First Patient Enrolled in Phase 2 Trial That Tests NSI-189 for Major Depressive Disorder

Neuralstem, Inc. has announced the enrollment of its first patient in its double-blind, placebo-controlled multi-center Phase 2 study of a compound called NSI-189 for the treatment of MDD (major depressive disorder).

MDD usually consists of a persistent feeling of sadness or loss of interest. MDD can also include an inability to sleep or concentrate on tasks, changes in appetite, decreased energy level, and even thoughts of suicide.

MDD is treated with a variety of psychological therapies, such as
cognitive behavioral therapy, Behavior therapy, and Psychotherapy. Cognitive behavioral therapy is a type of talk therapy that focuses on changing a person’s thoughts in order to change their behavior and feelings. Behavior therapy focuses on changing behavior to help people break unhealthy habits. Psychotherapy treats mental or behavioral disorders through talk therapy. A medical procedure called electroconvulsive therapy is also used for some patients. Medications include antipsychotic medicines such as Aripiprazole (Abilify), anxiolytics like buspirone (Buspar), and antidepressants such as Trazodone (Oleptro), Bupropion (Wellbutrin), Duloxetine (Cymbalta) and a host of others.

The medications used to treat MDD regulate the levels of particular neurotransmitters (small molecules used neurons use to communicate with each other) in the brain.

NSI-189 works rather different from these other medications. NSI-189 activates neurogenesis, or the production of new neurons. The drug also activates the formation of new synapses and increases the volume of the hippocampus. All of these processes are thought to play a role in reversing depression. Such neurological outcomes can also enhance cognition and promote neuroregeneration.


This phase 2 trial will randomize 220 patients, in three cohorts, two of whom will receive the drug (40 mg twice a day or 40 mg once a day) and another of which will receive the placebo. Twelve different sites will participate in this MDD trial, all under the direction of Maurizio Fava.

The primary efficacy endpoint is a reduction in depression symptoms. The Montgomery-Asberg Depression Rating Scale (MADRS) will be used to assess thee severity of depression symptoms. Other endpoints will examine cognitive improvement measures.

The trial will last for 12 weeks, with an additional observational follow-up period of six months in order to assess NSI-189 long-lasting durability of benefits.

Neuralstem expects to report the results of this trial in the second half of 2017.

“A new class of treatment is needed in major depression, where existing compounds are not effective for all patients and have high side effect profile, so patients discontinue treatment,” said Fava. “We were encouraged by the signs of improvement in the depression and cognitive symptoms of MDD patients, as witnessed in Phase I with NSI-189, and look forward to validating in Phase 2.”

As mentioned in this statement to the press by Fava, NSI-189 successfully completed a phase I clinical trial for MDD in 2011. In this trial, NSI-189 was administered to 41 healthy volunteers. A phase Ib clinical trial for treating MDD in 24 patients was started in 2012 and completed in July 2014, and the results of this trial were published in December 2015.

NSI-189 works via a new pathway that is different from current antidepressants in that it appears to create long-lasting, positive structural changes in the brain.

In animal experiments, rodents treated with NSI-189 showed significant increases in synaptogenesis, neurogenesis, and hippocampal volume.

In the Phase 1b trial, therapeutic effects were observed in patients after completion of the 28-day dosing, and these improvements persisted for an additional 56 days without the drug. This seems to support the hypothesis of a new mechanism of action that induces long-lasting structural changes in key areas of the brain. In this trial, NSU-189 was shown to be safe and demonstrated large treatment effects in two key depression outcome measures.

The Phase 1b study also showed significant improvement in cognitive symptoms (as measured by the Cognitive and Physical Functioning Questionnaire), compared to placebo.

Brain imaging with quantitative EEGs showed an increase in alpha brain waves in two parts of the brain (left posterior temporal and left parietal region), both of which are involved in depression and cognition, compared to placebo.

No significant adverse effects were observed.

This new clinical trial will test the efficacy of this new drug to treat moderate to severe clinical depression.

Epilepsy Reduces The Formation of New Neurons in the Brain

An ambitious, multidisciplinary project led by Amanda Sierra and Juan Manuel Encinas, Ikerbasque from the Achucarro centre (Achucarro Basque Center for Neuroscience) has discovered that epilepsy in a mouse model system reduces the production of new cells in the brain.

The hippocampus is a region of the brain involved in learning and memory and it is also the site of a robust neural stem cell population that generates new neurons. These hippocampal neural stem cells generate new neurons throughout the adult life of mammals. The cells generated by the hippocampal neural stem cells function in certain types of learning and memory and in responses to anxiety and stress.


This new research by Sierra and Encinas has revealed that in epileptic mice, hippocampal neural stem cells stop generating new neurons and are turn into reactive astrocytes. Reactive astrocytes promote inflammation and alter communication between neurons. Could manipulation of neural stem cells provide new ways to treat epilepsy?

Reactive Astrocyte
Reactive Astrocyte

This work has recently been published in the journal Cell Stem Cell.

The results of this research also confirms previous work by the same group that showed that epilepsy, which causes hyperexcitation of neurons but does not cause convulsions, activates neural stem cells, which leads to their premature exhaustion. Thus the generation of new neurons in the hippocampus ends is chronically reduced.

Juan Manuel Encinas, the leader of this study, highlighted the fact that “this discovery has enabled us to gain a better understanding about how neural stem cells function. We have shown that in addition to generating neurons and astrocytes, neural stem cells in the adult hippocampus can generate reactive astrocytes following an epileptic seizure.”

Encinas and his colleagues carried out this work in experimental animals that were genetically engineered to be epileptic. However, this discovery has clear implications in clinical practice and in the quest to develop new therapies for epilepsy, since the generation of new neurons (neurogenesis) is a process that is negatively affected in epileptic seizures in the hippocampus. Encinas pointed out, “If we can manage to preserve the population of neural stem cells and their capacity to generate new neurons in humans, it may be possible to prevent the development of certain symptoms associated with epilepsy and very likely to mitigate the damage that is caused in the hippocampus.”

In this project, Encinas and his colleagues collaborated with research groups attached to institutions such as the Baylor College of Medicine in Houston (United States), the Université Catholique de Louvain (Belgium), the Achucarro centre itself, and the UPV/EHU’s Genetic Expression Service.

New Neuron Formation Required for Maintenance of Olfactory Nerves in Mice

For many years, scientists and neurologists were convinced that neurons in the brain only formed during early development, and after that it was simply impossible for new neurons to be formed.  More recent work, however, has shown this to be largely untrue, since several regions of the brain possess resident stem cell populations that can divide to replenished damaged neurons and even augment learning and memory.  The capacity of neural stem cell populations to regenerate the central nervous system is a continuing field of intense research, and scientists at the National Institutes of Health (NIH) have reported one region of the central nervous system that can form new brain cells; the mouse olfactory system, which processes smells.  This work appeared in the October 8 issue of the Journal of Neuroscience.

“This is a surprising new role for brain stem cells and changes the way we view them,” said Leonardo Belluscio, Ph.D., a scientist at NIH’s National Institute of Neurological Disorders and Stroke (NINDS) and lead author of the study.

The olfactory bulb is at the front of the brain (shown as “A” in he picture below), and is rather small in humans, but somewhat larger in other animals.  This structure receives information directly from the nose about volatile odors.  Neurons in the olfactory bulb sort through this smelly information and relay neural signals to the rest of the brain.  This is the point at which we become aware of the smells in our surroundings.  The loss of the sense of smell is sometimes an early symptom in a variety of neurological disorders, including Alzheimer’s and Parkinson’s diseases.

Olfactory lobes in brain


Neurogenesis is the process by which neuroprogenitor cells are produced in the subventricular zone deep in the brain.  After birth, these cells migrate to the olfactory bulb, which becomes the final location of these cells.  Once they arrive at the olfactory bulb. the neuroprogenitor cells divide, differentiate, and form connections with existing cells to become integrated into the neural circuitry in the olfactory bulb and elsewhere.

Dr. Belluscio studies the olfactory system, and for this study, he collaborated with Heather Cameron, Ph.D., a neurogenesis researcher at the NIH’s National Institute of Mental Health.  The goal of this study was to better understand how the continuous addition of new neurons affects the neural organization of the olfactory bulb.  They used two different types of genetically engineered laboratory mice that had specifically genes knocked out.  Consequently, these mice lacked the specific stem cell populations that generate the new neurons during adulthood, without affecting the other olfactory bulb cells.  Previously, this remarkable level of specificity had not been achieved.

Belluscio and his coworkers had previously shown that plugging the nostrils of the animals so that they are not subject to olfactory stimulation causes the axonal extensions of the olfactory neurons to dramatically spread out and lose the precise network of connections with other cells that are normally observed under normal conditions.  They also showed that this widespread disrupted circuitry could re-organize itself and restore its original precision once the sensory deprivation was reversed.  Therefore, Belluscio and his team temporarily plugged a nostril in their lab animals to block olfactory sensory information from entering the brain.  However, if laboratory animals that do not produce new neuroprogenitors are subjected to this type of manipulation, once the nose is unblocked, new neurons are prevented from forming and entering the olfactory bulb, and, therefore, the neural circuits remain in disarray. “We found that without the introduction of the new neurons, the system could not recover from its disrupted state,” said Dr. Belluscio.

Further examination showed that elimination of the formation of adult-born neurons in mice that did not experience sensory deprivation also caused the organization of the olfactory bulb organization began to degenerate, eventually resembling the pattern observed in animals prevented from receiving sensory information from the nose.  Belluscio and his team also noticed that the extent of stem cell loss was directly proportional to the degree of disorganization in the olfactory bulb.

According to Belluscio, circuits of the adult brain are thought to be rather stable and that introducing new neurons alters the existing circuitry, causing it to re-organize. “However, in this case, the circuitry appears to be inherently unstable requiring a constant supply of new neurons not only to recover its organization following disruption but also to maintain or stabilize its mature structure. It’s actually quite amazing that despite the continuous replacement of cells within this olfactory bulb circuit, under normal circumstances its organization does not change,” he said.

Dr. Belluscio and his colleagues think that these new neurons in the olfactory bulb are important for the maintenance of activity-dependent changes in the brain, which help animals adapt to a constantly varying environment.

“It’s very exciting to find that new neurons affect the precise connections between neurons in the olfactory bulb. Because new neurons throughout the brain share many features, it seems likely that neurogenesis in other regions, such as the hippocampus, which is involved in memory, also produce similar changes in connectivity,” said Dr. Cameron.

The underlying basis of the connection between neurological disease and changes in the olfactory system is also unknown but may come from a better understanding of how the sense of smell works. “This is an exciting area of science,” said Dr. Belluscio, “I believe the olfactory system is very sensitive to changes in neural activity and given its connection to other brain regions, it could lend insight into the relationship between olfactory loss and many brain disorders.”

Fat-Based Stem Cells Support New Brain Cell Growth in Alzheimer’s Disease Mice

Alzheimer’s disease (AD) causes progressive death of brain cells and dementia. The loss of memory, coordination, and eventually motor function is relentless and horrific, and causes extensive suffering, financial pressures and loss. Stem cell treatments have been proposed as a treatment for AD, but such treatments have met resistance because of the complex pathology of AD. Introducing new neurons into the brain will do little good if cells are normally dying. However, some work with laboratory animals has suggested that stem cell treatments can benefit animals with conditions that approximately AD (see Kim S, et al., PLoS One. 2012;7(9):e45757; Bae JS, et al., Curr Alzheimer Res. 2013 Jun;10(5):524-31). However there are few studies that examine the therapeutic effect of mesenchymal stem cells from fat tissue or “adipose-derived stem cells” on mice with AD, and the effect of these cells on the oxidative injury that tends to accompany AD, and if these stem cells stimulate the generation of new neurons in the brains of AD mice.

Now we have evidence that transplantation of mesenchymal stem cells can stimulate for formation of new brain cells in adult rat or mouse models of AD and improve tissue structure and function after a stroke. Dr. Yufang Yan and her team from the School of Life Sciences at Tsinghua University, China transplanted adipose-derived stromal cells (ADSCs) into a part of the brain known as the hippocampus of mice that express the APP/PS1 transgene. Such mice show an AD-like disease, with memory loss and amyloid plaques that form in the brain.

Transplantation of ADSCs in these AD model mice decreased oxidative stress and promoted the growth of new neurons and glial cells in the subgranular and subventricular zones of the hippocampus, and, consequently improved the cognitive impairment in APP/PS1 transgenic AD mice.

These findings were published in Neural Regeneration Research (Vol. 9, No. 8, 2014), and provide theoretical and experimental evidence that ADSCs can be used to treat AD patients.

A Molecular Switch that Determines Stem Cell Or Neuron

A University of California, San Diego School of Medicine research team has provided new information about a well-known protein that provides the switch for cells to become neurons. This protein is part of a regulatory circuit that can push an immature neural cell to become a functional neuron.

Postdoctoral fellow Chih-Hong Lou and his colleagues worked with principal investigator Miles F. Wilkinson, who is a professor in the Department of Reproductive Medicine, and is also a member of the UC San Diego Institute for Genomic Medicine. These data were published in the February 13 online issue of the journal Cell Reports. These data may also elucidate a still poorly understood process – neuron specification – and might significantly accelerate the development of new therapies for specific neurological disorders, such as autism and schizophrenia.

Wilkinson, Lou and others discovered that the conversion of immature cells to neurons is controlled by a protein called UPF1. UPF1 works in a pathway called the “nonsense-mediated RNA decay” or NMD pathway. The NMD pathway provides a quality control mechanism that eliminates faulty messenger RNA (mRNA) molecules.

mRNA molecules are synthesized from DNA in the nucleus of cells and are exported to the cytoplasm where they are translated by ribosomes into protein. All proteins are encoded by stretches of DNA known as genes and the synthesis of an RNA copy of this stretch of DNA is called transcription. After the transcription of a messenger RNA molecule, is goes to the cytoplasm and is used as the template for the synthesis of a specific protein. Occasionally, mistakes are made in the transcription of mRNAs, and such aberrant mRNAs will either be translated into junk protein, or are so damaged that they cannot be recognized by ribosomes. Such junk mRNAs will gum up the protein synthesis machinery, but cells have the NMD pathway that degrades junk mRNAs to prevent the collapse of the protein synthesis machinery.

UPF1 mechanism

A second function for the NMD pathway is to degrade a specific group of normal mRNAs to prevent the production of particular proteins. This NMD function is physiologically important, but until now it had not been clear why it is important.

Wilkinson and others have discovered that UPF1, in combination with a particular class of microRNAs, acts as a molecular switch to determine when immature (non-functional) neural cells take the plunge and differentiate into non-dividing (functional) neurons. In particular, UPF1 directs the degradation of a specific mRNA that encodes for a protein in the TGF-beta signaling pathway, which promotes neural differentiation. The destruction of this mRNA prevents the proper functioning of the TGF-beta signaling pathway and neural differentiation fails to occur. Therefore, Wilkinson, Lou and co-workers identified, for the first time, a molecular pathway in which NMD drives a normal biological response.

NMD also promotes the decay of mRNAs that encode proliferation inhibitors, which Wilkinson said might explain why NMD stimulates the proliferative state characteristic of stem cells. There are many potential clinical ramifications for these findings,” Wilkinson said. “One is that by promoting the stem-like state, NMD may be useful for reprogramming differentiated cells into stem cells more efficiently.

Wilkinson continued: “Another implication follows from the finding that NMD is vital to the normal development of the brain in diverse species, including humans. Humans with deficiencies in NMD have intellectual disability and often also have schizophrenia and autism. Therapies to enhance NMD in affected individuals could be useful in restoring the correct balance of stem cells and differentiated neurons and thereby help restore normal brain function.”

Co-authors on this paper include Ada Shao, Eleen Y. Shum, Josh L. Espinoza and Rachid Karam, from the UCSD Department of Reproductive Medicine; and Lulu Huang, from Isis Pharmaceuticals.

Funding for this research came, in part, from National Institutes of Health (grant GM-58595) and the California Institute for Regenerative Medicine.

Teaching Old Neural Stem Cells New Tricks

In our brains, cells called neurons produce nerve impulses and are responsible for thinking, learning memory, reasoning, and so on. Neurons do not exist in isolation, but in combination with cells called glial cells that support the neurons, nourish them, and protects them from stress damage. Neurons and glial cells are replenished by brain-specific neural stem cell populations in the brain.

Unfortunately, the neural stem cell population in our brains tends to produce far fewer neurons as they age. This deficit of new neurons can play a role in the onset of neurodegenerative diseases such as Alzheimer’s disease and Parkinson’s disease. Also, our own “senior moments” when we forget where we placed our iPod or car keys comes from a loss of neurons as we age.

Fortunately, some recent research might change this trend. A team from Japan’s Keio University, and the Riken national research institute, has reported the discovery of a small RNA molecule (micro-RNA) that controls neuron production in young mice. When this micro-RNA was manipulated in older mice, their neural stem cells started to make neurons again. The Japanese team also has reasons to believe that the same mechanism is at work in human brains as well. This research was reported in the journal Proceedings of the National Academies of Science. The mechanism is believed to exist in humans as well.

Senior Author Hideyuki Okano said, “We observed the neurogenic-to-gliogenic switching in developing NSCs.” Translation: Okano and his team examined embryonic mouse brains and their neural stem cell (NSC) populations. They found what many other groups have previously observed: that the developing embryonic brain NSCs create neurons first, then switch over to making glial cells later. Okano’s team also discovered the microRNA-17/106-p38 axis that is responsible for this initial neuron-to-glial cell switch during embryonic development.

When they manipulated this embryonic microRNA-17/106-p38 pathway in older, post-natal NSCs in culture, these older post-natal NSCs switched from making glial cells to producing neurons.

In culture, NSCs are difficult to control, since getting large supplies of neurons from cell cultures that various research groups call NSCs is very difficult.

Nevertheless, “there is general agreement that neurogenesis (make neurons) largely precedes gliogenesis (making glial cells) during CNS development in vertebrates,” Okano explained. And adult NSCs, according to Okano, clearly can produce neurons in the body, “whereas they exhibit strong gliogenic characteristics under culture conditions in vitro (that is, in the laboratory).”

Adult NSCs in two regions of the brain—the subventricular zone and hippocampus—also “make neurons, even though transplant studies have shown us that the adult CNS is a gliogenic environment.”

Subventricular Zone

So it seems clear that old NSCs can make neurons, at least under certain conditions. However, it is very difficult to determine the age at which NSCs begin making substantially more glial cells than neurons. According to Okano, “It is difficult to clearly explain the association between total glial cell number and changes in NSC abilities. Moreover, there is less evidence about gliogenic ability of aged NSCs because most of studies about NSCs have mainly focused on the neurogenic ability. “

Still, Okano says: “There are some reports about decline of neurogenesis ability of NSCs with age. These reports indicate that reduction in paracrine Wnt3 factors, and increase of (chemokine) CCL11 concentration in blood, impaired adult neurogenesis in the hippocampus, for example.”

Could the group’s microRNA approach improve memory in humans? Okano believes so, but says more work needs to be done.

“We observed the neurogenic effect by overexpression of miR-17 in primary cultured neurospheres” – spheres of a variety of cells, including NSCs—“derived from the SVZ at postnatal day 30. Similar phenomenon by overexpression of miR-106b-25 cluster has been reported by another group.”

Okano also warns that his approach has only been attempted in cultured cells. He cautioned, “There is no evidence using knock-out mice. Therefore, the functions of them in adult neurogenesis and learning/memory functions are still unclear.”

Next, Okano’s group will develop “a useful method for precise manipulation of cytogenesis from NSCs. “

However, he says, “we think that further understanding of basic molecular mechanisms underlying the neural development is also an important issue.” He will study the ways in which his microRNA system interacts with other glia-producing genes. He wants to fully understand the mechanisms underlying “the end of neurogenic competence and acquisition of gliogenic competence.”

Finally, the group will “examine the significance of miR-17/p38 pathway in various somatic stem cells other than NSCs,” he says.

How Neural Stem Cells Become Neurons and Glia

How do neural stem cells differentiate into neurons or glia? A new paper from researchers at the University of California, Los Angeles (UCLA) seeks to explain this very phenomenon.

Neurons serve as the conductive cells of the nervous system. They transmit electrochemical signals from one neuron to another and provide signals to muscles, glands, and so on. They are responsible for consciousness, thought, learning and memory, and personality.

Despite their immense utility, neurons are not the only cells in the nervous system. Glial cells or just glia support neurons, hold them in place, and supply neurons with oxygen and nutrients and protect them from pathogens.

Glial Cells

When mouse neural stem cells were grown in culture, Wange Lu, associate professor of biochemistry and molecular biology at the Keck School of Medicine, and his colleagues came upon a protein called SMEK1 that promotes the differentiation of neural stem and progenitor cells. SMEK1 also keeps neural stem cells in check by preventing them from dividing uncontrollably.

When Lu and others took a more detailed look at the role of SMEK1, they discovered that it does not work alone, but in concert with a protein called Protein Phosphatase 4 (PP4) to suppress the function of a third protein called PAR3. PAR3 discourages the birth of new neurons (neurogenesis), and PAR3 inhibition leads to the differentiation of neural stem progenitor cells into neurons and glia.

“These studies reveal the mechanisms of how the brain keeps the balance of stem cells and neurons when the brain is formed,” said Wange. “If this process goes wrong, it leads to cancer, or mental retardation or other neurological diseases.”

Neural stem and progenitor cells offer tremendous promise as a future treatment for neurodegenerative disorders, and understanding their differentiation is the first step towards co-opting the therapeutic potential of these cells. This could offer new treatments for patients who suffer from Alzheimer’s, Parkinson’s and many other currently incurable diseases.

This work is interesting. It was published in Cell Reports 5, 593–600, November 14, 2013. My only criticism of some of the thinking in this paper is that neural stem cell lines are usually made from aborted fetuses. I realize that some of these neural stem cell lines come from medical abortions in which the baby had already died, but many of them come from aborted babies. If we are going to use neural stem cells for therapeutic purposes, then we should make them from induced pluripotent stem cells and take them from aborted babies.

Possible Treatment for Brain Disorders

Tuberous Sclerosis is a rare genetic disease that causes the growth of tumors in the brain and other vital organs and may also lead to other conditions such as autism, epilepsy, and cognitive impairment; all of which result from the abnormal generation of neurons.

Tuberous sclerosis is also called tuberous sclerosis complex or TSC, and it is a rare genetic disease that affects multiple organ systems. TSC causes the growth of benign tumors in the brain and on other vital organs such as the kidneys, heart, eyes, lungs, and skin. TSC typically affects the central nervous system and results in a combination of symptoms including seizures, developmental delay, behavioral problems, skin abnormalities, and kidney disease.

TSC affects as many as 25,000 to 40,000 individuals in the United States and about 1 to 2 million individuals worldwide. The estimated prevalence of this disease is one in 6,000 newborns, and it occurs in all races and ethnic groups, and in both genders.

TSC derives its name from the characteristic tuber or potato-like nodules in the brain. These growths calcify with age and become hard or sclerotic.

Many TSC patients show evidence of the disorder in the first year of life. However, clinical features can be subtle initially, and many signs and symptoms take years to develop. As a result, TSC can be unrecognized or misdiagnosed for years.

TSC is caused by defects, or mutations, on two genes-TSC1 and TSC2. Only one of the genes needs to be affected for TSC to be present. The TSC1 gene, discovered in 1997, is on chromosome 9 and produces a protein called Hamartin. The TSC2 gene, discovered in 1993, is on chromosome 16 and produces the protein Tuberin. These proteins combine to form a complex that suppresses cell growth by preventing activation of a master control protein called mTOR. Loss of regulation of mTOR occurs in cells lacking either Hamartin or Tuberin, and this leads to abnormal differentiation and development, and to the generation of enlarged cells, as are seen in TSC brain lesions.

Since Tuberous Sclerosis affect stem cell activity, scientists at Clemson University are examining how neurons are formed from neural stem cells and this research is vital to providing a treatment to Tuberous Sclerosis, which affects how neurons are formed in the brain.

David M Feliciano, assistant professor of biological sciences at Clemson University, said: “Current medicine is directed at inhibiting the mammalian target of rapamycin (mTOR), a common feature within these tumors that have abnormally high activity. However, current treatments have severe side effects, like due to mTOR’s many functions and playing an important role in cell survival, growth and migration.”

mTOR pathway

Feliciano continued: “Neural stem cells generate the primary communicating cells of the brain called neurons through the process of neurogenesis, yet how this is orchestrated is unknown.”

Neural stem cells lie at the very heart of brain development and repair, and alterations in the ability of these cells to self-renew and differentiate can have profound consequences for brain function at any stage of life, according to researchers.

In order to further elucidate the regulation of neurogenesis, Feliciano and his team delivered small pieces of DNA into the neural stem cells of the new-born mice. The team used electroporation to introduce the DNA into the mouse cells, and these small pieces of DNA allowed Feliciano’s team to express and control specific components of the mTOR pathway.

By using these tools, Feliciano and others showed that Increasing the activity of the mTOR pathway cause the neural stem cells to make more neurons at the expense of self-renewal. Increasing mTOR activity caused upregulation of 4E-BP2. 4E-BP2, also known as Eukaryotic translation initiation factor 4E-binding protein 2, binds to a component of the protein synthesis machinery and inhibits its function. Mice that lack functional EIF4EBP2 exhibit autism-like symptoms, including poor social interaction, altered communication and repetitive behaviors.

This work suggests that 4E-BP2 might be a new target for the treatment of TSC and that targeting this protein might cause fewer side effects than targeting mTOR. Future experiments hope to identify those proteins that are made due to the activation of this pathway in neural tissues.

Faulty Stem Cell Regulation Contributes to Down Syndrome Deficits

People who have three copies of chromosome 21 have a genetic condition known as Down Syndrome (DS). In particular, patients who have an extra copy of a small portion of chromosome 21 (q22.13–q22.2) known as the Down Syndrome Critical Region or DSCR have the symptoms of DS. The DSCR contains at least 30 genes or so and some of them tightly correlate to the pathology of DS. For example, the APP (amyloid protein precursor) gene accounts for the accumulation of amyloid protein in the brains of DS patients. DS patients develop Alzheimer disease-like pathology by the fourth decade of life, and the APP protein is overexpressed in the adult Down syndrome brain. Another gene found in the DSCR called DYRK1A (dual-specificity tyrosine phosphorylation-regulated kinase 1A) encodes a member of the dual-specificity tyrosine phosphorylation-regulated kinase family and this protein participates in various cellular processes. Overproduction of DYRK1A seems to cause the abnormal brain development observed in DS babies.

Another gene found in the DSCR is called USP16 and this gene encodes a protein that removes small peptides called ubiquitin from other proteins. Ubiquitin attachment marks a protein for degradation, but it can also mark a protein to do a specific job. USP16 removes ubiquitin an either stops the protein from acting or prevents the proteins from being degraded. Overexpression of UPS16 occurs in DS patients, and too much UPS16 protein affects stem cell function.

Michael Clarke, professor of cancer biology at the Stanford University School of Medicine, said, “There appear to be defects in the stem cells in all the tissues we tested, including the brain.” Clarke continued, “We believe USP16 overexpression is a major contributor to the neurological deficits seen in Down Syndrome.” Clarke’s laboratory conducted their experiments in mouse and human cells.

Additional work by Clarke and his colleagues showed that downregulation of USP16 partially rescues the stem cell proliferation defects found in DS patients.

Clarke’s study suggests that drugs that reduce the activity of USP16 could reduce the some of the most profound deficits in DS patients.

This paper also details some of the pathological mechanisms of DS. DS patients age faster and exhibit early Alzheimer’s disease. The reason for this seems to rely on the overexpression of UPS16, which accelerates the rate at which stem cells are used during early development. This accelerated rate of stem cell use burns out and exhausts the stem cell reserves and, consequently, the brains age faster and are susceptible to the early onset of neurodegenerative diseases.

After examining laboratory mice that had a rodent form of DS, Clarke and his coworkers turned their attention to USP16 overexpression in human cells. Clarke collaborated with a Stanford University neurosurgeon named Samuel Cheshier and their study showed that skin cells from normal volunteers grew much more slowly when the Usp16 gene was overexpressed. Furthermore, neural stem cells, which normally clump into little balls of cells called neurospheres, no longer formed these structures when Usp16 was overexpressed in them.

a, Proliferation analysis, as well as SA-βgal and p16Ink4a staining, of three control and four Down’s syndrome (DS) human fibroblast cultures show growth impairment and senescence of Down’s syndrome cells. b, c, Lentiviral-induced overexpression of USP16 decreases the proliferation of two different control fibroblast lines (b), whereas downregulation of USP16 in Down’s syndrome fibroblasts promotes proliferation (c). d, Overexpression of USP16 reduces the formation of neurospheres derived from human adult SVZ cells. The right panel quantifies the number of spheres in the first and second passages. P < 0.0001. All the experiments were replicated at least twice. Luc, luciferase.
a, Proliferation analysis, as well as SA-βgal and p16Ink4a staining, of three control and four Down’s syndrome (DS) human fibroblast cultures show growth impairment and senescence of Down’s syndrome cells. b, c, Lentiviral-induced overexpression of USP16 decreases the proliferation of two different control fibroblast lines (b), whereas downregulation of USP16 in Down’s syndrome fibroblasts promotes proliferation (c). d, Overexpression of USP16 reduces the formation of neurospheres derived from human adult SVZ cells. The right panel quantifies the number of spheres in the first and second passages. P < 0.0001. All the experiments were replicated at least twice. Luc, luciferase.

Conversely, when cultured cells from DS patients had their USP16 activity levels knocked down, their proliferation defects disappeared. In Clarke’s words, “This gene is clearly regulating processes that are central to aging in mice and humans, and stem cells are severely compromised. Reducing Usp16 expression gives an unambiguous rescue at the stem cell level. The fact that it’s also involved in this human disorder highlights how critical stem cells are to our well-being.”

How Neural Stem Cells Create New and Varied Neurons

A new study in fruit flies has elucidated a mechanism in neural stem cells by which these types of stem cells generate the wide range of neurons that they form.

Chris Doe, a professor of biology from the Institute of Neuroscience at the University of Oregon, and his co-authors have used the common fruit fly Drosophila melanogaster to investigate the cellular mechanism by which neural stem cells make their distinctive progeny.

As Doe put it, “The question we confronted was ‘How does a single kind of stem cell, like a neural stem cell, make all kinds of neurons?”

Researchers have known for some period of time that stem cells have the capacity to produce new cells, but the study by Doe’s group shows how a select group of stem cells can create progenitor cells that can generate numerous subtypes of cells.

Doe’s study builds on previous studies in which Doe and his colleagues identified the specific set of stem cells that generated neural precursors. These so-called “intermediate neural progenitors” or INPs can expand to form several different new cell types. However, this study did not account for the diversity of the cells generated even if it did account for the number of cells generated (see Boone JQ, Doe CQ, Dev Neurobiol. 2008 Aug;68(9):1185-95).

“While it’s been known that individual neural stem cells or progenitors could change over time to make different types of neurons and other types of cells in the nervous system, the full extent of this temporal patterning had not been described for large neural stem cell lineages, which contain several different kinds of neural progenitors,” according to this study’s first author Omar Bayraktar.

The cell types discovered in this study have analogs in the developing human brain and the research has potential applications for human biologists who want to know how neurons form in the human brain.

The paper from Doe’s lab was published along another study on the generation of diverse neurons by a group from New York University. These two papers provide new insight into the means by which neural stem cells generate the wide range of neurons found in the brains of fruit flies and humans.

In their study, Bayraktar and Doe specifically examined stem cells in fruit fly brains known as type II neuroblasts, which generate INPs. However, in this study, the type II neuroblasts were shown to generate INPs, which then go on to form distinct neural subtypes. Even though previous work showed that INPs went on to form about 100 new neurons, in this paper, the INPs were shown to make about 400-500 new neurons.

Another interesting finding was that the gene expression patterns of INPs, which began with three different transcription factors (Dichaete, Grainy Head, and Eyeless). These transcription factors lay the groundwork for INP differentiation, but once INP formation occurs, a new transcriptional program is extended that extends the types of neurons that INPs can form. Such nested transcriptional programs are also common during the specification of neural stem cell progeny in humans brains, with many of the same transcription factors playing a central role in neuron specification.

“If human biologists understand how the different types of neurons are made, if we can tell them ‘This is the pathway by which x, y, and Z neurons are made,’ then they may be able to reprogram and redirect stem cells to make these precise neurons,” Doe said.

However, the mechanism described in this paper has its limits. Eventually the process of generation new cells stops. One of the next questions to answer will be what makes the mechanism turn off, according to Doe.

“This vital research will no doubt capture the attention of human biologists,” said Kimberly Andrews Espy, who is vice-president for research and innovation and the dean of the UP graduate school. “Researchers at the University of Oregon continue to further our understanding of the processes that undergird development to improve the health and well-being of people throughout the world.”

See Bayraktar OA, Doe CQ. Combinatorial temporal patterning in progenitors expands neural diversity. Nature. 2013 Jun 27;498(7455):449-55. doi: 10.1038/nature12266.

Umbilical Cord Blood Stem Cells Revive Child From Persistent Vegetative State

Physicians from Ruhr-Universitaet-Bochum (RUB) have successfully treated cerebral palsy in a 2.5-year old boy with his own cord blood.

“Our findings, along with those from a Korean study, dispel the long-held doubts about the effectiveness of the new therapy,” says Dr. Arne Jensen of the Campus Clinic gynaecology. Jensen collaborated with his colleague Prof. Dr. Eckard Hamelmann of the Department of Pediatrics at the Catholic Hospital Bochum (University Clinic of the RUB). This case study was published in the journal Case Reports in Transplantation.

At the end of November 2008, a young child’s heart stopped (cardiac arrest), and his brain suffered oxygen deprivation, and, consequently, severe brain damage. He was in a persistent vegetative state, and his body was completely paralyzed. This condition, infantile cerebral palsy, until now, has no recognized treatment. Typically, the prognosis of children with infantile cerebral palsy is rather grim, since the chances of survival miniscule and months after suffering severe brain damage, the surviving children usually only exhibit minimal signs of consciousness. According to the physicians at RUB, “The prognosis for the little patient was threatening if not hopeless.”

However, this child’s persistent parents scoured the literature for alternative therapies to infantile cerebral palsy. Arne Jensen explains. “They contacted us and asked about the possibilities of using their son’s cord blood, frozen at his birth.”

Nine weeks after suffering brain damage, on 27 January 2009, Jensen and his colleagues administered the child’s prepared cord blood intravenously. They studied the child’s progressive recovery at 2, 5, 12, 24, 30, and 40 months after treatment.

After the cord blood therapy, the patient, however, recovered quickly. Within two months, the child’s spasms decreased significantly. He was able to see, sit, smile, and to speak simple words again. Forty months after treatment, the child was able to eat independently, walk with assistance, and form four-word sentences. “Of course, on the basis of these results, we cannot clearly say what the cause of the recovery is,” Jensen says. “It is, however, very difficult to explain these remarkable effects by purely symptomatic treatment during active rehabilitation.”

Just listen to the description of the child’s recovery from this paper:

After two years, there was independent eating and speech competence of eight words (pronunciation slurred, mimicking prosody) with broad understanding. The patient moved from a prone to a free sitting position and crawled without cross-pattern, but using the arms. Independent passive standing, walking with support, and independent locomotion in a gait trainer was possible (video S5). He played imaginative games, and recognized colours, animals, and objects, assigning them correctly. Fine motor control improved to such an extent that he managed to steer a remote control car (video S6). At 30 months, he formed two-word-sentences using 80 words.

After 40 months, there was further improvement in both receptive and expressive speech competence (four-word-sentences, 200 words), walking (Crocodile Retrowalker), crawling with cross-pattern, and getting into vertical position.

And this is from a child who was a in a persistent vegetative state, who could neither speak, nor eat on his own, nor talk.

In animal studies, scientists have examined the therapeutic potential of cord blood. In a previous study with rats, RUB researchers revealed that cord blood cells migrate to the damaged area of the brain in large numbers within 24 hours of administration.  Umbilical cord stem cells are also known to secrete gobs of neurotropic molecules that stimulate neuron growth and differentiation, promote neuron survival, quell inflammation, staunch star formation in the brain (gliosis), and stimulate the growth and formation of blood vessels.

In March 2013, in a controlled study of one hundred children, Korean doctors reported for the first time that they had successfully treated cerebral palsy with someone else’s cord blood.

These results show that cord blood has tremendous therapeutic potential for pediatric neurological conditions.  This remarkable recovery is seemingly miraculous.  Certainly this merits more work and excitement.

Human Brain Cells Made in the Lab that Grow in the Mouse Brain

The laboratory of Arnold Kriegstein, who serves as the director of the Broad Center of Regenerative Medicine and Stem Cell Research at UC San Francisco has made a vitally important type of brain cell from human pluripotent stem cells that smoothly integrates into the brains of laboratory animals. Such a discovery could potentially provide cells that could treat epileptics, Parkinson’s disease or even Alzheimer’s disease.

Medial ganglionic eminence (MGE) cells are a unique type of progenitor cell in the developing brain that guides cell and axon migration. The MGE is located between the thalamus and the caudate nucleus in the developing brain and it facilitates tangential cell migration during embryonic development in the brain.

In the developing brain, cells move radially, along special glial cells that act as tracts for the migrating cells, or tangentially between the radial glial cells. Those cells that move tangentially (perpendicular to the radial glial cells) are specially designated to form GABAminergic neurons; that is neurons that use gamma-amino-butyric acid as their neurotransmitter. However, the MGE also contributes cells to the basal ganglia, which helps control voluntary movement, and guides those axons that grow from the thalamus into the cerebral cortex, or, conversely, those axons that grow from the cerebral cortex to the thalamus. The MGE is a transient structure, and after one year of age, the MGE disappears.

medial ganglionic eminence

Making MGE cells from pluripotent stem cells has been one of the holy grails of developmental neurobiology. Now Kirgstein’s laboratory has succeeded in doing just that.   By subjecting human embryonic stem cells and induced pluripotent stem cells to a complex and extensive differentiation procedure, Kirgstein and his coworkers succeeded in producing large quantities of MGE progenitors that readily matured into forebrain interneurons.  They treated pluripotent stem cells with several growth factors, but more importantly, they timed the delivery of these factors to shape their developmental path.  By conducting neurophysiological experiments on the cells as they differentiated them, Kirgstein and coworkers discovered that they could effectively determine if they had properly derived GABAminergic interneurons.  Jiadong Chen in Kirgsteins’s laboratory showed that the MGE-like progenitors formed proper synapses or connections with other neurons and responded appropriately when stimulated.  Also, as the interneurons matured into more adult-like interneurons, their neurophysiology became more adult-like.  

When grown in the laboratory in culture or when injected into the brains of mice, these MGE-like cells developed into GABAergic interneuron subtypes that displayed the properties of mature GABAminergic neurons.  Also, the cells kept these properties for up to 7 months, and therefore, faithfully mimicked endogenous human neural development.

When injected into mouse brains, the MGE-like progenitors integrated into the brain and formed connections with existing cells.  According to Kirgstein, this property of these cells and their behavior in living tissue makes them prime candidates to test interneuron malfunction that is characteristic of human diseases.  They might also provide material to treat patients who suffer from neurological diseases that affect interneuron function.

According to Kirgstein, “We think that this one type of cell may be useful in treating several types of neurodevelopmental and neurodegenerative disorders in a targeted way.”

In earlier work, Kirgstein implanted mouse MGE cells into the spinal cord of mice that suffered from neuropathic pain.  The implanted cells reduced the pain of those mice, suggesting that they can be used to treat other neurological conditions such a spasticity, Parkinson’s disease and epilepsy.

The first author of this paper, Cory Nicholas said, “The hope is that we can deliver these cells to various places within the nervous system that have been overactive and that they will functionally integrate and provide regulated inhibition.”

Making New Neurons When You Need Them

Western societies are aging societies, and the incidence of dementias, Alzheimer’s disease, and other diseases of the aged are on the rise. Treatments for these conditions are largely supportive, but being able to make new neurons to replace the ones that have died is almost certainly where it’s at.

At INSERM and CEA in Marseille, France, researchers have shown that chemicals that block the activity of a growth factor called TGF-beta improves the generation of new neurons in aged mice. These findings have spurred new investigations into compounds that can enable new neuron production in order to mitigate the symptoms of neurodegenerative diseases. Such treatments could also restore the cognitive abilities of those who have suffered neuron loss as a result of radiation therapy or a stroke.

The brain forms new neurons regularly to maintain our cognitive abilities, but aging or radiation therapy to treat tumors can greatly perturb this function. Radiation therapy is the adjunctive therapy of choice for brain tumors in children and adults.

Various studies suggest that the reduction in our cache of neurons contributes to cognitive decline. For example, exposure of mice to 15 Grays of radiation is accompanied by disruption to the olfactory memory and reduction in neuron production. A similar event occurs as a result of aging, but in human patients undergoing radiation treatment, cognitive decline is accelerated and seems to result from the death of neurons.

How then, can we preserve the cache of neurons in our brains? The first step is to determine the factors responsible for the decline is neuron production. In contrast to contemporary theory, neither heavy doses of radiation nor aging causes completely destruction of the neural stem cells that can replenish neurons. Even after doses of radiation and aging, neuron stem cell activity remains highly localized in the subventricular zone (a paired brain structure located in the outer walls of the lateral ventricles), but they do not work properly.

Subventricular Zone
Subventricular Zone

Experiments at the INSERM and CEA strongly suggest that in response to aging and high doses of radiation, the brain makes high levels of a signaling molecule called TGF-beta, and this signaling molecule pushes neural stem cell populations into dormancy. This dormancy also increases the susceptibility of neural stem cells into apoptosis.

Marc-Andre Mouthon, one of the main authors of this research, explained his results in this manner: “Our study concluded that although neurogenesis is reduced in aging and after a high dose of radiation, many stem cells survive for several months, retaining their ‘stem’ characteristics.”

Part two of this project showed that blocking TGFbeta with drugs restored the production of new neurons in aging or irradiated mice.

Thus targeted therapies that block TGFbeta in the brains of older patients or cancer patients who have undergone high dose radiation for a brain tumor might reduce the impact of brain lesions caused by such events in elderly patients who show distinct signs of cognitive decline.