Memory-Boosting Chemical Is Identified in Mice

Memory improved in mice injected with a small, drug-like molecule discovered by UCSF San Francisco researchers studying how cells respond to biological stress.

The same biochemical pathway the molecule acts on might one day be targeted in humans to improve memory, according to the senior author of the study, Peter Walter, PhD, UCSF professor of biochemistry and biophysics and a Howard Hughes Investigator.

The discovery of the molecule and the results of the subsequent memory tests in mice were published in eLife, an online scientific open-access journal, on May 28, 2013.

In one memory test included in the study, normal mice were able to relocate a submerged platform about three times faster after receiving injections of the potent chemical than mice that received sham injections.

The mice that received the chemical also better remembered cues associated with unpleasant stimuli – the sort of fear conditioning that could help a mouse avoid being preyed upon.

Notably, the findings suggest that despite what would seem to be the importance of having the best biochemical mechanisms to maximize the power of memory, evolution does not seem to have provided them, Walter said.

“It appears that the process of evolution has not optimized memory consolidation; otherwise I don’t think we could have improved upon it the way we did in our study with normal, healthy mice,” Walter said.

The memory-boosting chemical was singled out from among 100,000 chemicals screened at the Small Molecule Discovery Center at UCSF for their potential to perturb a protective biochemical pathway within cells that is activated when cells are unable to keep up with the need to fold proteins into their working forms.

However, UCSF postdoctoral fellow Carmela Sidrauski, PhD, discovered that the chemical acts within the cell beyond the biochemical pathway that activates this unfolded protein response, to more broadly impact what’s known as the integrated stress response. In this response, several biochemical pathways converge on a single molecular lynchpin, a protein called eIF2 alpha.

Scientists have known that in organisms ranging in complexity from yeast to humans different kinds of cellular stress — a backlog of unfolded proteins, DNA-damaging UV light, a shortage of the amino acid building blocks needed to make protein, viral infection, iron deficiency — trigger different enzymes to act downstream to switch off eIF2 alpha.

“Among other things, the inactivation of eIF2 alpha is a brake on memory consolidation,” Walter said, perhaps an evolutionary consequence of a cell or organism becoming better able to adapt in other ways.

Turning off eIF2 alpha dials down production of most proteins, some of which may be needed for memory formation, Walter said. But eIF2 alpha inactivation also ramps up production of a few key proteins that help cells cope with stress.

Study co-author Nahum Sonenberg, PhD, of McGill University previously linked memory and eIF2 alpha in genetic studies of mice, and his lab group also conducted the memory tests for the current study.

The chemical identified by the UCSF researchers is called ISRIB, which stands for integrated stress response inhibitor. ISRIB counters the effects of eIF2 alpha inactivation inside cells, the researchers found.

“ISRIB shows good pharmacokinetic properties [how a drug is absorbed, distributed and eliminated], readily crosses the blood-brain barrier, and exhibits no overt toxicity in mice, which makes it very useful for studies in mice,” Walter said. These properties also indicate that ISRIB might serve as a good starting point for human drug development, according to Walter.

Walter said he is looking for scientists to collaborate with in new studies of cognition and memory in mouse models of neurodegenerative diseases and aging, using ISRIB or related molecules.

In addition, chemicals such as ISRIB could play a role in fighting cancers, which take advantage of stress responses to fuel their own growth, Walter said. Walter already is exploring ways to manipulate the unfolded protein response to inhibit tumor growth, based on his earlier discoveries.

At a more basic level, Walter said, he and other scientists can now use ISRIB to learn more about the role of the unfolded protein response and the integrated stress response in disease and normal physiology.

New Scientific Analysis Shines a Light on Ötzi the Iceman’s Dark Secrets

Protein investigation supports brain injury theory and opens up new research possibilities for mummies

After decoding the Iceman’s genetic make-up, a research team from the European Academy of Bolzano/Bozen (EURAC), Saarland University, Kiel University and other partners has now made another major breakthrough in mummy research: using just a pinhead-sized sample of brain tissue from the world-famous glacier corpse, the team was able to extract and analyse proteins to further support the theory that Ötzi suffered some form of brain damage in the final moments of his life.

Two dark coloured areas at the back of the Iceman’s cerebrum had first been mentioned back in 2007 during a discussion about the fracture to his skull. Scientists surmised from a CAT scan of his brain that he had received a blow to the forehead during his deadly attack that caused his brain to knock against the back of his head, creating dark spots from the bruising. Till now, this hypothesis had been left unexplored.

In 2010, with the help of computer-controlled endoscopy, two samples of brain tissue the size of a pinhead were extracted from the glacier mummy. This procedure was carried out via two tiny (previously existing) access holes and was thus minimally invasive. Microbiologist Frank Maixner (EURAC, Institute for Mummies and the Iceman) and his fellow scientist Andreas Tholey (Institute for Experimental Medicine, Kiel University) conducted two parallel, independent studies on the tiny bundles of cells. Tholey’s team provided the latest technology used in the study of complex protein mixtures known as “proteomes”. The various analyses were coordinated by Frank Maixner and Andreas Keller.

The protein research revealed a surprising amount of information. Scientists were able to identify numerous brain proteins, as well as proteins from blood cells. Microscopic investigation also confirmed the presence of astonishingly well-preserved neural cell structures and clotted blood cells. On the one hand, this led the scientists to conclude that the recovered samples did indeed come from brain tissue in remarkably good condition (the proteins contained amino acid sequence features specific to Ötzi). On the other hand, these blood clots in a corpse almost devoid of blood provided further evidence that Ötzi’s brain had possibly suffered bruising shortly before his death. Whether this was due to a blow to the forehead or a fall after being injured by the arrow remains unclear.

The discoveries represent a major breakthrough for the scientists. The research team emphasised that “the use of new protein-analysis methods has enabled us to pioneer this type of protein investigation on the soft tissue of a mummified human, extracting from the tiniest sample a vast quantity of data which in the future may well answer many further questions.” While many DNA samples from mummies are difficult or impossible to analyse because of natural biological decay, one can often still find proteins in tissue samples which allow a closer analysis and provide valuable information, explained Andreas Tholey: “Proteins are the decisive players in tissues and cells, and they conduct most of the processes which take place in cells. Identification of the proteins is therefore key to understanding the functional potential of a particular tissue. DNA is always constant, regardless of from where it originates in the body, whereas proteins provide precise information about what is happening in specific regions within the body.” Protein analysis of mummified tissue makes an especially valuable contribution to DNA research, Maixner added: “Investigating mummified tissue can be very frustrating. The samples are often damaged or contaminated and do not necessarily yield results, even after several attempts and using a variety of investigative methods. When you think that we have succeeded in identifying actual tissue changes in a human who lived over 5,000 years ago, you can begin to understand how pleased we are as scientists that we persisted with our research after many unsuccessful attempts. It has definitely proved worthwhile!”

The results of this joint study are published in the renowned journal “Cellular and Molecular Life Sciences”. Along with a sample taken from the Iceman´s stomach content, more than a dozen tissue samples from less well preserved mummies from all over the world will be submitted to this new protein-based research method and should provide insights which previously had not been possible.

Balancing mitochondrial dynamics in Alzheimer’s disease

Many diseases are multifactorial and can not be understood by simple molecular associations alone. Alzheimer’s disease (AD) is associated with toxic transformations in two classes of protein,amyloid beta and tau, but they do not explain the full underlying pathology. On the cellular scale, much of the real-time morphological changes in neurons can be attributed to their underlying mitochondrial dynamics—namely fission, fusion, and the motions between these events. Last year, researchers from Harvard Medical School made the intriguing discovery that alterations in tau could lead to a doubling in the length of mitochondria. This week, they published a review article in Trends in Neuroscience, in which they seek to explain the primary features of AD in terms of mitochondrial dynamics.

Together with a collaborator from the Queensland Brain Institute, the Harvard researchers arrive at the conclusion that, like many other neurological diseases, AD is fundamentally an energy problem. While some proteins, like APOE-ɛ4 can predispose one to AD, point defects in individual proteins can not account for AD in the same way that a single alteration in hemoglobin leads to sickle cell disease. Attempts to assign casual relations to the complex interactions of tau or amyloid, with hundreds of other proteins inside neurons have frequently served to cloud, rather than simplify the AD story.

In years gone by, it was possible to publish a paper about how phosphorylation at certain sites on proteins, like tau, could lead to any number of downstream events. Tau is one of many proteins that control the assembly and stability of microtubules, critical structures that are among those compromised in AD. The problem now, is that we know tau comes in so many flavors—it is a big family of different isoforms with different properties depending on how they are processed. As far as simple phosphorylation, tau has been found to have 79 potential sites, with at least 30 of them normally phosphorylated.

A welcome simplification to this situation of compounding molecular complexity, is that many pathways converge onto convenient pre-existing packets of time, space, and predictable molecular structure—the mitochondria. As opposed to massive cell-wide molecular accounting, describing a few sub-cellular morphological features may be a more tractable approach not only to capture disease etiology, but perhaps to treat it.

To this end, the researchers apply existing knowledge regarding some of the molecular players in AD, to a few of the well-established control points in mitochondrial dynamics. State transitions between fission and fusion are, at the moment at least, characterized by only a small handful of proteins. This simple formula might be prescribed as the following: molecular pathway locally effects the organelle dynamics, then, the dynamic behavior of organelle accounts for the disease. The imposition of this middleman can potentially simplify much of the vast body of fact and conjecture associated with the disease.

The elongation of mitochondria by tau can be caused by increasing fusion, decreasing fission, or both. One function of tau is to stabilize F-actin networks which prevents a key fission protein from ever reaching the mitochondria. Elongated mitochondria do not necessarily cause AD. In fact, amyloid beta, which is concentrated inside mitochondria, has been shown to cause increased fission and decreased fusion. When the balance between fission and fusion is pushed too far in either direction, the result is bad news for neurons. If there are defects in the transport of mitochondria, as seems to be the case in many neurological diseases, their redistribution is unable to compensate for this loss of balance.

Specific disease-associated isoforms and phosphorylation states of tau can lead to AD through the loss of mitochondria in axons. In studies of AD tissue, mitochondria have been found to be preferentially redistributed to the soma. These selective localizations can take place quickly, and are therefore difficult to quantify except by live videomicroscopy. In synapses, the mitochondria have been observed to be longer lived, and to play a more critical role in calcium regulation then those elsewhere. Disruption in the normal handling of calcium has been attributed to many aspects of AD, particularly synaptic pathology.

The canonical dogma that action potentials lead to vesicle fusion and transmitter release exclusively through the entry of extracellular calcium has recently been enhanced with the understanding that mitochondria contribute significantly to the synaptic calcium cycle. While mitochondria clearly do not depolarize as rapidly as whole spiking cells,(generally when mitochondria are depolarized there is some problem) their calcium transporters operate quickly to mop up and redistribute calcium. To say that mitochondria might single-handedly initiate vesicle fusion, or for that matter minipotentials or full-blown spikes, would await future experimental corroboration.

Countless scores of papers over the years have attempted to make sense of the myriad synaptic pathways underlying memory and LTP. They might be better understood when mitochondria are viewed as the primary authors of synaptic vesicle release probability, and by implication, “spontaneous” release (vesicle fusion in the absence of a spike). As in disease states, specific pathways, structures and organelles have significant roles to play in many aspects of brain function—but causally relating the motions and dynamics of mitochondria to these phenomena now gives the broadest interpretive power.

Finding a family for a pair of orphan receptors in the brain

Researchers at Emory University have identified a protein that stimulates a pair of “orphan receptors” found in the brain, solving a long-standing biological puzzle and possibly leading to future treatments for neurological diseases.

The results are published in the Proceedings of the National Academy of Sciences, Early Edition.

The human genome is littered with orphans: proteins that look like they will bind and respond to a hormone or a brain chemical, based on the similarity of their sequences to other proteins. However, scientists haven’t figured out what each orphan’s partner chemical is yet.

Orphans that look like GPCRs (G protein-coupled receptors) currently number about 100. GPCRs are the targets of many drugs and are involved in vision, smell and brain cells’ responses to a host of hormones and neurotransmitters. One orphan GPCR, called GPR37, has attracted interest from researchers because it is connected with an inherited form of Parkinson’s disease. It is abundant in the dopamine-producing neurons that degenerate in Parkinson’s. But its partner chemical, or “ligand,” has not been found.

"We reasoned that GPR37 had to be doing something important, besides becoming misfolded in some forms of Parkinson’s," says senior author Randy Hall, PhD, professor of pharmacology at Emory University School of Medicine.

Working with Hall, graduate student Rebecca Meyer devised a way to detect when cells producing GPR37 were reacting with GPR37’s ligand.

"Usually, cells remove GPCRs from their surfaces when they encounter their ligand," Meyer says. "So we set things up so that GPR37 would be labeled red on the surface of the cell, but would appear green once internalized."

They discovered that cells producing GPR37 – and also a close relative, GPR37L1 — respond to a protein known as prosaposin, which was discovered by John O’Brien of University of California San Diego in the 1990s.

Prosaposin is a growth factor for brain cells and protects them from stress. Scientists studying it had worked out that it stimulates cells via a GPCR – but which one was unclear until now. In animal models, prosaposin has shown potential for treating conditions such as stroke, Parkinson’s and neuropathic pain. An artificial fragment of prosaposin called prosaptide has been tested in clinical studies, but it quickly breaks down in the body.

"That’s the reason why it was so important to find the receptor," Hall says. "Then we can actually do some pharmacology."

Now, Hall’s laboratory is planning to look for other compounds that can activate GPR37 as well. These could be more stable in the body than the previously studied protein fragment and thus better potential drugs.

Doctors have reported a few cases of genetic deficiency in prosaposin, leading to severe neurodegeneration. Mice engineered to lack GPR37 have more subtle brain perturbations, so Hall also plans to test the hypothesis that prosaposin acts by both GPR37 and GPR37L1, by “knocking out” both in mice, potentially duplicating the same severe effects seen in the human cases of prosaposin deficiency.

Study Shows How Parkinson’s Disease Protein Acts like a Virus

A protein known to be a key player in the development of Parkinson’s disease is able to enter and harm cells in the same way that viruses do, according to a Loyola University Chicago Stritch School of Medicine study.

The protein is called alpha-synuclein. The study shows how, once inside a neuron, alpha synuclein breaks out of lysosomes, the digestive compartments of the cell. This is similar to how a cold virus enters a cell during infection. The finding eventually could lead to the development of new therapies to delay the onset of Parkinson’s disease or halt or slow its progression, researchers said.

The study by virologist Edward Campbell, PhD, and colleagues, was published April 25, 2013 in the journal PLOS ONE.

Alpha-synuclein plays a role in the normal functioning of healthy neurons. But in Parkinson’s disease patients, the protein turns bad, aggregating into clumps that lead to the death of neurons in the area of the brain responsible for motor control. Previous studies have shown that these protein aggregates can enter and harm cells. Campbell and colleagues showed how alpha synuclein can bust out of lysosomes, small structures that collectively serve as the cell’s digestive system. The rupture of these bubble-like structures, known as vesicles, releases enzymes that are toxic to the rest of the cell.

“The release of lysosomal enzymes is sensed as a ‘danger signal’ by cells, since similar ruptures are often induced by invading bacteria or viruses,” said Chris Wiethoff, a collaborator on the study. “Lysosomes are often described as ‘suicide bags’ because when they are ruptured by viruses or bacteria, they induce oxidative stress that often leads to the death of the affected cell.”

In a viral or bacterial infection, the deaths of such infected cells may overall be a good thing for the infected individual. But in Parkinson’s disease, this same protective mechanism may lead to the death of neurons and enhance the spread of alpha-synuclein between cells in the brain, Campbell said. “This might explain the progressive nature of Parkinson’s disease. More affected cells leads to the spread of more toxic alpha-synuclein aggregates in the brain,” Campbell said. “This is very similar to what happens in a spreading viral infection.”

Campbell stressed that these studies need to be followed up and confirmed in other models of Parkinson’s disease. “Using cultured cells, we have made some exciting observations. However, we need to understand how lysosomal rupture is affecting disease progression in animal models of Parkinson’s disease and, ultimately, the brains of people affected by Parkinson’s disease. Can we interfere with the ability of alpha-synuclein to rupture lysosomes in these settings? And will that have a positive effect on disease progression? These are the questions we are excited to be asking next.”

Jeffrey H. Kordower, PhD, professor of neurological sciences, professor of neurosurgery and director of the Research Center for Brain Repair at Rush University Medical Center, said the study “is an important finding by a group of investigators who are beginning to make their impact in the field of Parkinson’s disease. This paper adds to the growing concept that alpha-synuclein, a  main culprit in the cause of Parkinson’s disease, can transfer from cell to cell. This paper elegantly puts a mechanism behind such a transfer. The findings will help shape the direction of Parkinson’s disease research for years to come.”