, they reproduced the key benefits of the cardiac stem cells.

Apparently, this particular procedure could be ready for testing in human clinical studies as soon as next year.

Duchenne muscular dystrophy or DMD is a genetic disease that results from mutations in a gene found on the X chromosome in humans. DMD affects 1 in 3,600 boys and is a neuromuscular disease caused by abnormalities in a muscle protein called dystrophin.  Because dystrophin is an important structural protein for muscle that anchors muscle to other muscles and to the substratum, deficiencies for functional copies of the dystrophin protein cause progressive muscle wasting, destruction, and muscle weakness.

Dystrophin acts as an important link between the internal cytoskeleton and the extracellular matrix. Neuronal nitric oxide synthase (nNOS) binds to α-syntrophin but also has a binding site in repeat 17 of the rod domain of dystrophin (see Fig. 2A for details of dystrophin domains). αDG, α-dystroglycan; βDG, β-dystroglycan
Dystrophin acts as an important link between the internal cytoskeleton and the extracellular matrix. Neuronal nitric oxide synthase (nNOS) binds to α-syntrophin but also has a binding site in repeat 17 of the rod domain of dystrophin (see Fig. 2A for details of dystrophin domains). αDG, α-dystroglycan; βDG, β-dystroglycan.  See here

The majority of DMD patients lose their ability to walk by twelve years of age, although the severity of the disease varies from patient to patient. The average life expectancy is about 25, and the cause of death is usually heart failure. Dystrophin deficiency causes heart muscle weakness, and, ultimately, heart insufficiency, since the chronic weakness of the heart muscle prevents the heart from pumping enough blood to maintain a regular heart rhythm and provide for the needs of the rest of the body. Such a heart condition is called “cardiomyopathy.”

“Most research into treatments for Duchenne muscular dystrophy patients has focused on the skeletal muscle aspects of the disease, but more often than not, the cause of death has been the heart failure that affects Duchenne patients,” said Eduardo Marbán, MD, PhD, who is the director of the CSHI and the principal investigator of this particular study. “Currently, there is no treatment to address the loss of functional heart muscle in these patients.”

In 2009, Marbán and his team completed the world’s first procedure in which a patient’s own heart tissue was used to grow specialized heart stem cells. Stem cells from the heart were isolated, cultured, and then injected back into the patient’s heart in order to repair and regrow healthy heart muscle that had been injured by a heart attack. Results, Marbán and his colleagues published these results in The Lancet in 2012, and also demonstrated that one year after their patients had received the experimental stem cell treatment, they showed significant reductions in the size of the heart scar that had been produced by their heart attacks.

Earlier this year, CSHI researchers commenced a new clinical trial entitled “ALLSTAR,” which stands for Allogeneic Heart Stem Cells to Achieve Myocardial Regeneration (Clinical trial number NCT01458405). In this study, heart attack patients are given injections of stem cells from healthy donors, which should work better than the patient’s own stem cells, which were damaged by the heart attack.

CSHI has recently opened the nation’s first Regenerative Medicine Clinic, which is designed to match heart and vascular disease patients with the appropriate stem cell clinical trial being conducted at CSHI and other institutions.

“We are committed to thoroughly investigating whether stem cells could repair heart damage caused by Duchenne muscular dystrophy,” Marbán said.

The protocols for growing cardiac-derived stem cells were developed by Marbán when he was on the faculty of Johns Hopkins University. Johns Hopkins has filed for a patent on that intellectual property and has licensed it to Capricor, a company in which Cedars-Sinai and Marbán have a financial interest. Capricor is providing funds for the ALLSTAR clinical trial at Cedars-Sinai.

The Society for Neuroscience Meeting Continued


Glymphatics is a new subdiscipline in neuroscience that was essentially discovered by a Danish neuroscientist named Maiken Nedergaard. Dr. Nedergaard gave a fine seminar on this subject on Sunday.

Glymphatics consists of the system that removes waste products from the brain. Dr. Nedergaard showed movies that showed how the cerebrospinal fluid that bathes the periphery of the brain pulsates as it moves over the brain. When die molecules are injected into the cerebrospinal fluid, these dyes wend up in the blood system. How does this happen?

Nedergaard reasoned that diffusion of the fluid was far too slow for the dye to get to the blood system as fast as it does. Instead, she suspected that fluid moves by means of a “convection current.” How does this work? The blood vessels that feed the brain are surrounded by cells known as astrocytes. These astrocytes prevent molecules from entering the brain unless they can properly negotiate their way across these astrocytes, and this forms the basis for the blood-brain barrier. Cerebrospinal fluid moves across the cells of the brain and is removed by the astrocyte-surrounded vessels. This sink for the cerebrospinal fluid essentially pulls the cerebrospinal fluid across the brain cells and serves as the means by which the brain is cleansed of waste products.

This system, however, is subject to regulation, since the flow of fluid from the cerebrospinal fluid depends on the size of the spaces between brain cells. As it turns out, the spaces between brain cells in larger during sleep than when we are awake. Therefore, sleep seems to be the means by which our bodies clear the rubbish from our brains.

The molecule that controls the space between brain cells is norepinephrine. How it does that remains uncertain, but this is the molecule that is released during sleep to help clear out the garbage in the brain.

Since Alzheimer’s disease, Parkinson’s disease, other neurodegenerative diseases include the accumulation of protein aggregates in the brain, the removal of waste products in the brain would seem to be a rather important process. Also, when there is a head injury, surgeons sometimes leave the skull cap open while the brain heals. This, however, hamstrings the glymphatic system and surgeons should replace the skull cap so that the glymphatic system can do its job. Secondly, if norepinephrine can regulate this system, then this might be a way to increase clearance of waste products from the brain to reduce or delay the accumulation of protein aggregates in the brain.

Remarkable isn’t it?

Society for Neuroscience Meeting


I am in Washington DC at the Society for Neuroscience 2014 meeting. There is some incredible science here. Let me just share a few of the things I saw today:

The Thompson laboratory from UC Irvine (my alma mater – go anteaters!) made a model system for the blood-brain barrier from induced pluripotent stem cells. These scientists made iPSCs that had similar genetic defects to those observed in patients with Huntington’s disease. These iPSCs were then differentiated into blood-brain barrier cells and showed that these cells showed defects similar to those seen in patients with Huntington’s disease. The barrier leaked, which makes this a good model to study blood brain barrier defects in patients with neurological diseases.

Another poster described the use of vesicles from human fat-based stem cells to treat laboratory animals with a type of Huntington’s disease. These vesicles attenuated Huntington’s disease pathology and delayed its onset.

There were several other brilliant posters, and tomorrow, there will be even more. I will blog about those as time permits.

Growth Factor Delivery Stimulates Endogenous Heart Repair After Heart Attacks in Pigs


Steven Chamulean and his colleagues at the University Medical Center Utrecht in Holland have examined the use of growth factors to induce healing in the heart after a heart attack. Because simply applying growth factors to the heart will cause them to simply be washed out, Chamulean and his coworkers embedded the growth factors in a material called hydrogel. They were able to measure how long the implanted growth factors lasted. As it turns out, when the growth factors were embedded in the hydrogel, they lasted for four days, and the hydrogel caused the growth factors to spread out into heart tissue with a gradient with the highest concentration at the site of injection (see Bastings, et al., Advanced Healthcare Materials 2013 doi: 10.1002/adhm.201300076).

In his new publication in the Journal of Cardiovascular Translational Research, Chamulean and his group used a new hydrogen called UPy to into which they embedded their growth factors. UPy stands for ureido-pyrimidinone end-capped poly(ethylene glycol) polymer. At the pH of our bodies, UPy hydrogels form a gel-like material made of fibers. When the pH changes, the gel becomes liquid. They embedded the growth factors insulin-like growth factor-1 (IGF-1), and hepatocyte growth factor (HGF).

The experimental design of this paper used pigs that were given heart attacks and then reperfused 75 minutes later. One month later, the animals were broken into three groups: just hydrogel, hydrogel with growth factors embedded in it, and growth factors injected into other heart without hydrogel. One month later, the animals were examined for their heart function, and then the animals were sacrificed to examine their heart tissue.

In every case, the hearts treated with only the hydrogel did the poorest of the three groups. The animals injected with gel-less growth factors did better than the controls, but those animals treated with growth factors embedded in UPy hydrogel did the best. The physiological indicators of the hearts from the animals treated with UPy embedded with IGF-1 and HGF improved significantly more than the controls that were treated with only UPy hydrogel. The hearts from animals treated with IGF and HGF without hydrogels improved over controls, by not nearly as well as those treated with growth factor-embedded UPy hydrogels.

When the hearts were examined even more surprises were observed. The animals with hearts that had been treated with UPy + growth factors did not show the enlargement observed in the control hearts. This is significant, because enlargement of the heart is a side effect for a heart attack and is the sign of heart failure. The UPy + growth factor hearts also displayed many signs of dividing cells; far more than hearts from the other two groups. Since the heart has its own resident stem cell population, these growth factors stimulated these stem cells to divide and form new heart muscle, and new blood vessels. Blood vessel density was much higher in the UPy + growth factor group and the pressure against which blood flowed in these hearts was substantially less in this groups, demonstrating that not only was the blood vessel density higher, but blood flow through these vessel networks was much more efficient. There was also plentiful evidence of the formation of new muscle in the UPy + growth factor group. When these hearts were also stained for c-kit, which is a cell surface marker for cardiac stem cells, the UPy + growth factor hearts had lots of them – much more than the other two groups.

This paper reports significant findings because the resident stem cell population in the heart was actively mobilized without having to extract them by means of a biopsy. There is also evidence from Torella and others that IGF-1 and HGF can reactivate the sleeping cardiac stem cells of aged laboratory animals (Circulation Research 2004 94: 514-524). The UP{y hydrogels are well tolerated and are biodegradable. They provide a medium that stays in place and releases embedded growth factors in a sustained manner. The results in this paper provide the rationale to develop growth factor therapy for human patients.