Gene Therapy Sprouts New Neurons in Alzheimer’s Patients’ Brains


The journal JAMA Neurology has published a new study that describes an experimental gene therapy that reduces the rate at which nerve cells in the brains of Alzheimer’s patients degenerate and die. See Tuszynski, M. H., et al. (2015). Nerve Growth Factor Gene Therapy: Activation of Neuronal Responses in Alzheimer Disease. JAMA Neurology, published online August 24, 2015. DOI: 10.1001/jamaneurol.2015.1807.

In this study, targeted injections of a growth factor called “nerve growth factor” or NGF into the brain of patients rescued dying cells around the injection site, enhanced the growth of these cells and induced them to sprout new nerve fibers. Surprisingly, in some cases, these beneficial effects persisted for 10 years after the therapy was first delivered.

Alzheimer’s disease (AD) is the world’s leading form of dementia. It affects approximately 47 million people worldwide, and this number is expected to almost double every 20 years. Despite the huge amounts of time, effort, and money devoted to developing an effective cure, the vast majority of new drugs for AD have failed in clinical trials.

While these new results are preliminary findings, they come from the first human trials designed to test the potential benefits of NGF gene therapy for AD patients.

NGF was discovered in the 1940s by Rita Levi-Montalcini, who demonstrated, quite convincingly, that a small protein that she had isolated and purified promoted the survival of certain sub-types of sensory neurons during development of the nervous system. Since that time, others have shown that it also promotes the survival of neurons that produce acetylcholine in the basal forebrain; these cells die off at an alarming rate in AD patients.

In 2001, Mark Tuszynski and his coworkers at the University of California, San Diego School of Medicine initiated a clinical trial based on these laboratory findings. This trial was the first of its kind, and it was designed to investigate the ability of NGF gene therapy to slow or prevent the neuronal degeneration and cell death characteristic of AD.

In phase I of this trial, eight patients with mild AD received so-called “ex vivo” therapy to deliver the NGF gene directly into the brain. This trial extracted skin fibroblasts from the skin on the patient’s backs, and then genetically engineered those cells to express the NGF genes. These NGF-expressing cells were then implanted into the patients’ basal forebrain. Since NGF is too large to cross the blood-brain barrier, it had to be administered directly into the brain. Also, outside the brain, exogenous NGF can stimulate other nerve cells can cause unwanted side-effects such as pain and weight loss.

One of these patients died just 5 weeks after receiving the therapy. Tuszynski’s team secured permission to perform an autopsy of this patient, and in 2005 they reported that the treatment led to robust growth responses, and did not cause any adverse effects (Tuszynski, M. H., et al. (2005). A phase 1 clinical trial of nerve growth factor gene therapy for Alzheimer disease. Nature Medicine, 11: 551 – 555).

The latest results come from postmortem examination of these patients’ brains, all of whom had also been recruited in a safety trial between March 2001 and October 2012. Additionally, two other were included who had received in vivo therapy that included injecting a modified virus that carried the NGF gene into the basal forebrain.

Some of the participants died about one year after undergoing therapy, and others survived for 10 years after the treatment. These autopsies showed that all of them had responded to the treatment.

Essentially, all the brain tissue samples taken from around the implantation sites contained diseased neurons, as expected, but the cells were overgrown, and had sprouted axonal fibers that had grown towards the region into which NGF had been delivered. In contrast, samples taken from the untreated side of the brain exhibited no such response.

This trial was conducted to test the safety of the treatment and it did confirm that none of the patients experienced long-term adverse effects from the treatment, even after long periods of time. These results also suggest that NGF is successfully taken up by nerve cells following targeted delivery. Also the cells synthesize NGF protein so that its concentration dramatically increases in and around the delivery site. Probably the most exciting part of these findings is that the responses to NGF can persist for many years after the gene has been delivered into the brain.

Cholinergic Neuronal Hypertrophy and Sprouting Shown is labeling for p75, a neurotrophin receptor expressed on cholinergic neurons of the nucleus basalis of Meynert. Images were obtained 3 years after adeno-associated viral vectors (serotype 2)–nerve growth factor (AAV2-NGF) delivery (A-C) and 7 years after ex vivo gene transfer (D-F). A-C, Cholinergic neurons are labeled for p75 within the zone of transduction (A), 3 mm from the zone of transduction (B), and in a control Alzheimer disease (AD) brain of the same Braak stage (C). Cells near the NGF transduction region appear larger. The inset shows higher-magnification views of a typical neuron from each region. D, Shown is a graft of fibroblasts transduced to secrete NGF (yellow arrowhead) adjacent to the nucleus basalis of Meynert (red arrowheads). E, The graft (G) at higher magnification is densely penetrated by p75-labeled axons arising from the nucleus basalis of Meynert. These axons are sprouting toward the graft, a classic trophic response. F, Shown are p75-labeled axons from the nucleus basalis of Meynert sprouting toward the graft. Individual axons coursing toward the graft are evident (arrowheads). The bar represents 125 µm in A through C, 500 µm in D, and 100 µm in E and F.
Cholinergic Neuronal Hypertrophy and Sprouting
Shown is labeling for p75, a neurotrophin receptor expressed on cholinergic neurons of the nucleus basalis of Meynert. Images were obtained 3 years after adeno-associated viral vectors (serotype 2)–nerve growth factor (AAV2-NGF) delivery (A-C) and 7 years after ex vivo gene transfer (D-F). A-C, Cholinergic neurons are labeled for p75 within the zone of transduction (A), 3 mm from the zone of transduction (B), and in a control Alzheimer disease (AD) brain of the same Braak stage (C). Cells near the NGF transduction region appear larger. The inset shows higher-magnification views of a typical neuron from each region. D, Shown is a graft of fibroblasts transduced to secrete NGF (yellow arrowhead) adjacent to the nucleus basalis of Meynert (red arrowheads). E, The graft (G) at higher magnification is densely penetrated by p75-labeled axons arising from the nucleus basalis of Meynert. These axons are sprouting toward the graft, a classic trophic response. F, Shown are p75-labeled axons from the nucleus basalis of Meynert sprouting toward the graft. Individual axons coursing toward the graft are evident (arrowheads). The bar represents 125 µm in A through C, 500 µm in D, and 100 µm in E and F.

Now, does the observed cellular response to NGF alleviate disease symptoms? Although phase II trials testing the efficacy of the treatment are ongoing, preliminary findings from the initial study suggest that the therapy did indeed slow the rate at which mental function declined in one of the patients involved. These new results indicate that gene therapy is a viable strategy for treating Alzheimer’s and other neurodegenerative diseases, and warrants further research and development.

Using Human Stem Cells to Predict the Efficacy of Alzheimer’s Drugs


Scientists who work in the pharmaceutical industry have seen this time and time again: A candidate drug that works brilliantly in laboratory animals fails to work in human trials. So what’s up with this?

Now a research consortium from the University of Bonn and the biomedical company Life & Brain GmbH has shown that animal models of Alzheimer’s disease fail to recapitulate the results observed with cultured human nerve cells made from stem cells. Thus, they conclude that candidate Alzheimer’s disease drugs should be tested in human nerve cells rather than laboratory animals.

In the brains of patients with Alzheimer’s disease beta-amyloid protein deposits form that are deleterious to nerve cells. Scientists who work for drug companies are trying to find compounds that prevent the formation of these deposits. In laboratory mice that have a form of Alzheimer’s disease, over-the-counter drugs called NSAIDs (non-steroidal anti-inflammatory drugs), which include such population agents as aspirin, Tylenol, Advil, Nuprin and so on prevent the formation of beta-amyloid deposits. However in clinical trials, the NSAIDs royally flopped (see Jaturapatporn DIsaac MGMcCleery JTabet N. Cochrane Database Syst Rev. 2012 Feb 15;2:CD006378).

Professor Oliver Brüstle, the director of the Institute for Reconstructive Neurobiology at the University of Bonn and Chief Executive Officer of Life and Brain GmbH, said, “The reasons for these negative results have remained unclear for a long time.”

Jerome Mertens, a former member of Professor Brüstle’s research, and the lead author on this work, said, “Remarkably, these compounds were never tested directly on the actual target cells – the human neuron.”

The reason for this disparity is not difficult to understand because purified human neurons were very difficult to acquire. However, advances in stem cell biology have largely solved this problem, since patient-specific induced pluripotent stem cells can be grow in large numbers and differentiated into neurons in large numbers.

Using this technology, Brüstle and his collaborators from the University of Leuven in Belgium have made nerve cells from human patients. These cells were then used to test the ability of NSAIDs to prevent the formation of beta-amyloid deposits.

According to Philipp Koch, who led this study, “To predict the efficacy of Alzheimer drugs, such tests have to be performed directly on the affected human nerve cells.”

Nerve cells made from human induced pluripotent stem cells were completely resistant to NSAIDs. These drugs showed no ability to alter the biochemical mechanisms in these cells that eventually lead to the production of beta-amyloid.

Why then did they work in laboratory animals? Koch and his colleagues think that biochemical differences between laboratory mice and human cells allow the drugs to work in one but not in the other. In Koch’s words, “The results are simply not transferable.”

In the future, scientists hope to screen potential Alzheimer’s disease drugs with human cells made from the patient’s own cells.

“The development of a single drug takes an average of ten years,” said Brüstle. “By using patient-specific nerve cells as a test system, investments by pharmaceutical companies and the tedious search for urgently needed Alzheimer’s medications could be greatly streamlined.”

Understanding the Role of a Protein in Familial Alzheimer’s Disease


Lawrence Goldstein, director of the UC San Diego Stem Cell Program and a member of the Departments of Cellular and Molecular Medicine and Neurosciences, has an abiding interest in Alzheimer’s disease (AD).  To that end, he and his colleagues have used genetically engineered human induced pluripotent stem cells to determine the role a particular protein plays in the causation of familial AD.  Apparently, a simple loss-of-function model does not contribute to the inherited form of this disorder.  Goldstein hopes that his findings might be able to better explain the mechanisms behind AD and help drug makers design better drugs to treat this disease.

Familial AD is a subset of the larger group of conditions known as early-onset AD.  The vast majority of cases of AD are “sporadic” and do not have a precise known cause, even though age is a primary risk factor (an estimated 5.2 million Americans have AD).  Familial AD is causes by mutations in particular genes.  One of these genes, PS1, encodes a protein called “presenilin 1,” which acts as a protease (an enzyme that clips other proteins in half).  Presenilin 1 is the catalytic component of a protein complex called “gamma-secretase.”  Presenilin 1 forms a complex with three other proteins (Nicastrin, Aph1, Pen2) to form gamma-secretase, and this enzyme attacks specific proteins that are embedded in the cell membrane and clips them into smaller pieces.

gamma-secretase

By clipping these cell membrane proteins into smaller pieces, gamma-secretase helps the cell transport cellular material from one side of the cell membrane to the other side or form the outside of the cell to the inside.

One of the substrates of gamma-secretase is a protein called amyloid precursor protein (APP).  While the function of APP remains unknown, APP cleavage by the gamma-secretase produces small protein fragments known as amyloid beta.

A consensus among AD researchers is that the accumulation of specific forms of amyloid beta causes the formation of the amyloid plaques that kills off neurons and leads to the onset of AD.  The most abundant product of gamma-secretase cleavage of APP is a protein called “Aβ40.”  This protein is forty amino acids long and does not cause any brain damage.  However, a minority product of APP cleave by the gamma-secretase is “Aβ42,” which is 42 amino acids long and forms the amyloid plaques and neurofibillar tangles that are so characteristic of AD (see Scheuner, D., et al., Nat. Med. 2, 864–870).

According to Goldstein, most of the time, gamma-secretase clips APP without causing any problems, but some 20% of the time, the protein clips APP incorrectly and this results in the plaque-forming forms of amyloid beta.  Goldstein explained: “Our research demonstrates very precisely that mutations in PS1 double the frequency of bad cuts.”

To demonstrate this, Goldstein and his co-workers purchased human induced pluripotent stem cells and differentiated them into neurons.  These neurons contained different alleles (forms) of the PS1 gene, and some of these mutant forms of PS1 contained the types of mutations that cause familial AD.  Once PS1 allele in particular called PS1 ΔE9 increased the ratio of Aβ42 to Aβ40 dose-dependent manner.  Since the PS1 ΔE9 causes familial AD, this research elucidates precisely why it does so.

“We were able to investigate exactly how specific mutations and their frequency change the behavior of neurons.  We took finely engineered cells that we knew and understood and then looked how a single mutation causes changed in the molecular scissors and what happened next.”

Presenilin allele consequences

Goldstein further notes, “In some ways, this is a powerful technical demonstration of the promise of stem cells and genomics research in better understanding and ultimately treating AD.  We were able to identify and assign precise limits on how a mutations works in familial AD.  That’s an important step in advancing the science, in finding drugs and treatments that can slow, maybe reverse, the disease’s devastating effects.”

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.”

Why Do Some People Get Alzheimer’s Disease but Others Do Not?


Everyone has a brain that has the tools to develop Alzheimer’s disease. Why therefore do some people develop Alzheimer’s disease (AD) while others do not? An estimated five million Americans have AD – a number projected to triple by 2050– the vast majority of people do not and will not develop the devastating neurological condition. What is the difference between those whole develop AD and those who do not?

Subhojit Roy, associate professor in the Departments of Pathology and Neurosciences at the University of California, San Diego School of Medicine, asked this exact question. In a paper published in the journal Neuron, Roy and colleagues offer an explanation for this question. As it turns out, in most people there is a critical separation between a protein and an enzyme that, when combined, trigger the progressive cell degeneration and death characteristic of AD.

“It’s like physically separating gunpowder and match so that the inevitable explosion is avoided,” says principal investigator Roy, a cell biologist and neuropathologist in the Shiley-Marcos Alzheimer’s Disease Research Center at UC San Diego. “Knowing how the gunpowder and match are separated may give us new insights into possibly stopping the disease.”

Neurologists measure the severity of AD by the loss of functioning neurons. In terms of pathology, there are two tell-tale signs of AD: a) clumps of a protein called beta-amyloid “plaques” that accumulate outside neurons and, b) threads or “tangles” of ‘tau” protein found inside neurons. Most neuroscientists believe AD is caused by the accumulation of aggregates of beta-amyloid protein, which triggers a sequence of events that leads to impaired cell function and death. This so-called “amyloid cascade hypothesis” puts beta-amyloid protein at the center of AD pathology.

Creating beta-amyloid requires the convergence of a protein called amyloid precursor protein (APP) and an enzyme that cleaves APP into smaller toxic fragments called beta-secretase or BACE.

“Both of these proteins are highly expressed in the brain,” says Roy, “and if they were allowed to combine continuously, we would all have AD.”

It sounds inexorable, but it doesn’t always happen. Using cultured hippocampal neurons and tissue from human and mouse brains, Roy and Utpal Das, a postdoctoral fellow in Roy’s lab who was the first author of this paper, and other colleagues, discovered that healthy brain cells largely segregate APP and BACE-1 into distinct compartments as soon as they are manufactured, which ensures that these two proteins do not have much contact with each other.

“Nature seems to have come up with an interesting trick to separate co-conspirators,” says Roy.

What then brings APP and BACE together? Roy and his team found that those conditions that promote greater production of beta-amyloid protein also increase the convergence of APP and BACE. Specifically, an increase in neuronal electrical activity, which is known to increase the production of beta-amyloid, also increased the convergence of APP and BACE. Post-mortem examinations of AD patients have shown that the cellular locations of APP and BACE overlap, which lends credence to the pathophysiological significance of this phenomenon in human disease.

Neurons were cotransfected with APP:GFP and BACE-1:mCherry, neurons were stimulated with glycine or picrotoxin (PTX), and the colocalization of APP and BACE-1 fluorescence was analyzed (see Experimental Procedures for more details). (B) Note that stimulation with glycine greatly increased APP/BACE-1 colocalization in dendrites (overlaid images on right). (C and D) Quantification of APP/BACE-1 colocalization. Note that increases in glycine-induced APP/BACE-1 convergence can
Neurons were cotransfected with APP:GFP and BACE-1:mCherry, neurons were stimulated with glycine or
picrotoxin (PTX), and the colocalization of APP and BACE-1 fluorescence was analyzed (see Experimental Procedures for more details).
(B) Note that stimulation with glycine greatly increased APP/BACE-1 colocalization in dendrites (overlaid images on right).
(C and D) Quantification of APP/BACE-1 colocalization. Note that increases in glycine-induced APP/BACE-1 convergence can

Das says that their findings are fundamentally important because they elucidate some of the earliest molecular events triggering AD and show how a healthy brain naturally avoids them. In clinical terms, they point to a possible new avenue for ultimately treating or even preventing the disease.

“An exciting aspect is that we can perhaps screen for molecules that can physically keep APP and BACE-1 apart,” says Das. “It’s a somewhat unconventional approach.”

Getting Neurons Made From Stem Cells to Show Activity After Transplantation


Stem cells can differentiate into neurons, but can they integrate into the wider neural network and contribute to the function of the central nervous system? The evidence for this is scant. Even though transplanted stem cells can increase the function of the nervous system or decrease or halt the deterioration of the nervous system, there is little direct evidence that neurons made from stem cells can connect and signal to other neurons.

Until now.

The laboratory of clinical neurologist Stuart Lipton at the Sanford-Burnham Medical Research Institute has used embryonic stem cells for this experiment. They differentiated these stem cells into neurons and implanted them into the brains of laboratory rodents. However, transplanting them, they also genetically engineered these neurons so that they would express genes from bacteria that encode fast-acting light-activated ion channels. These ion channels would cause the neurons to activate a nerve impulse if a light was shined on them. This provided a way to artificially activate these neurons to determine if they were connected to other neurons and integrated into the central nervous system and it neural network.

This technique is called “optogenetics,” and it is a relatively new field of molecular biology. By using genes from particular species of bacteria that encode light-activated ion channels, cells that normally do not respond to light can be engineered to respond to particular frequencies of light. The use of optogenetics in stem cells is also novel, but this increasingly powerful technology is capturing the imaginations of more and more scientists every day.

To continue with our story, what happened to the implanted stem cell-derived neurons when they were illuminated? They made nerve impulses, but ion changes were detected in neurons that were located far from the implanted neurons. The only reasonable explanation for these observations is that the implanted neurons are forming proper neural connections with other neurons and any nerve impulses established in the implanted neurons stimulate nerve impulses in connected neurons that then activate neurons in all the neural pathways connected to them.

Lipton said of his work, “We showed for the first time that embryonic stem cells that we’ve programmed to become neurons can integrate into existing brain circuits and fire patterns of electrical activity that are critical for consciousness and neural network activity.”

Even more interestingly, Lipton and his team implanted neurons into a portion of the brain known as the “hippocampus.” This structure helps to consolidate information from short-term memory to long-term memory. It also helps with spatial navigation. Since the rate at which neurons generate or “fire” nerve impulses varies from one region of the brain to another, Lipton wanted to know if his stem cell-derived neurons would fire at the same rate as those native neurons in the hippocampus of the laboratory rodent. The answer was a clear yes. The implanted neurons fired at roughly the same rate as the surrounding, endogenous hippocampal neurons. This suggests that the implanted neurons adapt and ultimately become physiologically like those neurons around them.

hippocampus

Lipton sees great potential for clinical treatments in this work: “Based on these results, we might be able to restore brain activity – and thus restore motor and cognitive function – by transplanting easily manipulated neuronal cells derived from embryonic stem cells.”

Lipton’s optimism is infectious to one extent, but I think we must temper it by realizing that Lipton shined lights on his neurons, and that this is something that we cannot do to the brains of human beings. However, if neurons that respond to other neurons can be made and implanted into the brains of Alzheimer’s disease patients, for example, then this could definitely restore cognitive ability in patients with neurodegenerative diseases.