Archive for the ‘Ca2+ triggered activation’ Category

Lesson 6 of Cell Signaling & Motility – Cytoskeleton II: #TUBiol3373

Stephen J. Williams, Ph.D.

In this lesson we will go over the biochemical makeup and formation of various actin containing cellular structures involved in cellular motility, structure, as well as the dynamics of muscular contraction.  The lesson had been put on your Canvas and I am emailing you the Google Docs version.  If you are having problems downloading you can download here (I believe maybe the Canvas version had problems with embedding videos properly so that is why I am sending you also by email)

Download Below

cell signaling 6 lesson 2020

After opening the powerpoint (or Google Doc) please review with the following notes which highlight some concepts as well as some reviews and reminders of past lectures.  It may be handy to also have lecture 5 handy if you need to refer to it.  In between some sections there will be polls (really multiple choice quizzes DON’T WORRY you will not be graded on them but they are for your benefit.  There will also be a section under Comments all the way at the end and at the last quiz where you can also ask questions.

Remember you can always email me or Tweet me any questions @StephenJWillia2 using the hashtag #TUBiol3373.

In addition you can also leave comments at the very bottom which can be answered.

Slide 2 of lesson 6 is a refresher of the end of our last lecture, talking about Actin Binding Regulatory Proteins.



















The picture above shows a brief review of some of the structures and actin binding proteins involved in helping to form these actin filament structures (like filamin in cross linked structures, profilin which binds the actin monomers [G-actin] and helps with addition of these monomers to the leading plus end.

*** Remember G-actin (Globular Actin) is the monomer and F-actin (filamentious actin) is the polymerized actin strand [filament]

Also remember from the last lecture that G-Actin as monomer has affinity for ATP {Adenosine triphosphate} and these G-Actin-ATP will be able to polymerize to form the F-Actin form.  Also F-actin can then hydrolyze the ATP to ADP and inorganic phosphate.  At this point the actin-ADP unit looses affinity for the remaining F-Actin chain and depolymerization can occur


An event referred to as TREADMILLING or when the G actin units are removed from minus end and added to the plus (or growing barbed) end

Also remember that there is a critical concentration of G-Actin-ATP needed for bypassing the lag phase of nucleation before the elongation phase and the rate of addition to the plus end is faster than addition to minus end and greater than the rate of depolymerization at the minus end

Cell Structures That Involve Actin (see links for more information)

  1. filopodia
  2. parallel actin bundles
  3. actin cortex
  4. lamellipodia
  5. stress fibers
  6. microvilli
  7. contractile ring in cytokinesis



















Nucleating proteins Arp (actin related protein and Formins

Arp ====> formation of lamellipodia

Formins ====> formation of stress fibers

Process involving formins starts with a signaling event by activation of a G-protein, the GTP binding protein Rho

Rho is a subfamily member of the Ras superfamily.  The Rho family consists of cdc42, rac1, and RhoA (we will discuss at a later date).  Rho acts like G proteins, as a molecular switch.

Note that just like the Ras member of G-proteins and the Ras GTP/GDP cycle, the Rho activation, deactivation cycle also depends on GEFs [Guanine nucleotide exchange factors] and GAPs [GTPase activating proteins] and also GDIs [guanine nucleotide dissociation inhibitors which we will discuss later but involved in preventing Rho diffusion in the cell, acting as a tether].

Myosin and Motor (muscle) Function; Neuromuscular junctions, the sarcoplasmic reticulum and Ohhh the plethora of signaling events

In this section, from slides 29 to 54, we talk about myosin and the interactions between myosin and actin in formation of the contractile unit of the muscle (skeletal).

We also talk about some familiar signaling events, in particular the neuromuscular junction.

At this junction is a special type of acetylcholine receptor

Remember we talked about two types of acetylcholine receptors:

  1. muscarinic receptors – typical GPCRs that tranduce the signal via Gi or Gq depending on the muscarinic subtype
  2. nicotinic receptors – these are ligand {receptor} operated channels and when activated opens a Na+ channel which leads to depolarization


Now the depolarization activates another set of channels, the voltage operated calcium channels so we have two types of ion channels: Receptor {ligand} operated channels and Voltage operated channels.  These are sometimes abbreviated as ROCs and VOCs.

The unit of the myofibril on the contactile unit of the skeletal muscle is the sarcomere and upon the calcium transient, the sarcomere shortens with the two z-disks moving closer to each other as shown in the video in the lecture.

Also briefly review the introduction part on microtubules. We will finish that next week. Note that the microtubule is comprised of the protein tubulin, which is another GTP binding protein.

For other articles and more information please see

Lesson 5 Cell Signaling And Motility: Cytoskeleton & Actin: Curations and Articles of reference as supplemental information: #TUBiol3373

Role of Calcium, the Actin Skeleton, and Lipid Structures in Signaling and Cell Motility

Identification of Biomarkers that are Related to the Actin Cytoskeleton










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Synapse activity in neurotransmission

Larry H. Bernstein, MD, FCAP, Curator



The case of the silent synapses: Why are only 20% of synapses active during neurotransmission?

Unknown information coding in the brain?
February 26, 2016   http://www.kurzweilai.net/study-finds-only-a-small-portion-of-synapses-may-be-active-during-neurotransmission

Using a fluorescent molecule to track neurotransmission of dopamine in mouse synapses, scientists made a puzzling discovery. … (credit: Sulzer Lab/Columbia University Medical Center)

Columbia University scientists recently tested a new optical technique to study how information is transmitted in the brains of mice and made a surprising discovery: When stimulated electrically to release dopamine (a neurotransmitteror chemical released by neurons, or nerve cells, to send signals to other nerve cells), only about 20 percent of synapses — the connections between cells that control brain activity — were active at any given time.

The effect had never been noticed. “Older techniques only revealed what was going on in large groups of synapses,” explained David Sulzer, PhD, professor of neurobiology in Psychiatry, Neurology, and Pharmacology at Columbia University Medical Center (CUMC). “We needed a way to observe the neurotransmitter activity of individual synapses, to help us better understand their intricate behavior.”

So Sulzer’s team turned to Dalibor Sames, PhD, associate professor of chemistry at Columbia, to develop a novel compound called “fluorescent false neurotransmitter 200″ (FFN200). When added to brain tissue or nerve cells from mice, FFN200 mimicked the brain’s natural neurotransmitters, allowing the researchers to spy on chemical messaging in action, focusing on complex tasks such as learning and memory.


Only 20% of synapses (red) were observed to transmit dopamine. The rest (green) were found to be silent. (credit: Sulzer Lab/Columbia University Medical Center)

Silent synapses: unknown information coding?

Using a fluorescence microscope, the researchers were able for the first time to view the release and re-uptake of dopamine — a neurotransmitter involved in motor learning, habit formation, and reward-seeking behavior — in individual synapses.

When all the neurons were electrically stimulated in a sample of brain tissue, the researchers expected all the synapses to release dopamine. Instead, they found that less than 20 percent of dopaminergic synapses were active following a pulse of electricity.

One possibility: these silent synapses hint at a mechanism of information coding in the brain that’s yet to be revealed, the researchers hypothesize.

The study’s authors plan to pursue that hypothesis in future experiments and examine how other neurotransmitters behave. “If we can work this out, we may learn a lot more about how alterations in dopamine levels are involved in brain disorders such as Parkinson’s disease, addiction, and schizophrenia,” Sulzer said.

The study was published in the latest issue of Nature Neuroscience.

The authors note in the paper that “the state of silent vesicle clusters may be important in disorders such as schizophrenia, which show striatal hyperdopaminergia [excessive release of dopamine in the brain’s reward center] and cortical hypodopaminergia [low amounts of dopamine in the cortex] and processes of  ‘unsilencing’ may have clinical applications for diseases such as Parkinson’s disease.”

Columbia Medical | Study Finds Only a Small Portion of Synapses May Be Active During Neurotransmission

Abstract of Fluorescent false neurotransmitter reveals functionally silent dopamine vesicle clusters in the striatum

Neurotransmission at dopaminergic synapses has been studied with techniques that provide high temporal resolution, but cannot resolve individual synapses. To elucidate the spatial dynamics and heterogeneity of individual dopamine boutons, we developed fluorescent false neurotransmitter 200 (FFN200), a vesicular monoamine transporter 2 (VMAT2) substrate that selectively traces monoamine exocytosis in both neuronal cell culture and brain tissue. By monitoring electrically evoked Ca2+ transients with GCaMP3 and FFN200 release simultaneously, we found that only a small fraction of dopamine boutons that exhibited Ca2+ influx engaged in exocytosis, a result confirmed with activity-dependent loading of the endocytic probe FM1-43. Thus, only a low fraction of striatal dopamine axonal sites with uptake-competent VMAT2 vesicles are capable of transmitter release. This is consistent with the presence of functionally ‘silent’ dopamine vesicle clusters and represents, to the best of our knowledge, the first report suggestive of presynaptically silent neuromodulatory synapses.

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Reverse Engineering of Vision

Larry H. Bernstein, MD, FCAP, Curator



CMU announces research project to reverse-engineer brain algorithms, funded by IARPA

A Human Genome Project-level plan to make computers learn like humans
February 5, 2016   http://www.kurzweilai.net/cmu-announces-research-project-to-reverse-engineer-brain-algorithms-funded-by-iarpa


Individual brain cells within a neural network are highlighted in this image obtained using a fluorescent imaging technique (credit: Sandra Kuhlman/CMU)

Carnegie Mellon University is embarking on a five-year, $12 million research effort to reverse-engineer the brain and “make computers think more like humans,” funded by the U.S. Intelligence Advanced Research Projects Activity (IARPA). The research is led by Tai Sing Lee, a professor in the Computer Science Department and the Center for the Neural Basis of Cognition (CNBC).

The research effort, through IARPA’s Machine Intelligence from Cortical Networks (MICrONS) research program, is part of the U.S. BRAIN Initiative to revolutionize the understanding of the human brain.

A “Human Genome Project” for the brain’s visual system

“MICrONS is similar in design and scope to the Human Genome Project, which first sequenced and mapped all human genes,” Lee said. “Its impact will likely be long-lasting and promises to be a game changer in neuroscience and artificial intelligence.”

The researchers will attempt to discover the principles and rules the brain’s visual system uses to process information. They believe this deeper understanding could serve as a springboard to revolutionize machine learning algorithms and computer vision.

In particular, the researchers seek to improve the performance of artificial neural networks — computational models for artificial intelligence inspired by the central nervous systems of animals. Interest in neural nets has recently undergone a resurgence thanks to growing computational power and datasets. Neural nets now are used in a wide variety of applications in which computers can learn to recognize faces, understand speech and handwriting, make decisions for self-driving cars, perform automated trading and detect financial fraud.

How neurons in one region of the visual cortex behave

“But today’s neural nets use algorithms that were essentially developed in the early 1980s,” Lee said. “Powerful as they are, they still aren’t nearly as efficient or powerful as those used by the human brain. For instance, to learn to recognize an object, a computer might need to be shown thousands of labeled examples and taught in a supervised manner, while a person would require only a handful and might not need supervision.”

To better understand the brain’s connections, Sandra Kuhlman, assistant professor of biological sciences at Carnegie Mellon and the CNBC, will use a technique called “two-photon calcium imaging microscopy” to record signaling of tens of thousands of individual neurons in mice as they process visual information, an unprecedented feat. In the past, only a single neuron, or tens of neurons, typically have been sampled in an experiment, she noted.

“By incorporating molecular sensors to monitor neural activity in combination with sophisticated optical methods, it is now possible to simultaneously track the neural dynamics of most, if not all, of the neurons within a brain region,” Kuhlman said. “As a result we will produce a massive dataset that will give us a detailed picture of how neurons in one region of the visual cortex behave.”

A multi-institution research team

Other collaborators are Alan Yuille, the Bloomberg Distinguished Professor of Cognitive Science and Computer Science at Johns Hopkins University, and another MICrONS team at the Wyss Institute for Biologically Inspired Engineering, led by George Church, professor of genetics at Harvard Medical School.

The Harvard-led team, working with investigators at Cold Spring Harbor Laboratory, MIT, and Columbia University, is developing revolutionary techniques to reconstruct the complete circuitry of the neurons recorded at CMU. The database, along with two other databases contributed by other MICrONS teams, unprecedented in scale, will be made publicly available for research groups all over the world.

In this MICrONS project, CMU researchers and their collaborators in other universities will use these massive databases to evaluate a number of computational and learning models as they improve their understanding of the brain’s computational principles and reverse-engineer the data to build better computer algorithms for learning and pattern recognition.

“The hope is that this knowledge will lead to the development of a new generation of machine learning algorithms that will allow AI machines to learn without supervision and from a few examples, which are hallmarks of human intelligence,” Lee said.

The CNBC is a collaborative center between Carnegie Mellon and the University of Pittsburgh. BrainHub is a neuroscience research initiative that brings together the university’s strengths in biology, computer science, psychology, statistics and engineering to foster research on understanding how the structure and activity of the brain give rise to complex behaviors.

The MICrONS team at CMU allso includes Abhinav Gupta, assistant professor of robotics; Gary Miller, professor of computer science; Rob Kass, professor of statistics and machine learning and interim co-director of the CNBC; Byron Yu, associate professor of electrical and computer engineering and biomedical engineering and the CNBC; Steve Chase, assistant professor of biomedical engineering and the CNBC; and Ruslan Salakhutdinov, one of the co-creators of the deep belief network, a new model of machine learning that was inspired by recurrent connections in the brain, who will join CMU as an assistant professor of machine learning in the fall.

Other members of the team include Brent Doiron, associate professor of mathematics at Pitt, and Spencer Smith, assistant professor of neuroscience and neuro-engineering at the University of North Carolina.

Not all machine-intelligence experts are on board with reverse-engineering the brain. In a Facebook post today, Yann LeCun, Director of AI Research at Facebook and a professor at New York University, asked the question in a recent lecture, “Should we copy the brain to build intelligent machines?” “My answer was ‘no, because we need to understand the underlying principles of intelligence to know what to copy. But we should draw inspiration from biology.’”


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Graphene Interaction with Neurons

Larry H. Bernstein, MD, FCAP, Curator



Graphene Shown to Safely Interact with Neurons in the Brain

University of Cambridge

(Source: University of Cambridge)



Researchers have successfully demonstrated how it is possible to interface graphene – a two-dimensional form of carbon – with neurons, or nerve cells, while maintaining the integrity of these vital cells. The work may be used to build graphene-based electrodes that can safely be implanted in the brain, offering promise for the restoration of sensory functions for amputee or paralyzed patients, or for individuals with motor disorders such as epilepsy or Parkinson’s disease.

The research, published in the journal ACS Nano, was an interdisciplinary collaboration coordinated by the University of Trieste in Italy and the Cambridge Graphene Centre.

Previously, other groups had shown that it is possible to use treated graphene to interact with neurons. However the signal to noise ratio from this interface was very low. By developing methods of working with untreated graphene, the researchers retained the material’s electrical conductivity, making it a significantly better electrode.

“For the first time we interfaced graphene to neurons directly,” said Professor Laura Ballerini of the University of Trieste in Italy. “We then tested the ability of neurons to generate electrical signals known to represent brain activities, and found that the neurons retained their neuronal signaling properties unaltered. This is the first functional study of neuronal synaptic activity using uncoated graphene based materials.”

Our understanding of the brain has increased to such a degree that by interfacing directly between the brain and the outside world we can now harness and control some of its functions. For instance, by measuring the brain’s electrical impulses, sensory functions can be recovered. This can be used to control robotic arms for amputee patients or any number of basic processes for paralyzed patients – from speech to movement of objects in the world around them. Alternatively, by interfering with these electrical impulses, motor disorders (such as epilepsy or Parkinson’s) can start to be controlled.

Scientists have made this possible by developing electrodes that can be placed deep within the brain. These electrodes connect directly to neurons and transmit their electrical signals away from the body, allowing their meaning to be decoded.

However, the interface between neurons and electrodes has often been problematic: not only do the electrodes need to be highly sensitive to electrical impulses, but they need to be stable in the body without altering the tissue they measure.

Too often the modern electrodes used for this interface (based on tungsten or silicon) suffer from partial or complete loss of signal over time. This is often caused by the formation of scar tissue from the electrode insertion, which prevents the electrode from moving with the natural movements of the brain due to its rigid nature.

Graphene has been shown to be a promising material to solve these problems, because of its excellent conductivity, flexibility, biocompatibility and stability within the body.

Based on experiments conducted in rat brain cell cultures, the researchers found that untreated graphene electrodes interfaced well with neurons. By studying the neurons with electron microscopy and immunofluorescence the researchers found that they remained healthy, transmitting normal electric impulses and, importantly, none of the adverse reactions which lead to the damaging scar tissue were seen.

According to the researchers, this is the first step towards using pristine graphene-based materials as an electrode for a neuro-interface. In future, the researchers will investigate how different forms of graphene, from multiple layers to monolayers, are able to affect neurons, and whether tuning the material properties of graphene might alter the synapses and neuronal excitability in new and unique ways. “Hopefully this will pave the way for better deep brain implants to both harness and control the brain, with higher sensitivity and fewer unwanted side effects,” said Ballerini.

“We are currently involved in frontline research in graphene technology towards biomedical applications,” said Professor Maurizio Prato from the University of Trieste. “In this scenario, the development and translation in neurology of graphene-based high-performance biodevices requires the exploration of the interactions between graphene nano- and micro-sheets with the sophisticated signalling machinery of nerve cells. Our work is only a first step in that direction.”

“These initial results show how we are just at the tip of the iceberg when it comes to the potential of graphene and related materials in bio-applications and medicine,” said Professor Andrea Ferrari, Director of the Cambridge Graphene Centre. “The expertise developed at the Cambridge Graphene Centre allows us to produce large quantities of pristine material in solution, and this study proves the compatibility of our process with neuro-interfaces.”

The research was funded by the Graphene Flagship, a European initiative which promotes a collaborative approach to research with an aim of helping to translate graphene out of the academic laboratory, through local industry and into society.

Source: University of Cambridge


Remembering to Remember Supported by Two Distinct Brain Processes


To investigate how prospective memory is processed in the brain, psychological scientist Mark McDaniel of Washington University in St. Louis and colleagues had participants lie in an fMRI scanner and asked them to press one of two buttons to indicate whether a word that popped up on a screen was a member of a designated category.  In addition to this ongoing activity, participants were asked to try to remember to press a third button whenever a special target popped up. The task was designed to tap into participants’ prospective memory, or their ability to remember to take certain actions in response to specific future events.

When McDaniel and colleagues analyzed the fMRI data, they observed that two distinct brain activation patterns emerged when participants made the correct button press for a special target.

When the special target was not relevant to the ongoing activity—such as a syllable like “tor”—participants seemed to rely on top-down brain processes supported by the prefrontal cortex. In order to answer correctly when the special syllable flashed up on the screen, the participants had to sustain their attention and monitor for the special syllable throughout the entire task. In the grocery bag scenario, this would be like remembering to bring the grocery bags by constantly reminding yourself that you can’t forget them.

When the special target was integral to the ongoing activity—such as a whole word, like “table”—participants recruited a different set of brain regions, and they didn’t show sustained activation in these regions. The findings suggest that remembering what to do when the special target was a whole word didn’t require the same type of top-down monitoring. Instead, the target word seemed to act as an environmental cue that prompted participants to make the appropriate response—like reminding yourself to bring the grocery bags by leaving them near the front door.

“These findings suggest that people could make use of several different strategies to accomplish prospective memory tasks,” says McDaniel.

McDaniel and colleagues are continuing their research on prospective memory, examining how this phenomenon might change with age.

Co-authors on this research include Pamela LaMontagne, Michael Scullin, Todd Braver of Washington University in St. Louis; and Stefanie Beck of Technische Universität Dresden.

This research was funded by the National Institute on Aging, the Washington University Institute of Clinical and Translation Sciences, the National Center for Advancing Translational Sciences, and the German Science Foundation.

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The transport of molecules across membranes

Larry H. Bernstein, MD, FCAP, Curator



Cellular Transport and the Nobel Prize for Medicine

Extracted  from October 8, 2013 | by

The 2013 Nobel Prize in Physiology or Medicine was  awarded to  to Randy W. Schekman, at the University of California at Berkeley; James E. Rothman,  at Yale University in New Haven, Connecticut; and Thomas C. Südhof,  at Stanford University, for their discoveries of machinery regulating vesicle traffic, a major transport system in cells.three U.S. scientists for their work on how the cell coordinates its transport system to shuttle proteins and other molecules from one location to another.

The organization and transport of molecules across cellular mmembranes is accomplished via vesicles that shuttle cargo between organelles or fuse to other structures to release their cargo outside the cell. The vesicle transport system is critical for a variety of physiological processes, ranging from signaling in the brainto release of hormones and immune cytokines.

Schekman identified three classes of genes that control different facets of the cell’s transport system.

Vesicle fusion


This was followed by James Rothman’s discovery that a protein complex enables vesicles to fuse with their target membranes (pictured in orange above). This lock and key mechanism ensures that the vesicle fuses at the right location and that cargo molecules are delivered to the correct destination.

Also in the 1990s, Thomas Südhof was studying how nerve cells communicate in the brain. Calcium ions were known to be involved in vesicle cargo release, and Südhof searched for calcium sensitive proteins in nerve cells. He identified the molecular machinery (pictured in purple above) that responds to an influx of calcium ions (Ca2+) and triggers vesicle fusion.

Extracellular vesicles are  participate in the pathogenesis of various diseases, most notably neurodegenerative disorders, and extracellular vesicles are likely to have therapeutic applications in large-molecule drug delivery.


  1. The Nobel Prize in Physiology or Medicine 2013 – Press Release. Nobelprize.org. 7 Oct 2013.
  2. Andaloussi et al. Extracellular vesicles: biology and emerging therapeutic opportunities. Nature Reviews Drug Discovery 2013 Vol: 12(5):347-357. DOI: 10.1038/nrd3978
    View abstract
  3. Anderson et al. Role of extracellular membrane vesicles in the pathogenesis of various diseases, including cancer, renal diseases, atherosclerosis, and arthritis. Lab Invest. 2010 Nov;90(11):1549-57. DOI: 10.1038/labinvest.2010.152. Epub 2010 Aug 30.
    View abstract


Machinery Regulating Vesicle Traffic, A Major Transport System in our Cells


Together, Rothman, Schekman and Südhof have transformed the way we view transport of molecular cargo to specific destinations inside and outside the cell. Their discoveries explain a long-standing enigma in cell biology and also shed new light on how disturbances in this machinery can have deleterious effects and contribute to conditions such as neurological diseases, diabetes, and immunological disorders.

Eukaryotic cells differ from prokaryotic cells by their more complex intracellular organization. In eukaryotes, specific cellular functions are compartmentalized into the cell nucleus and organelles surrounded by intracellular membranes. This compartmentalization vastly improves the efficiency of many cellular functions and prevents potentially dangerous molecules from roaming freely within the cell. But when distinct cellular processes are compartmentalized, a problem emerges. Different compartments need to exchange specific molecules (Figure 1). Furthermore, certain molecules need to be exported to the cell exterior. Most molecules are too large to directly pass through membranes, thus a mechanism that ensures specific delivery of this molecular cargo is required.

Figure 1: Each cell in the body has a complex organization where specific cellular functions are separated into different compartments called organelles. Molecules produced in the cell are packaged in vesicles and transported with special and temporal precision to the correct locations within and outside the cell.

Mysteries of cellular compartmentalization have long intrigued scientists. Improved light microscopy techniques aided in the understanding of intracellular organization in eukaryotic cells, but the advent of electron microscopy and new staining techniques, combined with subcellular fractionation assays using differential ultracentrifugation procedures, led to a deeper understanding of the cell’s inner life. Albert Claude, George Palade and Christian de Duve, who received the Nobel Prize in Physiology or Medicine 1974*, were pioneers in this area and have shed light on how the cell is organized and compartmentalized. Secretory proteins were shown to be produced on ribosomes in the endoplasmic reticulum (ER) and trafficked to the Golgi complex (named after the 1906 Nobel Laureate Camillo Golgi) (Figure 1). Progress was also made in deciphering how proteins find their appropriate destination. Günter Blobel was awarded the 1999 Nobel Prize in Physiology or Medicine* for his discoveries that proteins have intrinsic signals that govern their transport and localization in the cell. Yet, a lingering question remained. How are molecules, including hormones, transport proteins, and neurotransmitters, correctly routed to their appropriate destination? From the work of Palade, the traffic of secretory proteins from the ER was understood to be carried out using small membrane-surrounded vesicles that bud from one membrane and fuse with another, but how precision could be acquired in this process remained enigmatic.

The work of  Rothman, Schekman and Südhof represents a paradigm shift in our understanding of how the eukaryotic cell, with its complex internal compartmentalization, organizes the routing of molecules packaged in vesicles to various intracellular destinations, as well as to the outside of the cell. Specificity in the delivery of molecular cargo is essential for cell function and survival. This specificity is required for the release of neurotransmitters into the presynaptic region of a nerve cell to transmit a signal to a neighboring nerve cell. Likewise, specificity is required for the export of hormones such as insulin to the cell surface. While vesicles within the cell were long known to be critical components of this transportation scheme, the precise mechanism by which these vesicles found their correct destination and how they fused with organelles or the plasma membrane to deliver the cargo remained mysterious. The work of the three 2013 Laureates radically altered our understanding of this aspect of cell physiology. Randy W. Schekman used yeast genetics to identify a set of genes critical for vesicular trafficking. He showed that these genes were essential for life and could be classified into three categories regulating different aspects of vesicle transport. James E. Rothman embarked on a biochemical approach and identified proteins that form a functional complex controlling cell fusion. Proteins on the vesicle and target membrane sides bind in specific combinations, ensuring precise delivery of molecular cargo to the right destination. Thomas C. Südhof became interested in how vesicle fusion machinery was controlled. He unraveled the mechanism by which calcium ions trigger release of neurotransmitters, and identified key regulatory components in the vesicle fusion machinery.

Schekman discovered genes encoding proteins that are key regulators of vesicle traffic. Comparing normal with genetically mutated yeast cells in which vesicle traffic was disturbed, he identified genes that control transport to different compartments and to the cell surface

Rothman published a series of papers where he reconstituted the intracellular transport of the VSV-G protein within the Golgi complex. He then used the assay to study both vesicle budding and fusion, and purified proteins from the cytoplasm that were required for transport. The first protein to be purified was the Nethylmaleimide-sensitive factor (NSF). Rothman’s discovery of NSF paved the way for the subsequent identification of other proteins important for the control of vesicle fusion, and the next one in line was SNAP (soluble NSFattachment protein). SNAPs bind to membranes and assist in the recruitment of NSF.

One of the yeast mutants, sec18, corresponded to NSF, which also revealed that the vesicle fusion machinery was evolutionarily ancient. Furthermore, Rothman and Schekman collaboratively cloned sec17 and provided evidence of its functional equivalence to SNAP. Other sec genes were shown to correspond to genes encoding fusion proteins were identified by other methods.

Using the NSF and SNAP proteins as bait, Rothman next turned to brain tissue, from which he purified proteins that he later named SNAREs (soluble NSF-attachment protein receptors). Intriguingly, three SNARE proteins, VAMP/Synaptobrevin, SNAP-25 and syntaxin, were found in stoichiometric amounts, which suggested to Rothman that they functioned together in the vesicle and target membranes. The three proteins had previously been identified by several scientists, including Richard Scheller, Kimio Akagawa, Reinhard Jahn and Pietro de Camilli, and localized to the presynaptic region, but their function was largely unknown. VAMP/Synaptobrevin resided on the vesicle, whereas SNAP-25 and syntaxin were found at the plasma membrane. This prompted Rothman to propose a hypothesis – the SNARE hypothesis – which stipulated that target and vesicle SNAREs (t-SNAREs and v-SNAREs) were critical for vesicle fusion through a set of sequential steps of synaptic docking, activation and fusion.

Thomas C. Südhof originally trained at the Georg-August-Universität and the Max-Planck Institute for Biophysical Sciences in Göttingen, Germany, and was a postdoctoral fellow with Michael Brown and Joseph Goldstein (Nobel Prize 1985) at University of Texas Southwestern Medical School in Dallas. As a junior group leader, he set out to study how synaptic vesicle fusion was controlled. Rothman and Schekman had provided fundamental machinery for vesicle fusion, but how vesicle fusion was temporally controlled remained enigmatic. Vesicular fusions in the body need to be kept carefully in check, and in some cases vesicle fusion has to be executed with high precision in response to specific stimuli. This is the case for example for neurotransmitter release in the brain and for insulin secretion from the endocrine pancreas.

The neurophysiology field was electrified by the discoveries of Bernard Katz, Ulf von Euler and Julius Axelrod who received the Nobel Prize in Physiology or Medicine 1970* for their discoveries concerning the humoral transmittors in the nerve terminals and the mechanism for their storage, release and inactivation. Südhof was intrigued by the rapid exocytosis of synaptic vesicles, which is under tight temporal control and regulated by the changes in the cytoplasmic free calcium concentration. Südhof elucidated how calcium regulates neurotransmitter release in neurons and discovered that complexin and synaptotagmin are two critical proteins in calcium-mediated vesicle fusion.

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Serum Folate and Homocysteine, Mood Disorders, and Aging

Larry H. Bernstein, MD, FCAP, Curator



Dietary Folate and the Risk of Depression in Finnish MiddleAged Men

Tolmunen T, et al.
PSYCHOTHER AND PSYCHOSOM · OCT 2004; 73:334-339    DOI: http://dx.doi.org:/10.1159/000080385


Serum Folate, Vitamin B-12, and Homocysteine and Their Association With Depressive Symptoms Among U.S. Adults

PSYCHOSOM MED · NOV 2010;             DOI: http://dx.doi.org:/10.1097/PSY.0b013e3181f61863

Objective: To examine, in a nationally representative sample of U.S. adults, the associations of serum folate, vitamin B-12, and total homocysteine (tHcy) levels with depressive symptoms. Several nutritional and physiological factors have been linked to depression in adults, including low folate and vitamin B-12 and elevated tHcy levels.
Methods: Data on U.S. adults (age, 20–85 years; n 2524) from the National Health and Nutrition Examination Survey during the period 2005 to 2006 were used. Depressive symptoms were measured with the Patient Health Questionnaire (PHQ), and elevated symptoms were defined as a PHQ total score of 10. Serum folate, vitamin B-12, and tHcy were mainly expressed as tertiles. Multiple ordinary least square (OLS), logistic, and zero-inflated Poisson regression models were conducted in the main analysis.
Results: Overall, mean PHQ score was significantly higher among women compared with men. Elevated depressive symptoms (PHQ score of 10) were inversely associated with folate status, particularly among women (fully adjusted odds ratio [tertiles T3 versus T1] 0.37; 95% confidence interval, 0.17–0.86), but not significantly related to tHcy or vitamin B-12. No interaction was noted between the three exposures in affecting depressive symptoms. In older adults (50 years) and both sexes combined, tHcy was positively associated with elevated depressive symptoms (fully adjusted odds ratio [tertiles T2 versus T1] 3.01; 95% confidence interval, 1.01–9.03), although no significant dose-response relationship was found. Conclusions: Future interventions to improve mental health outcomes among U.S. adults should take into account dietary and other factors that would increase levels of serum folate.
Key words: depression, folate, vitamin B-12, homocysteine, adults.


Relationship of homocysteine, folic acid and vitamin B12 depression in a middle-aged community sample

P.S. SACHDEV, et al.   PSYCHOL MED · MAY 2005;   35, 529–538         http://dx.doi.org: /10.1017/S0033291704003721 

Background. Case control studies have supported a relationship between low folic acid and vitamin B12 and high homocysteine levels as possible predictors of depression. The results from epidemiological studies are mixed and largely from elderly populations.
Method. A random subsample of 412 persons aged 60–64 years from a larger community sample underwent psychiatric and physical assessments, and brain MRI scans. Subjects were assessed using the PRIME-MD Patient Health Questionnaire for syndromal depression and severity of depressive symptoms. Blood measures included serum folic acid, vitamin B12, homocysteine and creatinine levels, and total antioxidant capacity. MRI scans were quantified for brain atrophy, subcortical atrophy, and periventricular and deep white-matter hyperintensity on T2-weighted imaging.
Results. Being in the lowest quartile of homocysteine was associated with fewer depressive symptoms, after adjusting for sex, physical health, smoking, creatinine, folic acid and B12 levels. Being in the lowest quartile of folic acid was associated with increased depressive symptoms, after adjusting for confounding factors, but adjustment for homocysteine reduced the incidence rate ratio for folic acid to a marginal level. Vitamin B12 levels did not have a significant association with depressive symptoms. While white-matter hyperintensities had significant correlations with both homocysteine and depressive symptoms, the brain measures and total antioxidant capacity did not emerge as significant mediating variables. Conclusions. Low folic acid and high homocysteine, but not low vitamin B12 levels, are correlates of depressive symptoms in community-dwelling middle-aged individuals. The effects of folic acid and homocysteine are overlapping but distinct.


Association of folate intake with the occurrence of depressive episodes in middle-aged French men and women

P. Astorg, et al.    BRIT J  NUTR · AUG 2008; 100, 183–18       http://dx.doi.org:/10.1017/S0007114507873612

A low folate intake or a low folate status have been found to be associated with a higher frequency of depression in populations, but the existence and the direction of a causal link between folate intake or status and depression is still uncertain. The aim of this study was to seek the relation between the habitual folate intake in middle-aged men and women and the occurrence of depressive episodes. In a subsample of 1864 subjects (809 men and 1055 women) from the French SU.VI.MAX cohort, dietary habits have been measured at the beginning of the follow-up (six 24 h records) and declarations of antidepressant prescription, taken as markers of depressive episodes, have been recorded during the 8-year follow-up. No significant association was observed between folate intake and the risk of any depressive episode or of a single depressive episode during the follow-up, in both men and women. In contrast, the risk of experiencing recurrent depressive episodes (two or more) during the follow-up was strongly reduced in men with high folate intake (OR 0·25 (95% CI 0·06, 0·98) for the highest tertile v. the lowest, P for trend 0·046). This association was not observed in women. These results suggest that a low folate intake may increase the risk of recurrent depression in men.   Folate: Depression: Cohort studies


Homocysteine, vitamin B12, and folic acid levels in Alzheimer’s disease, mild cognitive impairment, and healthy elderly: baseline characteristics in subjects of the Australian Imaging Biomarker Lifestyle study.

Faux NG1, Ellis KA, Porter L, Fowler CJ,…, Ames D, Masters CL, Bush AI.
J Alzheimers Dis. 2011; 27(4):909-22.    http://dx.doi.org:/10.3233/JAD-2011-110752.

There is some debate regarding the differing levels of plasma homocysteine, vitamin B12 and serum folate between healthy controls (HC), mild cognitive impairment (MCI), and Alzheimer’s disease (AD). As part of the Australian Imaging Biomarker Lifestyle (AIBL) study of aging cohort, consisting of 1,112 participants (768 HC, 133 MCI patients, and 211 AD patients), plasma homocysteine, vitamin B12, and serum and red cell folate were measured at baseline to investigate their levels, their inter-associations, and their relationships with cognition. The results of this cross-sectional study showed that homocysteine levels were increased in female AD patients compared to female HC subjects (+16%, p-value < 0.001), but not in males. Red cell folate, but not serum folate, was decreased in AD patients compared to HC (-10%, p-value = 0.004). Composite z-scores of short- and long-term episodic memory, total episodic memory, and global cognition all showed significant negative correlations with homocysteine, in all clinical categories. Increasing red cell folate had a U-shaped association with homocysteine, so that high red cell folate levels were associated with worse long-term episodic memory, total episodic memory, and global cognition. These findings underscore the association of plasma homocysteine with cognitive deterioration, although not unique to AD, and identified an unexpected abnormality of red cell folate.


Homocysteine and folate as risk factors for dementia and Alzheimer disease1,2,3

Giovanni RavagliaPaola FortiFabiola Maioli, …., Nicoletta BrunettiElisa Porcellini, and Federico Licastro
Am J Clin Nutr Sept 2005; 82(3): 636-643


Background: In cross-sectional studies, elevated plasma total homocysteine (tHcy) concentrations have been associated with cognitive impairment and dementia. Incidence studies of this issue are few and have produced conflicting results.

Objective: We investigated the relation between high plasma tHcy concentrations and risk of dementia and Alzheimer disease (AD) in an elderly population.

Design: A dementia-free cohort of 816 subjects (434 women and 382 men; mean age: 74 y) from an Italian population-based study constituted our study sample. The relation of baseline plasma tHcy to the risk of newly diagnosed dementia and AD on follow-up was examined. A proportional hazards regression model was used to adjust for age, sex, education, apolipoprotein E genotype, vascular risk factors, and serum concentrations of folate and vitamin B-12.

Results: Over an average follow-up of 4 y, dementia developed in 112 subjects, including 70 who received a diagnosis of AD. In the subjects with hyperhomocysteinemia (plasma tHcy > 15 μmol/L), the hazard ratio for dementia was 2.08 (95% CI: 1.31, 3.30; P = 0.002). The corresponding hazard ratio for AD was 2.11 (95% CI: 1.19, 3.76; P = 0.011). Independently of hyperhomocysteinemia and other confounders, low folate concentrations (≤11.8 nmol/L) were also associated with an increased risk of both dementia (1.87; 95% CI: 1.21, 2.89; P = 0.005) and AD (1.98; 95% CI: 1.15, 3.40; P = 0.014), whereas the association was not significant for vitamin B-12.

Conclusions: Elevated plasma tHcy concentrations and low serum folate concentrations are independent predictors of the development of dementia and AD.


In Western societies, the prevalence and economic costs of Alzheimer disease (AD) are soaring in step with the increased number of elders in the population (1). Therefore, it is important to identify modifiable risk factors for this disease. The sulfur amino acid homocysteine is a unique candidate for this role because of its direct neurotoxicity (24) and its association with cerebrovascular disease (5), which is currently believed to play a significant role in AD etiology (6). Moreover, elevated concentrations of plasma total homocysteine (tHcy) are an indicator of inadequate folate and vitamin B-12 status (7) and can directly affect brain function via altered methylation reactions (8).

An association between AD and elevated tHcy concentrations has been reported in case-control (9, 10) and cross-sectional (11, 12) studies. Moreover, in nondemented elderly populations, plasma tHcy is inversely associated with poor performance at simultaneously performed tests of global cognitive function (1315) and specific cognitive skills (13, 16). However, cross-sectional studies cannot determine causality. Only 2 longitudinal studies investigated the relation between hyperhomocysteinemia and risk of incident AD, but their results were inconsistent; the Framingham Study reported a strong association (17), and the Washington Heights–Inwood Columbia Ageing Project (WHICAP) reported no association (18). Clarification of this issue is important because consistent evidence of a prospective association between homocysteine and AD would more strongly support the need for intervention trials testing the effectiveness of homocysteine-lowering vitamin therapy in preventing dementia.

Therefore, we examined baseline plasma tHcy in relation to risk of incident dementia and AD in the Conselice Study of Brain Aging (CSBA), an Italian population-based study of older persons.

Study population

The CSBA is a population-based survey, already described in detail elsewhere (19,20), the principal aim of which is to provide data about epidemiology and risk factors for dementia in the elderly. Its design includes both cross-sectional and longitudinal components. The study was approved by the Institutional Review Board of the Department of Internal Medicine, Cardioangiology, and Hepatology, University of Bologna, and written informed consent was obtained from all participants.

Briefly, in 1999–2000, 1016 (75%) of the 1353 individuals aged ≥65 y residing in the Italian municipality of Conselice (province of Ravenna, Emilia Romagna region) participated in the prevalence study. Data on cognitive status at the follow-up examination in 2003–2004 were collected for 861 of the 937 participants free of dementia at baseline. A flow chart detailing the derivation of the incidence sample used in this study is reported in Figure 1.

This prospective population-based study was the first to replicate previous findings from the Framingham Study (17), indicating that hyperhomocysteinemia doubles the risk of developing dementia and AD independently of several major confounders. Our results disagree with the negative findings recently reported in the WHICAP study (18). Possible explanations for this difference are the acknowledged insufficient statistical power of the WHICAP study, the rather homogeneously high tHcy concentrations of its sample—which did not permit enough variability to detect an association—and methodologic issues related to the prolonged time between blood sample collection and processing, which could have affected tHcy measurements.

Inconsistent results were also given by the only 2 studies that examined the association between homocysteine and cognitive decline at follow-up as measured with the MMSE (30, 31). These studies, however, differed in sample size and in which confounders were taken into account. Moreover, MMSE is a reliable global screening measure of cognitive function but was not developed to estimate changes in cognitive function or to diagnose dementia (32).

The substantial evidence that tHcy is an independent vascular risk factor (5) supports the role of hyperhomocysteinemia in AD. Subjects with vascular risk factors and cerebrovascular disease have an increased risk of AD (6), and hyperhomocysteinemia has been related to cerebral macro- and microangiopathy, endothelial dysfunction, impaired nitric oxide activity, and increased oxidative stress (3335). Moreover, as shown in cell cultures, homocysteine can directly cause brain damage through several mechanisms: increased glutamate excitoxicity via activation of N-methyl-D-aspartate receptors (2), enhancement of β-amyloid peptide generation (4), impairment of DNA repair, and sensitization of neurons to amyloid toxicity (3).

On the basis of cross-sectional observations, some authors have suggested that elevated plasma tHcy concentrations are not a causative factor in dementia and AD but are only a marker for concomitant vascular disease, independently of cognitive status (36, 37). Results from other cross-sectional investigations (9, 12, 38), as well as those from the present investigation and the Framingham Study (17), argue against this interpretation, but only intervention trials can give the ultimate proof of a causal relation between hyperhomocysteinemia and AD.

In contrast with both the Framingham (17) and WHICAP (18) studies, we also found that, independent of homocysteine and other confounders (including vitamin B-12), low serum folate is associated with an increased risk of incident dementia and AD. Mandatory folate fortification of food might partially explain the negative results of the US studies, whereas in Italy, where folate fortification is not practiced, relative folate deficiency may be endemic among the elderly population. Nondemented patients with poor cognitive performance and AD patients often exhibit poor folate status (reviewed in 8), but only one study specifically examined B vitamins in relation to incident dementia. In a selected sample of nondemented Swedish elderly participants in the Kungsholmen Study, low serum folate and vitamin B-12 were predictive of AD at 3 y of follow-up (39). The sample, however, was small (370 subjects), and a clear association was detected only when both vitamins were taken into account.

Biologic explanatory mechanisms relating folate deficiency to dementia include impaired methylation reactions in the central nervous system, with a consequent insufficient supply of methyl groups, which are required for the synthesis of myelin, neurotransmitters, membrane phospholipids, and DNA (8). However, because of the study design and the relatively short follow-up time, we cannot definitely establish whether the independent association between low folate and dementia risk indicates an actual effect of folate status on cognitive function or, on the contrary, that subtle functional alterations may affect the dietary intake of folate in the early preclinical stages of dementia.


Neurotoxicity associated with dual actions of homocysteine at the N-methyl-D-aspartate receptor

Stuart A. Lipton*Won-Ki KimYun-Beom Choi*,…, Derrick R. Arnelle§, and Jonathan S. Stamler
NAS 1997; 94(11):5923–5928    http://www.pnas.org/content/94/11/5923.abstract

Severely elevated levels of total homocysteine (approximately millimolar) in the blood typify the childhood disease homocystinuria, whereas modest levels (tens of micromolar) are commonly found in adults who are at increased risk for vascular disease and stroke. Activation of the coagulation system and adverse effects of homocysteine on the endothelium and vessel wall are believed to underlie disease pathogenesis. Here we show that homocysteine acts as an agonist at the glutamate binding site of the N-methyl-D-aspartate receptor, but also as a partial antagonist of the glycine coagonist site. With physiological levels of glycine, neurotoxic concentrations of homocysteine are on the order of millimolar. However, under pathological conditions in which glycine levels in the nervous system are elevated, such as stroke and head trauma, homocysteine’s neurotoxic (agonist) attributes at 10–100 μM levels outweigh its neuroprotective (antagonist) activity. Under these conditions neuronal damage derives from excessive Ca2+ influx and reactive oxygen generation. Accordingly, homocysteine neurotoxicity through overstimulation of N-methyl-D-aspartate receptors may contribute to the pathogenesis of both homocystinuria and modest hyperhomocysteinemia.


Vitamin B12 and folate in relation to the development of Alzheimer’s disease

H-X. Wang, Å. WahlinH. Basun, …, B. Winblad, and L. Fratiglioni
Neurology May 8, 2001; 56(9):1188-1194    http:/​/​dx.​doi.​org/​10.​1212/​WNL.​56.​9.​1188

Objective: To explore the associations of low serum levels of vitamin B12 and folate with AD occurrence.

Methods: A population-based longitudinal study in Sweden, the Kungsholmen Project. A random sample of 370 nondemented persons, aged 75 years and older and not treated with B12 and folate, was followed for 3 years to detect incident AD cases. Two cut-off points were used to define low levels of vitamin B12 (≤150 and ≤250 pmol/L) and folate (≤10 and ≤12 nmol/L), and all analyses were performed using both definitions. AD and other types of dementia were diagnosed by specialists according to DSM-III-R criteria.

Results: When using B12 ≤150pmol/L and folate ≤10 nmol/L to define low levels, compared with people with normal levels of both vitamins, subjects with low levels of B12or folate had twice higher risks of developing AD (relative risk [RR] = 2.1, 95% CI = 1.2 to 3.5). These associations were even stronger in subjects with good baseline cognition (RR = 3.1, 95% CI = 1.1 to 8.4). Similar relative risks of AD were found in subjects with low levels of B12or folate and among those with both vitamins at low levels. A comparable pattern was detected when low vitamin levels were defined as B12 ≤250 pmol/L and folate ≤12 nmol/L.

Conclusions: This study suggests that vitamin B12 and folate may be involved in the development of AD. A clear association was detected only when both vitamins were taken into account, especially among the cognitively intact subjects. No interaction was found between the two vitamins. Monitoring serum B12 and folate concentration in the elderly may be relevant for prevention of AD.


Assessing the association between homocysteine and cognition: reflections on Bradford Hill, meta-analyses, and causality

Hyperhomocysteinemia is a recognized risk factor for cognitive decline and incident dementia in older adults. Two recent reports addressed the cumulative epidemiological evidence for this association but expressed conflicting opinions. Here, the evidence is reviewed in relation to Sir Austin Bradford Hill’s criteria for assessing “causality,” and the latest meta-analysis of the effects of homocysteine-lowering on cognitive function is critically examined. The meta-analysis included 11 trials, collectively assessing 22 000 individuals, that examined the effects of B vitamin supplements (folic acid, vitamin B12, vitamin B6) on global or domain-specific cognitive decline. It concluded that homocysteine-lowering with B vitamin supplements has no significant effect on cognitive function. However, careful examination of the trials in the meta-analysis indicates that no conclusion can be made regarding the effects of homocysteine-lowering on cognitive decline, since the trials typically did not include individuals who were experiencing such decline. Further definitive trials in older adults experiencing cognitive decline are still urgently needed.
Mouse model for deficiency of methionine synthase reductase exhibits short-term memory impairment and disturbances in brain choline metabolism
, , , , , , ,
Biochem. J. 2014 461: 205212    http://dx.doi.org:/10.1042/BJ20131568
Hyperhomocysteinaemia can contribute to cognitive impairment and brain atrophy. MTRR (methionine synthase reductase) activates methionine synthase, which catalyses homocysteine remethylation to methionine. Severe MTRR deficiency results in homocystinuria with cognitive and motor impairments. An MTRR polymorphism may influence homocysteine levels and reproductive outcomes. The goal of the present study was to determine whether mild hyperhomocysteinaemia affects neurological function in a mouse model with Mtrr deficiency. Mtrr+/+, Mtrr+/gt and Mtrrgt/gtmice (3 months old) were assessed for short-term memory, brain volumes and hippocampal morphology. We also measured DNA methylation, apoptosis, neurogenesis, choline metabolites and expression of ChAT (choline acetyltransferase) and AChE (acetylcholinesterase) in the hippocampus. Mtrrgt/gt mice exhibited short-term memory impairment on two tasks. They had global DNA hypomethylation and decreased choline, betaine and acetylcholine levels. Expression of ChAT and AChE was increased and decreased respectively. At 3 weeks of age, they showed increased neurogenesis. In the cerebellum, mutant mice had DNA hypomethylation, decreased choline and increased expression of ChAT. Our work demonstrates that mild hyperhomocysteinaemia is associated with memory impairment. We propose a mechanism whereby a deficiency in methionine synthesis leads to hypomethylation and compensatory disturbances in choline metabolism in the hippocampus. This disturbance affects the levels of acetylcholine, a critical neurotransmitter in learning and memory.

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Muscular dystrophy has deficient stem cell dystrophin

Larry H. Bernstein, MD, FCAP, Curator



Dystrophin Deficient Stem Cell Pathology


Muscular Dystrophy is a Stem Cell-Based Disease

Because DMD results from mutations in the dystrophin gene, the vast majority of muscular dystrophy research was based on a simple model in which the Dystrophin protein played a structural role in the structural integrity of muscle fibers. Abnormal versions of the Dystrophin protein caused the muscle fibers to become damaged and die as a result of contraction.  Dystrophin anchors the cytoskeleton of the muscle fibers, which are essential for muscle contraction, to the muscle cell membrane, and then to the extracellular matrix outside the cell that serves as a foundation upon which the muscle cells are built.


However in this current study, Rudnicki and his team discovered that muscle stem cells also express the dystrophin protein. This is a revelation because Dystrophin was thought to be protein that ONLY appeared in mature muscle. However, in this study, it became exceedingly clear that in the absence of Dystrophin, muscle stem cells generated ten-fold fewer muscle precursor cells, and, consequently, far fewer functional muscle fibers. Dystrophin is also a component of a signal transduction pathway that allows muscle stem cells to properly ascertain if they need to replace dead or dying muscle.  Muscle stem cells repair the muscle in response to injury or exercise by dividing to generate precursor cells that differentiate into muscle fibers.

Even though Rudnicki used mice as a model system in these experiments, the Dystrophin protein is highly conserved in most vertebrate animals. Therefore, it is highly likely that these results will also apply to human muscle stem cells.

Gene therapy experiments and trials are in progress and even show some promise, but Rudnicki’s work tells us that gene therapy approaches must target muscle stem cells as well as muscle fibers if they are to work properly.

“We’re already looking at approaches to correct this problem in muscle stem cells,” said Dr. Rudnicki.

This paper has received high praise from the likes of Ronald Worton, who was one of the co-discovers of the dystrophin gene with Louis Kunkel in 1987.

Early pathogenesis of Duchenne muscular dystrophy modelled in patient-derived human induced pluripotent stem cells

Emi Shoji, Hidetoshi Sakurai, Tokiko Nishino, Tatsutoshi Nakahata, Toshio Heike, Tomonari Awaya, Nobuharu Fujii, Yasuko Manabe, Masafumi Matsuo & Atsuko Sehara-Fujisawa

Scientific Reports 5, Article number: 12831 (2015)   http://dx.doi.org:/10.1038/srep12831

Duchenne muscular dystrophy (DMD) is a progressive and fatal muscle degenerating disease caused by a dystrophin deficiency. Effective suppression of the primary pathology observed in DMD is critical for treatment. Patient-derived human induced pluripotent stem cells (hiPSCs) are a promising tool for drug discovery. Here, we report an in vitro evaluation system for a DMD therapy using hiPSCs that recapitulate the primary pathology and can be used for DMD drug screening. Skeletal myotubes generated from hiPSCs are intact, which allows them to be used to model the initial pathology of DMD in vitro. Induced control and DMD myotubes were morphologically and physiologically comparable. However, electric stimulation of these myotubes for in vitro contraction caused pronounced calcium ion (Ca2+) influx only in DMD myocytes. Restoration of dystrophin by the exon-skipping technique suppressed this Ca2+ overflow and reduced the secretion of creatine kinase (CK) in DMD myotubes. These results suggest that the early pathogenesis of DMD can be effectively modelled in skeletal myotubes induced from patient-derived iPSCs, thereby enabling the development and evaluation of novel drugs.

Duchenne muscular dystrophy (DMD) is characterised by progressive muscle atrophy and weakness that eventually leads to ambulatory and respiratory deficiency from early childhood1. It is an X-linked recessive inherited disease with a relatively high frequency of 1 in 3500 males1,2.DMD, which is responsible for DMD, encodes 79 exons and produces dystrophin, which is one of the largest known cytoskeletal structural proteins3. Most DMD patients have various types of deletions or mutations in DMD that create premature terminations, resulting in a loss of protein expression4. Several promising approaches could be used to treat this devastating disease, such as mutation-specific drug exon-skipping5,6, cell therapy7, and gene therapy1,2.

Myoblasts from patients are the most common cell sources for assessing the disease phenotypes of DMD11,12. …Previous reports have shown that muscle cell differentiation from DMD patient myoblasts is delayed and that these cells have poor proliferation capacity compared to those of healthy individuals11,12. Our study revealed that control and DMD myoblasts obtained by activating tetracycline-dependent MyoD transfected into iPS cells (iPStet-MyoD cells) have comparable growth and differentiation potential and can produce a large number of intact and homogeneous myotubes repeatedly.

The pathogenesis of DMD is initiated and progresses with muscle contraction. The degree of muscle cell damage at the early stage of DMD can be evaluated by measuring the leakage of creatine kinase (CK) into the extracellular space15. Excess calcium ion (Ca2+) influx into skeletal muscle cells, together with increased susceptibility to plasma membrane injury, is regarded as the initial trigger of muscle damage in DMD19,20,21,22,23,24. Targeting these early pathogenic events is considered essential for developing therapeutics for DMD.

In this study, we established a novel evaluation system to analyse the cellular basis of early DMD pathogenesis by comparing DMD myotubes with the same clone but with truncated dystrophin-expressing DMD myotubes, using the exon-skipping technique. We demonstrated through in vitro contraction that excessive Ca2+ influx is one of the earliest events to occur in intact dystrophin-deficient muscle leading to extracellular leakage of CK in DMD myotubes.

Generation of tetracycline-inducible MyoD-transfected DMD patient-derived iPSCs (iPStet-MyoD cells)

Figure 1: Generation and characterization of control and DMD patient-derived Tet-MyoD-transfected hiPS cells.   Full size image

Morphologically and physiologically comparable intact myotubes differentiated from control and DMD-derived hiPSCs

Figure 2: Morphologically and physiologically comparable skeletal muscle cells differentiated from Control-iPStet-MyoD and DMD-iPStet-MyoD.   Full size image

Exon-skipping with AO88 restored expression of Dystrophin in DMD myotubes differentiated from DMD-iPStet-MyoD cells

Figure 3: Restoration of dystrophin protein expression by AO88.   Full size image

Restored dystrophin expression attenuates Ca2+ overflow in DMD-Myocytes

Figure 4: Restored expression of dystrophin diminishes Ca2+ influx in DMD muscle in response to electric stimulation.   Full size image

Ca2+ influx provokes skeletal muscle cellular damage in DMD muscle

Figure 5: Ca2+ influx induces prominent skeletal muscle cellular damage in DMD-Myocytes.   Full size image

Skeletal muscle differentiation in myoblasts from DMD patients is generally delayed compared to that in healthy individuals11,36,37.  Our differentiation system successfully induced the formation of myotubes from DMD patients, and the myotubes displayed analogous morphology and maturity compared with control myotubes (Fig. 2a–c).  Comparing myotubes generated from patient-derived iPS cells with those derived from the same DMD clones but expressing dystrophin by application of the exon-skipping technique enabled us to demonstrate the primary cellular phenotypes in skeletal muscle solely resulting from the loss of the dystrophin protein (Fig. 4b).  Our results demonstrate that truncated but functional dystrophin protein expression improved the cellular phenotype of DMD myotubes.

In DMD, the lack of dystrophin induces an excess influx of Ca2+ , leading to pathological dystrophic changes22. We consistently observed excess Ca2+ influx in DMD-Myocytes compared to Control-Myocytes (Supplementary Figure S3a and S3b) in response to electric stimulation. TRP channels, which are mechanical stimuli-activated Ca2+ channels40that are expressed in skeletal muscle cells41, can account for this pathogenic Ca2+ influx…

In conclusion, our study revealed that the absence of dystrophin protein induces skeletal muscle damage by allowing excess Ca2+ influx in DMD myotubes. Our experimental system recapitulated the early phase of DMD pathology as demonstrated by visualisation and quantification of Ca2+ influx using intact myotubes differentiated from hiPS cells.  This evaluation system significantly expands prospective applications with regard to assessing the effectiveness of exon-skipping drugs and also enables the discovery of drugs that regulate the initial events in DMD.

Duchenne muscular dystrophy affects stem cells, University of Ottawa study finds  

New treatments could one day be available for the most common form of muscular dystrophy after a study suggests the debilitating genetic disease affects the stem cells that produce healthy muscle fibres.

The findings are based on research from the University of Ottawa and The Ottawa Hospital, published Monday in the journal Nature Medicine.

For nearly two decades, doctors had thought the muscular weakness that is the hallmark of the disease was due to problems with human muscle fibers, said Dr. Michael Rudnicki, the study’s senior author.

The new research shows the specific protein characterized by its absence in Duchenne muscular dystrophy normally exists in stem cells.

Dystrophin protein found in stem cells

“The prevailing notion was that the protein that’s missing in Duchenne muscular dystrophy — a protein called dystrophin — was not involved at all in the function of the stem cells.”


When the genetic mutations caused by Duchenne muscular dystrophy inhibit the production of dystrophin in stem cells, those stem cells produce significantly fewer precursor cells — and thus fewer properly functioning muscle fibres.  Further, stem cells need dystrophin to sense their environment to figure out if they need to divide to produce more stem cells or perform muscle repair work.

Genetic repair might treat Duchenne muscular dystrophy

July 25, 2011|By Thomas H. Maugh II, Los Angeles Times

A genetic technique that allows the body to work around a crucial mutation that causes Duchenne muscular dystrophy increased the mass and function of muscles in a small group of patients with the devastating disease, paving the way for larger clinical trials of the drug. The study in a handful of boys age 5 to 15 showed that patients receiving the highest level of the drug, called AVI-4658 or eteplirsen, had a significant increase in production of a missing protein and increases in muscle fibers. The study demonstrated that the drug is safe in the short term. Results were reported Sunday in the journal Lancet.

Duchenne muscular dystrophy affects about one in every 3,500 males worldwide. It is caused by any one of several different mutations that affect production of a protein called dystrophin, which is important for the production and maintenance of muscle fibers. Affected patients become unable to walk and must use a wheelchair by age 8 to 12. Deterioration continues through their teens and 20s, and the condition typically proves fatal as muscle failure impairs their ability to breathe.

This study is designed to assess the efficacy, safety, tolerability, and pharmacokinetics (PK) of AVI-4658 (eteplirsen) in both 50.0 mg/kg and 30.0 mg/kg doses administered over 24 weeks in subjects diagnosed with Duchenne muscular dystrophy (DMD).


Condition Intervention Phase
Duchenne Muscular Dystrophy Drug: AVI-4658 (Eteplirsen)
Other: Placebo
Phase 2


Study Type: Interventional
Study Design: Allocation: Randomized
Endpoint Classification: Safety/Efficacy Study
Intervention Model: Parallel Assignment
Masking: Double Blind (Subject, Caregiver, Investigator, Outcomes Assessor)
Primary Purpose: Treatment
Official Title: A Randomized, Double-Blind, Placebo-Controlled, Multiple Dose Efficacy, Safety, Tolerability and Pharmacokinetics Study of AVI-4658(Eteplirsen),in the Treatment of Ambulant Subjects With Duchenne Muscular Dystrophy
Resource links provided by NLM:
Dystrophin expression in muscle stem cells regulates their polarity and asymmetric division

Nature Medicine(2015)   http://dx.doi.org:/10.1038/nm.3990

Dystrophin is expressed in differentiated myofibers, in which it is required for sarcolemmal integrity, and loss-of-function mutations in the gene that encodes it result in Duchenne muscular dystrophy (DMD), a disease characterized by progressive and severe skeletal muscle degeneration. Here we found that dystrophin is also highly expressed in activated muscle stem cells (also known as satellite cells), in which it associates with the serine-threonine kinase Mark2 (also known as Par1b), an important regulator of cell polarity. In the absence of dystrophin, expression of Mark2 protein is downregulated, resulting in the inability to localize the cell polarity regulator Pard3 to the opposite side of the cell. Consequently, the number of asymmetric divisions is strikingly reduced in dystrophin-deficient satellite cells, which also display a loss of polarity, abnormal division patterns (including centrosome amplification), impaired mitotic spindle orientation and prolonged cell divisions. Altogether, these intrinsic defects strongly reduce the generation of myogenic progenitors that are needed for proper muscle regeneration. Therefore, we conclude that dystrophin has an essential role in the regulation of satellite cell polarity and asymmetric division. Our findings indicate that muscle wasting in DMD not only is caused by myofiber fragility, but also is exacerbated by impaired regeneration owing to intrinsic satellite cell dysfunction.

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The Reconstruction of Life Processes requires both Genomics and Metabolomics to explain Phenotypes and Phylogenetics

Writer and Curator: Larry H. Bernstein, MD, FCAP 






This discussion that completes and is an epicrisis (summary and critical evaluation) of the series of discussions that preceded it.

  1. Innervation of Heart and Heart Rate
  2. Action of hormones on the circulation
  3. Allogeneic Transfusion Reactions
  4. Graft-versus Host reaction
  5. Unique problems of perinatal period
  6. High altitude sickness
  7. Deep water adaptation
  8. Heart-Lung-and Kidney
  9. Acute Lung Injury

The concept inherent in this series is that the genetic code is an imprint that is translated into a message.  It is much the same as a blueprint, or a darkroom photographic image that has to be converted to a print. It is biologically an innovation of evolutionary nature because it establishes a simple and reproducible standard for the transcription of the message through the transcription of the message using strings of nucleotides (oligonucleotides) that systematically transfer the message through ribonucleotides that communicate in the cytoplasm with the cytoskeleton based endoplasmic reticulum (ER), composing a primary amino acid sequence.  This process is a quite simple and convenient method of biological activity.  However, the simplicity ends at this step.  The metabolic components of the cell are organelles consisting of lipoprotein membranes and a cytosol which have particularly aligned active proteins, as in the inner membrane of the mitochondrion, or as in the liposome or phagosome, or the structure of the  ER, each of which is critical for energy transduction and respiration, in particular, for the mitochondria, cellular remodeling or cell death, with respect to the phagosome, and construction of proteins with respect to the ER, and anaerobic glycolysis and the hexose monophosphate shunt in the cytoplasmic domain.  All of this refers to structure and function, not to leave out the membrane assigned transport of inorganic, and organic ions (electrolytes and metabolites).

I have identified a specific role of the ER, the organelles, and cellular transactions within and between cells that is orchestrated.  But what I have outlined is a somewhat limited and rigid model that does not reach into the dynamics of cellular transactions.  The DNA has expression that may be old, no longer used messages, and this is perhaps only part of a significant portion of “dark matter”.  There is also nuclear DNA that is enmeshed with protein, mRNA that is a copy of DNA, and mDNA  is copied to ribosomal RNA (rRNA).  There is also rDNA. The classic model is DNA to RNA to protein.  However, there is also noncoding RNA, which plays an important role in regulation of transcription.

This has been discussed in other articles.  But the important point is that proteins have secondary structure through disulfide bonds, which is determined by position of sulfur amino acids, and by van der Waal forces, attraction and repulsion. They have tertiary structure, which is critical for 3-D structure.  When like subunits associate, or dissimilar oligomers, then you have heterodimers and oligomers.  These constructs that have emerged over time interact with metabolites within the cell, and also have an important interaction with the extracellular environment.

When you take this into consideration then a more complete picture emerges. The primitive cell or the multicellular organism lives in an environment that has the following characteristics – air composition, water and salinity, natural habitat, temperature, exposure to radiation, availability of nutrients, and exposure to chemical toxins or to predators.  In addition, there is a time dimension that proceeds from embryonic stage to birth in mammals, a rapid growth phase, a tapering, and a decline.  The time span is determined by body size, fluidity of adaptation, and environmental factors.  This is covered in great detail in this work.  The last two pieces are in the writing stage that completes the series. Much content has already be presented in previous articles.

The function of the heart, kidneys and metabolism of stressful conditions have already been extensively covered in http://pharmaceuticalintelligence.com  in the following and more:

The Amazing Structure and Adaptive Functioning of the Kidneys: Nitric Oxide – Part I


Nitric Oxide and iNOS have Key Roles in Kidney Diseases – Part II


The pathological role of IL-18Rα in renal ischemia/reperfusion injury – Nature.com


Summary, Metabolic Pathways



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Summary of Cell Structure, Anatomic Correlates of Metabolic Function

Author and Curator: Larry H. Bernstein, MD, FCAP  


This chapter has been concerned with the subcellular ultrastructure of organelles, and importantly, their function.  There is no waste in the cell structure. The nucleus has the instructions necessary to carry out the cell’s functions.  In the Eukaryotic cell there is significant differentiation so that the cells are regulated for the needs that they uniquely carry out.  When there is disregulation, it leads to remodeling or to cell death.

Here I shall note some highlights of this chapter.

  1. In every aspect of cell function, proteins are involved embedded in the structure, for most efficient functioning.
  2. Metabolic regulation is dependent on pathways that are also linkages of proteins.
  3. Energy utilization is dependent on enzymatic reactions, often involving essential metal ions of high valence numbers, which facilitates covalent and anion binding, and has an essential role in allostericity.





Mitochondria range from 0.5 to 1.0 micrometer (μm) in diameter. These structures are sometimes described as “cellular power plants” because they generate most of the cell’s supply of adenosine triphosphate (ATP), used as a source of chemical energy. In addition to supplying cellular energy, mitochondria are involved in other tasks such as signaling, cellular differentiation, cell death, as well as the control of the cell cycle and cell growth. Mitochondria have been implicated in several human diseases, including mitochondrial disorders and cardiac dysfunction.

The number of mitochondria in a cell can vary widely by organism, tissue, and cell type. For instance, red blood cells have no mitochondria, whereas liver cells can have more than 2000. The organelle is composed of compartments that carry out specialized functions. These compartments or regions include the outer membrane, the intermembrane space, the inner membrane, and the cristae and matrix. Mitochondrial proteins vary depending on the tissue and the species. The mitochondrial proteome is thought to be dynamically regulated. Although most of a cell’s DNA is contained in the cell nucleus, the mitochondrion has its own independent genome. Further, its DNA shows substantial similarity to bacterial genomes.

In 1913 particles from extracts of guinea-pig liver were linked to respiration by Otto Heinrich Warburg, which he called “grana”. Warburg and Heinrich Otto Wieland, who had also postulated a similar particle mechanism, disagreed on the chemical nature of the respiration. It was not until 1925 when David Keilin discovered cytochromes that the respiratory chain was described.  In 1939, experiments using minced muscle cells demonstrated that one oxygen atom can form two adenosine triphosphate molecules, and, in 1941, the concept of phosphate bonds being a form of energy in cellular metabolism was developed by Fritz Albert Lipmann. In the following years, the mechanism behind cellular respiration was further elaborated, although its link to the mitochondria was not known. The introduction of tissue fractionation by Albert Claude allowed mitochondria to be isolated from other cell fractions and biochemical analysis to be conducted on them alone. In 1946, he concluded that cytochrome oxidase and other enzymes responsible for the respiratory chain were isolated to the mitchondria.

The first high-resolution micrographs appeared in 1952, replacing the Janus Green stains as the preferred way of visualising the mitochondria. This led to a more detailed analysis of the structure of the mitochondria, including confirmation that they were surrounded by a membrane. It also showed a second membrane inside the mitochondria that folded up in ridges dividing up the inner chamber and that the size and shape of the mitochondria varied from cell to cell.  In 1967, it was discovered that mitochondria contained ribosomes. In 1968, methods were developed for mapping the mitochondrial genes, with the genetic and physical map of yeast mitochondria being completed in 1976.

A mitochondrion contains outer and inner membranes composed of phospholipid bilayers and proteins. The two membranes have different properties. Because of this double-membraned organization, there are five distinct parts to a mitochondrion. They are:

  1. the outer mitochondrial membrane,
  2. the intermembrane space (the space between the outer and inner membranes),
  3. the inner mitochondrial membrane,
  4. the cristae space (formed by infoldings of the inner membrane), and
  5. the matrix (space within the inner membrane).

Mitochondria stripped of their outer membrane are called mitoplasts.




Mitochondrion ultrastructure (interactive diagram) A mitochondrion has a double membrane; the inner one contains its chemiosmotic apparatus and has deep grooves which increase its surface area. While commonly depicted as an “orange sausage with a blob inside of it” (like it is here), mitochondria can take many shapes and their intermembrane space is quite thin.

The intermembrane space is the space between the outer membrane and the inner membrane. It is also known as perimitochondrial space. Because the outer membrane is freely permeable to small molecules, the concentrations of small molecules such as ions and sugars in the intermembrane space is the same as the cytosol. However, large proteins must have a specific signaling sequence to be transported across the outer membrane, so the protein composition of this space is different from the protein composition of the cytosol. One protein that is localized to the intermembrane space in this way is cytochrome c.

The inner mitochondrial membrane contains proteins with five types of functions:

  1. Those that perform the redox reactions of oxidative phosphorylation
  2. ATP synthase, which generates ATP in the matrix
  3. Specific transport proteins that regulate metabolite passage into and out of the matrix
  4. Protein import machinery.
  5. Mitochondria fusion and fission protein.

It contains more than 151 different polypeptides, and has a very high protein-to-phospholipid ratio (more than 3:1 by weight, which is about 1 protein for 15 phospholipids). The inner membrane is home to around 1/5 of the total protein in a mitochondrion. In addition, the inner membrane is rich in an unusual phospholipid, cardiolipin. This phospholipid was originally discovered in cow hearts in 1942, and is usually characteristic of mitochondrial and bacterial plasma membranes. Cardiolipin contains four fatty acids rather than two, and may help to make the inner membrane impermeable. Unlike the outer membrane, the inner membrane doesn’t contain porins, and is highly impermeable to all molecules. Almost all ions and molecules require special membrane transporters to enter or exit the matrix. Proteins are ferried into the matrix via the translocase of the inner membrane (TIM) complex or via Oxa1. In addition, there is a membrane potential across the inner membrane, formed by the action of the enzymes of the electron transport chain.

The inner mitochondrial membrane is compartmentalized into numerous cristae, which expand the surface area of the inner mitochondrial membrane, enhancing its ability to produce ATP. For typical liver mitochondria, the area of the inner membrane is about five times as large as the outer membrane. This ratio is variable and mitochondria from cells that have a greater demand for ATP, such as muscle cells, contain even more cristae. These folds are studded with small round bodies known as F1 particles or oxysomes. These are not simple random folds but rather invaginations of the inner membrane, which can affect overall chemiosmotic function. One recent mathematical modeling study has suggested that the optical properties of the cristae in filamentous mitochondria may affect the generation and propagation of light within the tissue.




The matrix is the space enclosed by the inner membrane. It contains about 2/3 of the total protein in a mitochondrion. The matrix is important in thThe MAM is enriched in enzymes involved in lipid biosynthesis, such as phosphatidylserine synthase on the ER face and phosphatidylserine decarboxylase on the mitochondrial face.[28][29] Because mitochondria are dynamic organelles constantly undergoing fission and fusion events, they require a constant and well-regulated supply of phospholipids for membrane integrity.[30][31] But mitochondria are not only a destination for the phospholipids they finish synthesis of; rather, this organelle also plays a role in inter-organelle trafficking of the intermediates and products of phospholipid biosynthetic pathways, ceramide and cholesterol metabolism, and glycosphingolipid anabolisme production of ATP with the aid of the ATP synthase contained in the inner membrane. The matrix contains a highly concentrated mixture of hundreds of enzymes, special mitochondrial ribosomes, tRNA, and several copies of the mitochondrial DNA genome. Of the enzymes, the major functions include oxidation of pyruvate and fatty acids, and the citric acid cycle.

Purified MAM from subcellular fractionation has shown to be enriched in enzymes involved in phospholipid exchange, in addition to channels associated with Ca2+ signaling. The mitochondria-associated ER membrane (MAM) is another structural element that is increasingly recognized for its critical role in cellular physiology and homeostasis. Once considered a technical snag in cell fractionation techniques, the alleged ER vesicle contaminants that invariably appeared in the mitochondrial fraction have been re-identified as membranous structures derived from the MAM—the interface between mitochondria and the ER. Physical coupling between these two organelles had previously been observed in electron micrographs and has more recently been probed with fluorescence microscopy. Such studies estimate that at the MAM, which may comprise up to 20% of the mitochondrial outer membrane, the ER and mitochondria are separated by a mere 10–25 nm and held together by protein tethering complexes.

Such trafficking capacity depends on the MAM, which has been shown to facilitate transfer of lipid intermediates between organelles. In contrast to the standard vesicular mechanism of lipid transfer, evidence indicates that the physical proximity of the ER and mitochondrial membranes at the MAM allows for lipid flipping between opposed bilayers. Despite this unusual and seemingly energetically unfavorable mechanism, such transport does not require ATP. Instead, in yeast, it has been shown to be dependent on a multiprotein tethering structure termed the ER-mitochondria encounter structure, or ERMES, although it remains unclear whether this structure directly mediates lipid transfer or is required to keep the membranes in sufficiently close proximity to lower the energy barrier for lipid flipping.

A critical role for the ER in calcium signaling was acknowledged before such a role for the mitochondria was widely accepted, in part because the low affinity of Ca2+ channels localized to the outer mitochondrial membrane seemed to fly in the face of this organelle’s purported responsiveness to changes in intracellular Ca2+ flux. But the presence of the MAM resolves this apparent contradiction: the close physical association between the two organelles results in Ca2+ microdomains at contact points that facilitate efficient Ca2+ transmission from the ER to the mitochondria. Transmission occurs in response to so-called “Ca2+ puffs” generated by spontaneous clustering and activation of IP3R, a canonical ER membrane Ca2+ channel.

The properties of the Ca2+ pump SERCA and the channel IP3R present on the ER membrane facilitate feedback regulation coordinated by MAM function. In particular, clearance of Ca2+ by the MAM allows for spatio-temporal patterning of Ca2+ signaling because Ca2+ alters IP3R activity in a biphasic manner. SERCA is likewise affected by mitochondrial feedback: uptake of Ca2+ by the MAM stimulates ATP production, thus providing energy that enables SERCA to reload the ER with Ca2+ for continued Ca2+ efflux at the MAM. Thus, the MAM is not a passive buffer for Ca2+ puffs; rather it helps modulate further Ca2+ signaling through feedback loops that affect ER dynamics.

Regulating ER release of Ca2+ at the MAM is especially critical because only a certain window of Ca2+ uptake sustains the mitochondria, and consequently the cell, at homeostasis. Sufficient intraorganelle Ca2+ signaling is required to stimulate metabolism by activating dehydrogenase enzymes critical to flux through the citric acid cycle. However, once Ca2+ signaling in the mitochondria passes a certain threshold, it stimulates the intrinsic pathway of apoptosis in part by collapsing the mitochondrial membrane potential required for metabolism.  Studies examining the role of pro- and anti-apoptotic factors support this model; for example, the anti-apoptotic factor Bcl-2 has been shown to interact with IP3Rs to reduce Ca2+ filling of the ER, leading to reduced efflux at the MAM and preventing collapse of the mitochondrial membrane potential post-apoptotic stimuli. Given the need for such fine regulation of Ca2+ signaling, it is perhaps unsurprising that dysregulated mitochondrial Ca2+ has been implicated in several neurodegenerative diseases, while the catalogue of tumor suppressors includes a few that are enriched at the MAM.



Lysosome and Apoptosis

Role of autophagy in cancer

R Mathew, V Karantza-Wadsworth & E White

Nature Reviews Cancer 7, 961-967 (Dec 2007) |  http://dx.doi.org:/10.1038/nrc2254

Autophagy is a cellular degradation pathway for the clearance of damaged or superfluous proteins and organelles. The recycling of these intracellular constituents also serves as an alternative energy source during periods of metabolic stress to maintain homeostasis and viability. In tumour cells with defects in apoptosis, autophagy allows prolonged survival. Paradoxically, autophagy defects are associated with increased tumorigenesis, but the mechanism behind this has not been determined. Recent evidence suggests that autophagy provides a protective function to limit tumour necrosis and inflammation, and to mitigate genome damage in tumour cells in response to metabolic stress.

Sustained Activation of mTORC1 in Skeletal Muscle Inhibits Constitutive and Starvation-Induced Autophagy and Causes a Severe, Late-Onset Myopathy

P Castets, S Lin, N Rion, S Di Fulvio, et al.
cell-metabolism 7 May, 2013; 17(5): p731–744   http://dx.doi.org/10.1016/j.cmet.2013.03.015

  • mTORC1 inhibition is required for constitutive and starvation-induced autophagy
  • Sustained activation of mTORC1 causes a severe myopathy due to autophagy impairment
  • TSC1 depletion is sufficient to activate mTORC1 irrespective of other stimuli
  • mTORC1 inactivation is sufficient to trigger LC3 lipidation

Autophagy is a catabolic process that ensures homeostatic cell clearance and is deregulated in a growing number of myopathological conditions. Although FoxO3 was shown to promote the expression of autophagy-related genes in skeletal muscle, the mechanisms triggering autophagy are unclear. We show that TSC1-deficient mice (TSCmKO), characterized by sustained activation of mTORC1, develop a late-onset myopathy related to impaired autophagy. In young TSCmKO mice,

  • constitutive and starvation-induced autophagy is blocked at the induction steps via
  • mTORC1-mediated inhibition of Ulk1, despite FoxO3 activation.

Rapamycin is sufficient to restore autophagy in TSCmKO mice and

  • improves the muscle phenotype of old mutant mice.

Inversely, abrogation of mTORC1 signaling by

  • depletion of raptor induces autophagy regardless of FoxO inhibition.

Thus, mTORC1 is the dominant regulator of autophagy induction in skeletal muscle and

  • ensures a tight coordination of metabolic pathways.

These findings may open interesting avenues for therapeutic strategies directed toward autophagy-related muscle diseases.

Histone deacetylases 1 and 2 regulate autophagy flux and skeletal muscle homeostasis in mice

Viviana Moresi, et al.   PNAS Jan 31, 2012; 109(5): 1649-1654

HDAC1 activates FoxO and is both sufficient and required for skeletal muscle atrophy

Beharry, PB. Sandesara, BM. Roberts, et al.
J. Cell Sci. Apr 2014 127 (7) 1441-1453   http://dx.doi.org:/10.1242/​jcs.136390

The Forkhead box O (FoxO) transcription factors are activated, and necessary for the muscle atrophy, in several pathophysiological conditions, including muscle disuse and cancer cachexia. However, the mechanisms that lead to FoxO activation are not well defined. Recent data from our laboratory and others indicate that

  • the activity of FoxO is repressed under basal conditions via reversible lysine acetylation,
  • which becomes compromised during catabolic conditions.

Therefore, we aimed to determine how histone deacetylase (HDAC) proteins contribute to

  • activation of FoxO and induction of the muscle atrophy program.

Through the use of various pharmacological inhibitors to block HDAC activity, we demonstrate that

  • class I HDACs are key regulators of FoxO and the muscle-atrophy program
  • during both nutrient deprivation and skeletal muscle disuse.

Furthermore, we demonstrate, through the use of wild-type and dominant-negative HDAC1 expression plasmids,

  • that HDAC1 is sufficient to activate FoxO and induce muscle fiber atrophy in vivo and
  • is necessary for the atrophy of muscle fibers that is associated with muscle disuse.

The ability of HDAC1 to cause muscle atrophy required its deacetylase activity and

  • was linked to the induction of several atrophy genes by HDAC1,
  • including atrogin-1, which required deacetylation of FoxO3a.

Moreover, pharmacological inhibition of class I HDACs during muscle disuse, using MS-275,

  • significantly attenuated both disuse muscle fiber atrophy and contractile dysfunction.

Together, these data solidify the importance of class I HDACs in the muscle atrophy program and

  • indicate that class I HDAC inhibitors are feasible countermeasures to impede muscle atrophy and weakness.

Autophagy and thyroid carcinogenesis: genetic and epigenetic links
F Morani, R Titone, L Pagano, et al.  Endocr Relat Cancer Feb 1, 2014 21 R13-R29

Autophagy is a vesicular process for the lysosomal degradation of protein aggregates and

  • of damaged or redundant organelles.

Autophagy plays an important role in cell homeostasis, and there is evidence that

  • this process is dysregulated in cancer cells.

Recent in vitro preclinical studies have indicated that autophagy is

  • involved in the cytotoxic response to chemotherapeutics in thyroid cancer cells.

Indeed, several oncogenes and oncosuppressor genes implicated in thyroid carcinogenesis

  • also play a role in the regulation of autophagy.

In addition, some epigenetic modulators involved in thyroid carcinogenesis also influence autophagy. In this review, we highlight the genetic and epigenetic factors that

  • mechanistically link thyroid carcinogenesis and autophagy, thus substantiating the rationale for
  • an autophagy-targeted therapy of aggressive and radio-chemo-resistant thyroid cancers.

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Compilation of References in Leaders in Pharmaceutical Intelligence about proteomics, metabolomics, signaling pathways, and cell regulation

Compilation of References in Leaders in Pharmaceutical Intelligence about
proteomics, metabolomics, signaling pathways, and cell regulation

Curator: Larry H. Bernstein, MD, FCAP



  1. The Human Proteome Map Completed
    Reporter and Curator: Larry H. Bernstein, MD, FCAP
  1. Proteomics – The Pathway to Understanding and Decision-making in Medicine
    Author and Curator, Larry H Bernstein, MD, FCAP
  1. Advances in Separations Technology for the “OMICs” and Clarification of Therapeutic Targets
    Author and Curator, Larry H Bernstein, MD, FCAP
  1. Expanding the Genetic Alphabet and Linking the Genome to the Metabolome
    Author and Curator, Larry H Bernstein, MD, FCAP
  1. Synthesizing Synthetic Biology: PLOS Collections
    Reporter: Aviva Lev-Ari



  1. Extracellular evaluation of intracellular flux in yeast cells
    Larry H. Bernstein, MD, FCAP, Reviewer and Curator
  2. Metabolomic analysis of two leukemia cell lines. I.
    Larry H. Bernstein, MD, FCAP, Reviewer and Curator
  3. Metabolomic analysis of two leukemia cell lines. II.
    Larry H. Bernstein, MD, FCAP, Reviewer and Curator
  4. Metabolomics, Metabonomics and Functional Nutrition: the next step in nutritional metabolism and biotherapeutics
    Reviewer and Curator, Larry H. Bernstein, MD, FCAP
  5. Buffering of genetic modules involved in tricarboxylic acid cycle metabolism provides homeomeostatic regulation
    Larry H. Bernstein, MD, FCAP, Reviewer and curator


Metabolic Pathways

  1. Pentose Shunt, Electron Transfer, Galactose, more Lipids in brief
    Reviewer and Curator: Larry H. Bernstein, MD, FCAP
  2. Mitochondria: More than just the “powerhouse of the cell”
    Reviewer and Curator: Ritu Saxena
  3. Mitochondrial fission and fusion: potential therapeutic targets?
    Reviewer and Curator: Ritu saxena
  4. Mitochondrial mutation analysis might be “1-step” away
    Reviewer and Curator: Ritu Saxena
  5. Selected References to Signaling and Metabolic Pathways in PharmaceuticalIntelligence.com
    Curator: Larry H. Bernstein, MD, FCAP
  6. Metabolic drivers in aggressive brain tumors
    Prabodh Kandal, PhD
  7. Metabolite Identification Combining Genetic and Metabolic Information: Genetic association links unknown metabolites to functionally related genes
    Author and Curator: Aviva Lev-Ari, PhD, RD
  8. Mitochondria: Origin from oxygen free environment, role in aerobic glycolysis, metabolic adaptation
    Author and curator:Larry H Bernstein, MD, FCAP
  9. Therapeutic Targets for Diabetes and Related Metabolic Disorders
    Reporter, Aviva Lev-Ari, PhD, RD
  10. Buffering of genetic modules involved in tricarboxylic acid cycle metabolism provides homeomeostatic regulation
    Larry H. Bernstein, MD, FCAP, Reviewer and curator
  11. The multi-step transfer of phosphate bond and hydrogen exchange energy
    Curator:Larry H. Bernstein, MD, FCAP,
  12. Studies of Respiration Lead to Acetyl CoA
    Author and Curator: Larry H. Bernstein, MD, FCAP
  13. Lipid Metabolism
    Author and Curator: Larry H. Bernstein, MD, FCAP
  14. Carbohydrate Metabolism
    Author and Curator: Larry H. Bernstein, MD, FCAP
  15. Prologue to Cancer – e-book Volume One – Where are we in this journey?
    Author and Curator: Larry H. Bernstein, MD, FCAP
  16. Introduction – The Evolution of Cancer Therapy and Cancer Research: How We Got Here?
    Author and Curator: Larry H. Bernstein, MD, FCAP
  17. Inhibition of the Cardiomyocyte-Specific Kinase TNNI3K
    Author and Curator: Larry H. Bernstein, MD, FCAP
  18. The Binding of Oligonucleotides in DNA and 3-D Lattice Structures
    Author and Curator: Larry H. Bernstein, MD, FCAP
  19. Mitochondrial Metabolism and Cardiac Function
    Author and Curator: Larry H. Bernstein, MD, FCAP
  20. How Methionine Imbalance with Sulfur-Insufficiency Leads to Hyperhomocysteinemia
    Curator: Larry H. Bernstein, MD, FCAP
  21. AMPK Is a Negative Regulator of the Warburg Effect and Suppresses Tumor Growth In Vivo
    Author and Curator: SJ. Williams
  22. A Second Look at the Transthyretin Nutrition Inflammatory Conundrum
    Author and Curator: Larry H. Bernstein, MD, FCAP
  23. Overview of Posttranslational Modification (PTM)
    Writer and Curator: Larry H. Bernstein, MD, FCAP
  24. Malnutrition in India, high newborn death rate and stunting of children age under five years
    Writer and Curator: Larry H. Bernstein, MD, FCAP
  25. Update on mitochondrial function, respiration, and associated disorders
    Writer and Curator: Larry H. Bernstein, MD, FCAP
  26. Omega-3 fatty acids, depleting the source, and protein insufficiency in renal disease
    Larry H. Bernstein, MD, FCAP, Curator
  27. Late Onset of Alzheimer’s Disease and One-carbon Metabolism
    Reporter and Curator: Dr. Sudipta Saha, Ph.D.
  28. Problems of vegetarianism
    Reporter and Curator: Dr. Sudipta Saha, Ph.D.


Signaling Pathways

  1. Introduction to e-Series A: Cardiovascular Diseases, Volume Four Part 2: Regenerative Medicine
    Larry H. Bernstein, MD, FCAP, writer, and Aviva Lev- Ari, PhD, RN  https://pharmaceuticalintelligence.com/2014/04/27/larryhbernintroduction_to_cardiovascular_diseases-translational_medicine-part_2/
  2. Epilogue: Envisioning New Insights in Cancer Translational Biology
    Series C: e-Books on Cancer & Oncology
    Author & Curator: Larry H. Bernstein, MD, FCAP, Series C Content Consultant
  3. Ca2+-Stimulated Exocytosis:  The Role of Calmodulin and Protein Kinase C in Ca2+ Regulation of Hormone and Neurotransmitter  Writer and Curator: Larry H Bernstein, MD, FCAP and Curator and Content Editor: Aviva Lev-Ari, PhD, RN
  4. Cardiac Contractility & Myocardial Performance: Therapeutic Implications of Ryanopathy (Calcium Release-related Contractile Dysfunction) and Catecholamine Responses
    Author, and Content Consultant to e-SERIES A: Cardiovascular Diseases: Justin Pearlman, MD, PhD, FACC
    Author and Curator: Larry H Bernstein, MD, FCAP and Article Curator: Aviva Lev-Ari, PhD, RN
  5. Role of Calcium, the Actin Skeleton, and Lipid Structures in Signaling and Cell Motility
    Author and Curator: Larry H Bernstein, MD, FCAP Author: Stephen Williams, PhD, and Curator: Aviva Lev-Ari, PhD, RN
  6. Identification of Biomarkers that are Related to the Actin Cytoskeleton
    Larry H Bernstein, MD, FCAP, Author and Curator
  7. Advanced Topics in Sepsis and the Cardiovascular System at its End Stage
    Author and Curator: Larry H Bernstein, MD, FCAP
  8. The Delicate Connection: IDO (Indolamine 2, 3 dehydrogenase) and Cancer Immunology
    Demet Sag, PhD, Author and Curator
  9. IDO for Commitment of a Life Time: The Origins and Mechanisms of IDO, indolamine 2, 3-dioxygenase
    Demet Sag, PhD, Author and Curator
  10. Confined Indolamine 2, 3 dioxygenase (IDO) Controls the Homeostasis of Immune Responses for Good and Bad
    Author and Curator: Demet Sag, PhD, CRA, GCP
  11. Signaling Pathway that Makes Young Neurons Connect was discovered @ Scripps Research Institute
    Reporter: Aviva Lev-Ari, PhD, RN
  12. Naked Mole Rats Cancer-Free
    Writer and Curator: Larry H. Bernstein, MD, FCAP
  13. Amyloidosis with Cardiomyopathy
    Writer and Curator: Larry H. Bernstein, MD, FCAP
  14. Liver endoplasmic reticulum stress and hepatosteatosis
    Larry H Bernstein, MD, FACP
  15. The Molecular Biology of Renal Disorders: Nitric Oxide – Part III
    Curator and Author: Larry H Bernstein, MD, FACP
  16. Nitric Oxide Function in Coagulation – Part II
    Curator and Author: Larry H. Bernstein, MD, FCAP
  17. Nitric Oxide, Platelets, Endothelium and Hemostasis
    Curator and Author: Larry H Bernstein, MD, FACP
  18. Interaction of Nitric Oxide and Prostacyclin in Vascular Endothelium
    Curator and Author: Larry H Bernstein, MD, FACP
  19. Nitric Oxide and Immune Responses: Part 1
    Curator and Author:  Aviral Vatsa PhD, MBBS
  20. Nitric Oxide and Immune Responses: Part 2
    Curator and Author:  Aviral Vatsa PhD, MBBS
  21. Nitric Oxide and iNOS have Key Roles in Kidney Diseases – Part II
    Curator and Author: Larry H Bernstein, MD, FACP
  22. New Insights on Nitric Oxide donors – Part IV
    Curator and Author: Larry H Bernstein, MD, FACP
  23. Crucial role of Nitric Oxide in Cancer
    Curator and Author: Ritu Saxena, Ph.D.
  24. Nitric Oxide has a ubiquitous role in the regulation of glycolysis -with a concomitant influence on mitochondrial function
    Curator and Author: Larry H Bernstein, MD, FACP
  25. Nitric Oxide and Immune Responses: Part 2
    Author and Curator: Aviral Vatsa, PhD, MBBS
  26. Mitochondrial Damage and Repair under Oxidative Stress
    Author and Curator: Larry H. Bernstein, MD, FCAP
  27. Is the Warburg Effect the cause or the effect of cancer: A 21st Century View?
    Curator and Author: Larry H Bernstein, MD, FACP
  28. Targeting Mitochondrial-bound Hexokinase for Cancer Therapy
    Curator and Author: Ziv Raviv, PhD, RN 04/06/2013
  29. Ubiquinin-Proteosome pathway, autophagy, the mitochondrion, proteolysis and cell apoptosis
    Curator and Author: Larry H Bernstein, MD, FACP
  30. Ubiquitin-Proteosome pathway, Autophagy, the Mitochondrion, Proteolysis and Cell Apoptosis: Part III
    Curator and Author: Larry H Bernstein, MD, FACP
  31. Biochemistry of the Coagulation Cascade and Platelet Aggregation – Part I
    Curator and Author: Larry H Bernstein, MD, FACP


Genomics, Transcriptomics, and Epigenetics

  1. What is the meaning of so many RNAs?
    Writer and Curator: Larry H. Bernstein, MD, FCAP
  2. RNA and the transcription the genetic code
    Larry H. Bernstein, MD, FCAP, Writer and Curator
  3. A Primer on DNA and DNA Replication
    Writer and Curator: Larry H. Bernstein, MD, FCAP
  4. Pathology Emergence in the 21st Century
    Author and Curator: Larry Bernstein, MD, FCAP
  5. RNA and the transcription the genetic code
    Writer and Curator, Larry H. Bernstein, MD, FCAP
  6. Commentary on Biomarkers for Genetics and Genomics of Cardiovascular Disease: Views by Larry H Bernstein, MD, FCAP
    Author: Larry H Bernstein, MD, FCAP
  7. Observations on Finding the Genetic Links in Common Disease: Whole Genomic Sequencing Studies
    Author an Curator: Larry H Bernstein, MD, FCAP
  8. Silencing Cancers with Synthetic siRNAs
    Larry H. Bernstein, MD, FCAP, Reviewer and Curator
  9. Cardiometabolic Syndrome and the Genetics of Hypertension: The Neuroendocrine Transcriptome Control Points
    Reporter: Aviva Lev-Ari, PhD, RN
  10. Developments in the Genomics and Proteomics of Type 2 Diabetes Mellitus and Treatment Targets
    Larry H. Bernstein, MD, FCAP, Reviewer and Curator
  11. CT Angiography & TrueVision™ Metabolomics (Genomic Phenotyping) for new Therapeutic Targets to Atherosclerosis
    Reporter: Aviva Lev-Ari, PhD, RN
  12. CRACKING THE CODE OF HUMAN LIFE: The Birth of BioInformatics & Computational Genomics
    Genomics Curator, Larry H Bernstein, MD, FCAP
  13. Big Data in Genomic Medicine
    Author and Curator, Larry H Bernstein, MD, FCAP
  14.  From Genomics of Microorganisms to Translational Medicine
    Author and Curator: Demet Sag, PhD
  15.  Summary of Genomics and Medicine: Role in Cardiovascular Diseases
    Author and Curator, Larry H Bernstein, MD, FCAP

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