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How does the PrP scrapie protein maintain its confirmation while going through the GI tract? How does the PrP scrapie protein cross the blood brain barrier?
Wouldn't the host immune system recognize the prion as "not self" and attack it?
I am not sure if a perfect answer is possible owing to the fact that Prion diseases are still incompletely understood, and are actively under research. On a preliminary search, I found some papers which address the issue (cited at the end).
- Since $PrP^C$ is normally found in the body, its role and its transport is also relevant to the issue. It seems to undergo bidirectional transport across the BBB using a saturable mechanism, mostly a specific transporter (1). It is taken up peripherally, and by several regions of the brain with the possibility that it is involved in various brain functions including modulating the BBB itself. Amyloid Beta-protein, with several kinetic and dynamic similarities with $PrP^C$, is shown to have BBB altering effects. (2) How far do the pathological prions follow this is seemingly unknown.
- Autonomic nerves seem to play a role in their transport.(3) When challenged with prions, sympathectomy delays or even prevents the disease, whereas sympathetic hyperinnervation accelerates prion pathogenesis. There have been studies to elucidate the transport via the nerves, such as (4) and (5).
There are several other studies on this issue, and following the cited references might help you find them.
Reference number 3 also has information on the immunological response to prions.
- Banks WA, Robinson SM, Diaz-Espinoza R, Urayama A, Soto C. Transport of prion protein across the blood-brain barrier. Experimental neurology. 2009 Jul 31;218(1):162-7.
- Giri R, Shen Y, Stins M, Du Yan S, Schmidt AM, Stern D, Kim KS, Zlokovic B, Kalra VK: Am J Physiol Cell Physiol. 2000 Dec; 279(6):C1772-81.
- Aguzzi A, Zhu C. Five questions on prion diseases. PLoS Pathog. 2012 May 3;8(5):e1002651.
- McBride PA, Schulz-Schaeffer WJ, Donaldson M, Bruce M, Diringer H, Kretzschmar HA, Beekes M. Early spread of scrapie from the gastrointestinal tract to the central nervous system involves autonomic fibers of the splanchnic and vagus nerves. Journal of virology. 2001 Oct 1;75(19):9320-7.
- Glatzel M, Heppner FL, Albers KM, Aguzzi A. Sympathetic innervation of lymphoreticular organs is rate limiting for prion neuroinvasion. Neuron. 2001 Jul 19;31(1):25-34.
Deadly proteins: prions
Since the epidemic of ‘mad cow disease’ in the 1980s and 90s, and the emergence of its human equivalent, variant Creutzfeld-Jacob disease, there has been a great deal of research into prions, the causative agents. Mico Tatalovic reviews the current state of knowledge.
Image courtesy of spooh
There may be a hidden epidemic waiting to occur, with millions of people already infected we cannot prevent or cure it, and we cannot even diagnose it until the fatal symptoms appear.
The disease is variant Creutzfeld-Jacob disease (vCJD), one of a group of diseases known as transmissible spongiform encephalopathies (TSEs), which are caused and transmitted by abnormal forms of prion proteins. Examples of TSEs include not only vCJD, but also scrapie in sheep, bovine spongiform encephalopathy (BSE or mad cow disease) in cattle and kuru w1 in humans. These diseases create large fluid-filled holes in the brain tissue because the accumulation of aberrant prions causes the neurons (nerve cells) to die. It is these characteristic holes that give the diseases their names: spongiform (sponge-like) encephalopathies (brain diseases).
TSEs affect the central nervous system, with symptoms including problems with co-ordination and balance, shakiness and uncontrollable jerking movements. In humans, TSEs also cause personality changes and depression, and sufferers may experience confusion, memory problems and insomnia. As the diseases progress, most mental functions are lost, including the ability to speak. All TSEs are fatal, and as yet, there is no cure.
Colour-enhanced image of prion
proteins (orange) from an animal
infected with scrapie
Image courtesy of R. Dourmashkin
/ Wellcome Images
Prions are specific proteins found mainly in the nervous system, where – in their normal forms – they may have important functions. For example, studies on sea slugs, Aplysia, suggest that prions have a crucial role in memory formation (Si et al., 2010). Infectious prions are abnormal (aberrant) forms of prion proteins that replicate inside the host by forcing normal proteins of the same type to adopt the aberrant structure. This has a domino effect whereby a small number of aberrant prions can affect many normal ones and eventually lead to disease. As the aberrant prions form amyloids – aggregates of protein – in the cells, the cells die, creating holes in the brain.
Prions are the only known case of self-propagating pathogenic (disease-causing) proteins, and they are able to cause severe illness even though they seem to be just protein molecules: unlike bacteria, viruses or other known pathogens, they have no information encoded in nucleic acids (DNA or RNA) about how to invade and replicate within the host. There is still a veil of mystery around prions and exactly how they replicate, cross the blood-brain barrier and cross the species barrier – i.e. infect different species of host.
It was in the 1960s that investigators first found that TSE disease-causing agents appeared to lack nucleic acids Tikvah Alper suggested that the agent was a protein. This idea sounded heretical because all other known disease-causing agents contained nucleic acids and their virulence and pathogenesis were genetically determined.
Portrait of bacteriologist
Robert Koch (1843-1910)
However, three decades of subsequent investigations, pursued most notably by Stanley Prusiner, who was awarded the Nobel Prize in Physiology or Medicine in 1997 for his work with prions and TSEs, resulted in the wide acceptance of this ‘protein-only hypothesis’ w2 .
Nevertheless, there are still those who believe that prion diseases are actually caused by unconventional viruses and that prion proteins are just part of this mysterious virus. Koch’s postulates describe what is needed to prove that a certain agent causes a particular disease one of the necessary steps is to use that agent to induce the disease in a healthy organism. To prove that a TSE is indeed caused by the prion protein itself, the isolated, purified aberrant prions must be used to transmit the disease. It wasn’t until February 2010 that exactly this was done, adding further substantial evidence for the protein-only hypothesis (Wang et al., 2010).
Repairing leaky blood-brain barrier may rejuvenate brain function
New research in mice questions the idea that “you can’t teach an old dog new tricks.” The answer may lie in preserving the blood-brain barrier, which tends to become leaky with age.
Share on Pinterest New research looks at the decline of brain functions that accompanies aging.
The blood-brain barrier is a complex set of blood vessel characteristics that help shield the brain from potentially harmful substances in the bloodstream.
In a recent Science Translational Medicine study, scientists describe how the breakdown of the blood-brain barrier can trigger brain inflammation and cognitive impairment in aging mice.
The international team found that the breakdown of the blood-brain barrier activates a signaling protein in brain cells called astrocytes.
The researchers then developed and tested a drug that blocked the signaling protein, which goes by the name transforming growth factor-beta (TGF-beta).
After treatment with the drug, the mice showed fewer signs of brain inflammation and an improved ability to learn new tasks that matched the performance of much younger mice.
“We tend to think about the aged brain in the same way we think about neurodegeneration: Age involves loss of function and dead cells,” says co-senior study author Daniela Kaufer, a professor of integrative biology at the University of California, Berkeley.
“But our new data tell a different story about why the aged brain is not functioning well: It is because of this ‘fog’ of inflammatory load,” she adds.
Prof. Kaufer explains that within days of abolishing the “inflammatory fog,” the aged brain starts to function more like a young brain.
The findings should help scientists better understand the decline of brain functions involving inflammation that can accompany aging and conditions such as dementia.
An increasing body of research — including imaging studies by co-senior study author Alon Friedman, of Ben-Gurion University of the Negev in Israel and Dalhousie University in Canada — shows that the blood-brain barrier becomes less efficient with age.
The leakier the blood-brain barrier becomes, the easier it is for substances that cause inflammation to cross over from the bloodstream into brain tissue and damage cells.
Kaufer and Friedman are also co-senior authors of another recent Science Translational Medicine study that took a closer look at inflammatory fog in leaky blood-brain barriers.
People with Alzheimer’s disease may frequently experience epileptic events, but they and their doctors are not necessarily aware of them.
Advancing age is a risk factor for both Alzheimer’s and epilepsy, and experimental and clinical data support the idea of a link between the two conditions.
For the second study, the team analyzed EEG readings from people with Alzheimer’s disease and found an EEG signature for what they describe as “paroxysmal slow wave events (PSWEs).”
From the EEGs, they saw how the rate of PSWEs seemed to match the level of cognitive impairment of the individuals.
In EEGs of people with epilepsy, they found that PSWEs that occurred between seizures matched areas of leaky blood-brain barrier. They found the same match in aged mice, mice prone to Alzheimer’s disease, and rats with induced epilepsy.
Additional tests in young rats revealed that it was possible to impair the blood-brain barrier by introducing the protein albumin into the brain. This led to a higher rate of PSWEs.
In earlier research, Friedman and Kaufer had shown that albumin can leak into the brain following trauma. The protein attaches itself to the TGF-beta receptor of astrocytes.
By binding to astrocytes’ TGF-beta receptors, the protein sets off a chain of inflammation events that damage brain cells and circuits.
The damage increases the likelihood of seizures by disrupting the balance between excitation and inhibition of neurons.
The team concludes that the findings point to a leaky blood-brain barrier as a potential cause of nonconvulsive seizures in people with Alzheimer’s disease. It may also offer a potential treatment target.
The researchers suggest that the two sets of findings offer two new biomarkers that could help doctors identify individuals who might have a blood-brain barrier problem: one using MRI (which can detect leaky barriers), and the other using EEG (which can detect abnormal brain rhythms).
There is also scope to develop the drug that they used as a way to repair a leaky blood-brain barrier to slow and perhaps even reverse some of the problems that it can cause.
“ We now have two biomarkers that tell you exactly where the blood-brain barrier is leaking, so you can select patients for treatment and make decisions about how long you give the drug.”
Prof. Daniela Kaufer
Experts commenting on the two studies generally welcome the findings but warn against jumping to the conclusion that they describe ways to reverse dementia in humans.
“Overall,” notes Diego Gomez-Nicola, an associate professor of neuroscience at the University of Southampton in the United Kingdom, “these studies add to a body of knowledge supporting the impact of inflammation on dementia, and provide promising targets for future clinical studies.”
Drugs that quell brain inflammation reverse dementia
Drugs that tamp down inflammation in the brain could slow or even reverse the cognitive decline that comes with age.
In a publication appearing today in the journal Science Translational Medicine, University of California, Berkeley, and Ben-Gurion University scientists report that senile mice given one such drug had fewer signs of brain inflammation and were better able to learn new tasks, becoming almost as adept as mice half their age.
“We tend to think about the aged brain in the same way we think about neurodegeneration: Age involves loss of function and dead cells. But our new data tell a different story about why the aged brain is not functioning well: It is because of this “fog” of inflammatory load,” said Daniela Kaufer, a UC Berkeley professor of integrative biology and a senior author, along with Alon Friedman of Ben-Gurion University of the Negev in Israel and Dalhousie University in Canada. “But when you remove that inflammatory fog, within days the aged brain acts like a young brain. It is a really, really optimistic finding, in terms of the capacity for plasticity that exists in the brain. We can reverse brain aging.”
The successful treatment in mice supports a radical new view of what causes the confusion and dementia that often accompany aging. More and more research shows that, with age, the filtration system that prevents molecules or infectious organisms in the blood from leaking into the brain — the so-called blood-brain barrier — becomes leaky, letting in chemicals that cause inflammation and a cascade of cell death. After age 70, nearly 60% of adults have leaky blood- brain barriers, according to Friedman’s magnetic resonance imaging (MRI) studies.
An accompanying paper by the two researchers and Dan Milikovsky of Ben-Gurion University shows that the inflammatory fog induced by a leaky blood-brain barrier alters the mouse brain’s normal rhythms, causing microseizure-like events — momentary lapses in the normal rhythm within the hippocampus — that could produce some of the symptoms seen in degenerative brain diseases like Alzheimer’s disease. Electroencephalograms (EEGs) revealed similar brain wave disruption, or paroxysmal slow wave events, in humans with epilepsy and with cognitive dysfunction, including Alzheimer’s and mild cognitive impairment (MCI).
Dynamic contrast-enhanced MRI (DCE-MRI) scans show that with age, the blood-brain barrier becomes leakier. This dysfunction in shown in both humans and mice. A leaky BBB triggers a cascade of cell death that may be the cause of age-related cognitive decline. (Images by Alon Friedman and Daniela Kaufer)
Together, the papers give doctors two biomarkers — leaky barriers detectable by MRI and abnormal brain rhythms detectable by EEG — that can be used to flag people with blood-brain barrier problems, as well as a potential drug to slow or reverse the consequences.
“We now have two biomarkers that tell you exactly where the blood-brain barrier is leaking, so you can select patients for treatment and make decisions about how long you give the drug,” said Kaufer, a member of UC Berkeley’s Helen Wills Neuroscience Institute. “You can follow them, and when the blood-brain barrier is healed, you no longer need the drug.”
Scientists have long suspected that a leaky blood-brain barrier causes at least some of the tissue damage after brain injury and some of the mental decline that comes with age. But no one knew how.
A patient with a very leaky blood-brain barrier as measured by MRI (left, with red indicating the highest permeability) also has more seizure-like events (paroxysmal slow wave events) per minute as measured by EEG (red indicates more frequent events). (Image courtesy of Kaufer lab)
In 2007, however, Friedman and Kaufer linked these problems to a blood protein, albumin. In 2009, they showed that when albumin leaks into the brain after trauma, it binds to the TGF-β (TGF-beta) receptor in brain cells called astrocytes. This triggers a cascade of inflammatory responses that damage other brain cells and neural circuits, leading to decreased inhibition and increased excitation of neurons and a propensity toward seizures.
They also showed in mice that blocking the receptor with an antihypertension drug, losartan, prevented the development of epilepsy after brain trauma. Epilepsy is a frequent consequence of concussions like those sustained by soldiers from roadside bombs.
Subsequent studies revealed leakiness in the barrier after stroke, traumatic brain injury and football concussions, solidly linking albumin and an overexcited TGF-β receptor to the damage resulting from these traumas.
In their new studies, Kaufer and Friedman showed that introducing albumin into the brain can, within a week, make the brains of young mice look like those of old mice, in terms of hyperexcitability and their susceptibility to seizures. These albumin-treated mice also navigated a maze as poorly as aged mice.
“When we infused albumin into the brains of young mice, we recapitulated aging of the brain: the gene expression, the inflammatory response, resilience to induced seizures and mortality after seizures, performance in a maze. And when we recorded their brain activity, we found these paroxysmal slow wave events,” Kaufer said. “And all were specific to the site we infused. So, doing this is sufficient to get an aged phenotype of this very young brain.”
Alon Friedman and Daniela Kaufer, who have worked together for more than 20 years to pin down the role of the blood-brain barrier in brain disorders, including aging.
When they genetically engineered mice so that they could knock out the TGF-β receptor in astrocytes after they’d reached old age, the senile mouse brains looked young again. The mice were as resistant to induced seizures as a young mouse, and they learned a maze like a young mouse.
Serendipitously, a Palo Alto, California, medicinal chemist, Barry Hart, offered to synthesize a small-molecule drug that blocks the TGF-β receptor in astrocytes only, and that could traverse the blood-brain barrier. When they gave the drug, called IPW, to mice in doses that lowered the receptor activity level to that found in young mice, the brains of the aged mice looked younger, too. They showed young brain-like gene expression, reduced inflammation and improved rhythms — that is, reduced paroxysmal slow wave events — as well as reduced seizure susceptibility. They also navigated a maze or learned a spatial task like a young mouse.
In analyzing brain tissue from humans, Kaufer found evidence of albumin in aged brains and increased neuroinflammation and TGF-β production with age. Friedman developed a special type of MRI imaging — dynamic contrast-enhanced (DCE) imaging — to detect leakage in the blood-brain barrier and found more leakage in people with greater cognitive dysfunction.
Altogether, the evidence points to a dysfunction in the brain’s blood filtration system as one of the earliest triggers of neurological aging, Kaufer said.
Kaufer, Friedman and Hart have started a company to develop a drug to heal the blood-brain barrier for clinical treatment and hope that the drug will help reduce brain inflammation — and, thus, permanent damage — after stroke, concussion or traumatic brain injury, and eventually help older adults with dementia or Alzheimer’s disease who have demonstrated leakage of the blood-brain barrier.
“We got to this through this back door we started with questions about plasticity having to do with the blood-brain barrier, traumatic brain injury and how epilepsy develops,” Kaufer said. “But after we’d learned a lot about the mechanisms, we started thinking that maybe in aging it is the same story. This is new biology, a completely new angle on why neurological function deteriorates as the brain ages.”
This work was supported by the National Institutes of Health (R01NS066005, R56NS066005), European Union’s Seventh Framework Program, Israel Science Foundation and United States-Israel Binational Science Foundation.
Morphine and metabolites
The major morphine metabolite, morphine-3-glucuronide (M3G), although analgesically inactive, has been reported to antagonize morphine and to produce stimulatory effects, such as myoclonus, seizure and allodynia (Smith 2000). On the other hand, the minor metabolite, morphine-6-glucuronide (M6G), is much more analgesic than morphine.
Morphine is primarily metabolized in the liver by uridine-5′-diphosphate (UDP) glucoronosyltransferase, with specific affinity for the UGT2B7 isoenzyme. This isoenzyme is responsible for the formation of both glucuronide species, but at different amounts (5 times more M3G than M6G). Researchers postulated the presence of another metabolic isoenzyme that largely forms M3G. Although in vitro results have indicated a possible role of UGT1A1 in the formation of M3G, in vivo UGT2B7 isoenzyme remains the primary metabolic enzyme for morphine (Stone et al. 2003). The different formation of M3G and M6G metabolites (Fig. 1 ) is likely due to physicochemical and steric issues that affect the binding of morphine to the phase II enzyme (Coffman et al. 1998).
UGT2B7 is also responsible for the metabolism of several endogenous and exogenous compounds, especially steroid hormones and bilirubin in the newborn. These compounds are competitive substrates for UGT2B7 and can reduce the formation of morphine-conjugate metabolites.
In addition to the liver, human brain homogenates have been shown to metabolize morphine at nanomolar concentrations to M3G and M6G therefore, M6G can be formed directly in the CNS and seems to penetrate the BBB at a greater rate than the M6G produced in the liver (Yamada et al. 2003). Interestingly, the M3G/M6G ratio produced by the brain homogenates has been found to be directly associated with morphine concentration. Lower concentrations of morphine corresponded to a lower M3G/M6G ratio, perhaps due to the preferential formation of M6G by UGT2B7 (Yamada et al. 2003). Although UGT2B7 appears to play a role in M6G formation from endogenous morphine, the enzymology of this metabolism in the brain needs to be more thoroughly elucidated. Moreover, the brain UGT isoforms responsible for morphine glucuronidation may be different from the hepatic ones.
Several studies have been performed on morphine-metabolizing enzymes and on the μ-opioid receptor, in order to detect genetic variants possibly contributing to interindividual variability in morphine pharmacology. The UGT2B7 H288Y polymorphism does not seem to account for the significant variations in glucuronide-to-morphine ratio seen in cancer patients (Coughtrie et al. 1989). On the contrary, global UGT activity is apparently modulated by a series of genetic polymorphisms (Coffman et al. 1998). Variants in μ-opioid receptor gene (OPRM1) play an important role in mediating morphine activity: this gene is highly polymorphic, and the rs1799971 SNP in exon 1 seems to be associated with a decreased therapeutic efficacy. In particular, the 118 G homozygotes require higher morphine doses for pain relief than heterozygotes or non-carriers, showing increased risks of intoxication and respiratory depression. The explanation could be related to clinically measurable differences in M6G effects (Lötsch et al. 2002a, b Lötsch and Geisslinger 2005 Romberg et al. 2005). Similarly, COMT (Catechol-O-MethylTransferase) polymorphisms have to be taken into account for morphine dose modulation: the enzyme is responsible for dopamine, norepinephrine and epinephrine metabolism. Its Val158Met polymorphism (rs4680) is related with enzyme activity since methionine causes a three times reduction of activity, contributing to alterations in pain perception. In particular, patients with Val/Val homozygous genotype require more morphine than Val/Met heterozygous or Met/Met homozygous (reviewed by Allegri et al. 2010).
Future directions for currently unsolved questions include answering questions about whether oxytocin transport is increased in the excess of sRAGE or esRAGE via mRAGE. We would also like to know what extent does RAGE-dependent oxytocin transport rely on other transport means, such as the direct intranasal pathway or trigeminal nerve transport 26 . It will be important to follow up results on additional oxytocin binding proteins in plasma. The molecular weight of sRAGE/esRAGE is approximately 50 kDa. However, it is known that there is an oxytocin binding protein with a molecular weight of 10 kDa 30 . It has frequently been demonstrated that no oxytocin transport to the brain occurs in rats and it would be interesting to know if this could be due to lack of RAGE function. Another question is whether oxytocin recruited in cortical regions that are far from the hypothalamus with no axon collaterals from oxytocinergic neurons 2 . With regards to behavior, there is one hypothesis that personal differences in social behavior in humans depends on plasma oxytocin levels. Given our data, it is reasonable to ask whether molecular variants or single nucleotide polymorphisms of the AGER gene coding RAGE contribute to this phenomenon. Further, can orally administered oxytocin reach the brain? This is particularly relevant for oxytocin in milk. We also need to determine the precise bioavailability of oxytocin from the intestine and the blood. Several questions arise about oxytocin movement. Can oxytocin enter the lymph and intraperitoneal space, and tight junctions? Can oxytocin be absorbed from mouse epithelial cells and esophageal epithelial cells in a RAGE-dependent fashion across epithelial barriers? Overall, it will be important to determine how oxytocin is transported intracellularly, transcellularly and intercellularly 25 .
To summarize, although we need to determine the pharmacokinetics of oxytocin, our data present a better understanding of how exogenous oxytocin from the intestine or blood can influence the brain targets of oxytocin, which results in social behavioral improvement in humans with or without social impairments.
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Prion Diseases Biology & Genetics
Scientists at NIAID’s Rocky Mountain Laboratories (RML) in Hamilton, Montana, have studied prion diseases since the 1960s when Dr. William Hadlow spearheaded work on the sheep brain disease known as scrapie, which was later shown to be a prion disease. RML is one of the world's premier laboratories for studying prion diseases. Primary to their mission is understanding how abnormal prion protein cause disease at the molecular, biochemical, cellular, and animal-model levels.
NIAID scientists at RML are studying how cells in the nervous system interact with prion protein and whether those interactions affect disease progression. These studies have shown how prions move through the brain and how cells in the brain called microglia help slow down disease.
Other studies at RML have looked at different types of CJD in human tissue and how different types of CJD may occur. These studies have shown how human prions interact with cells. They have also shown that prions derived from slightly different forms of the prion protein gene can influence how prions accumulate in the brain.
NIAID scientists at RML also have shown that, in response to damage to the brain, normal prion protein acquires properties similar to those of infectious prion protein. These studies show that prion protein, like the tau protein in chronic traumatic encephalopathy (CTE), acts abnormally following brain injury. They also suggest that damage to the brain might be a way in which CJD infection starts in some people.
RML chronic wasting disease studies have focused on whether infectious prions can cross species from cervids, like deer and elk, into people. During research that took 13 years of observation, NIAID scientists published a series of studies, the latest in 2018, showing that CWD from deer or elk does not cause disease in prion models that are susceptible to infection by human prions. These studies reinforce the belief that a strong barrier to CWD infection exists between cervids and people.
Studies of prion disease infection of cerebral organoid (“minibrain”) cultures in incubators began at RML in 2017. These studies could provide a new model for scientists to study how prion diseases affect the human brain. Cerebral organoids are small balls of human brain cells – developed from skin stem cells – that range in size from a poppy seed to a small pea. Their organization, structure, and electrical signaling mimic some aspects of brain tissue.
How Listeria crosses the placental barrier to infect the fetus
A study conducted by the group directed by Marc Lecuit (Avenir Inserm / Group Microorganisms and barriers host the Pasteur Institute), at the Inserm unit U604 directed by Pascale Cossart, has uncovered how the bacterium responsible for Listeriosis (Listeria monocytogenes) can cross the placenta of pregnant women to cause serious fetal infections —even death—, premature birth and infections in newborns. This is the first time the molecular mechanism allowing a pathogenic bacterium to cross the placenta in vivo is discovered. This work is published on September 17 on Nature's website.
Press release Paris, september 17, 2008
Listeriosis is a bacterial infection caused by food-borne Listeria monocytogenes. Widespread in nature (water, soil, plants, animals) that bacteria can contaminate many foods: raw vegetables, ready-to-eat cooked dishes, cheese, meats and threatens primarily pregnant women, children and unborn newborns. The elderly and immunocompromised are also at risk. Among them listeriosis is responsible for septicemia, meningitis and encephalitis. Antibiotics are in most cases effective but the infection is nevertheless still lethal in 20 to 30% of infected individuals. In healthy adults, symptoms are generally less severe and can result in a simple gastroenteritis.
The infection begins with ingestion of food contaminated with Listeria monocytogenes, which can then cross the intestinal barrier and reach the bloodstream. The bacterium is then able to cross the barrier between the blood vessels of the brain (blood-brain barrier) or cross the placenta to disseminate to the fetus in pregnant women.
Marc Lecuit, Pascale Cossart and their colleagues at the Institut Pasteur, INSERM and INRA have developed the first two animal models for human listeriosis allowing to study the crossing of the placental barrier in vivo: the gerbil, a rodent naturally susceptible to Listeria monocytogenes, and a new mouse by genetically modified by "knock-in". This mouse expresses a human adhesion protein on the surface of epithelial cells called E-cadherin. With these two animal models, researchers were able to identify that two Listeria proteins, called InlA and InlB, interact with specific receptors, respectively E-cadherin and Met, which allow them to adhere to the placenta and cross it to reach the fetus.
Researchers had previously studied in vitro interactions of these proteins with their receptors and showed (Science, 2001) how Listeria crosses the intestinal barrier, but this is the first time the molecular mechanism allowing a pathogenic bacterium to cross the placental barrier in vivo is discovered.
This work greatly improves our understanding of the pathogenicity of Listeria monocytogenes and may lead to the development of molecules capable of preventing the spread of Listeria to the fetus, by inhibiting host-bacterial interactions. Understanding the mechanism of crossing the placenta could also help deliver therapeutic molecules across the placental barrier.
The teams of Marc Lecuit and Pascale Cossart work together to understand the mechanism of the crossing the blood-brain barrier by Listeria monocytogenes. Their working hypothesis is that these same proteins could also play a role in the infection of the central nervous system.
“Conjugated action of two species-specific invasion proteins for fetoplacental listeriosis”, Nature, 17 septembre 2008.
Olivier Disson 1,2*, Solène Grayo 1,2,3*, Eugénie Huillet 4,5,6*, Georgios Nikitas 1,2, Francina Langa-Vives 7, Olivier Dussurget 4,5,6, Marie Ragon 3, Alban Le Monnier 3, Charles Babinet 8‡, Pascale Cossart 4,5,6 & Marc Lecuit 1,2,3,9
1 Institut Pasteur, Groupe Microorganismes et Barrières de l’Hôte, Unité des Interactions Bactéries-Cellules, F-75015, Paris, France.
2 Inserm Avenir U604, F-75015, Paris, France.
3 Institut Pasteur, Centre de Référence des Listeria, F-75015, Paris, France.
4 Institut Pasteur, Unité des Interactions Bactéries-Cellules, F-75015, Paris, France.
5 Inserm U604, F-75015, Paris, France.
6 INRA USC2020, F-75015, Paris, France.
7 Institut Pasteur, Centre d’Ingénierie Génétique Murine, F-75015, Paris, France.
8 Institut Pasteur, Unité de Biologie du Développement, F-75015, Paris, France.
9 Centre d’Infectiologie Necker-Pasteur, Service des Maladies Infectieuses et Tropicales, Hôpital Necker-Enfants malades, Assistance Publique-Hôpitaux de Paris, Université Paris Descartes, F-75015, Paris, France.
Delivering Genes Across the Blood-Brain Barrier
Caltech biologists have modified a harmless virus in such a way that it can successfully enter the adult mouse brain through the bloodstream and deliver genes to cells of the nervous system. The virus could help researchers map the intricacies of the brain and holds promise for the delivery of novel therapeutics to address diseases such as Alzheimer's and Huntington's. In addition, the screening approach the researchers developed to identify the virus could be used to make additional vectors capable of targeting cells in other organs.
"By figuring out a way to get genes across the blood-brain barrier, we are able to deliver them throughout the adult brain with high efficiency," says Ben Deverman, a senior research scientist at Caltech and lead author of a paper describing the work in the February 1 online publication of the journal Nature Biotechnology.
The blood-brain barrier allows the body to keep pathogens and potentially harmful chemicals circulating in the blood from entering the brain and spinal cord. The semi-permeable blockade, composed of tightly packed cells, is crucial for maintaining a controlled environment to allow the central nervous system to function properly. However, the barrier also makes it nearly impossible for many drugs and other molecules to be delivered to the brain via the bloodstream.
To sneak genes past the blood-brain barrier, the Caltech researchers used a new variant of a small, harmless virus called an adeno-associated virus (AAV). Over the past two decades, researchers have used various AAVs as vehicles to transport specific genes into the nuclei of cells once there, the genes can be expressed, or translated, from DNA into proteins. In some applications, the AAVs carry functional copies of genes to replace mutated forms present in individuals with genetic diseases. In other applications, they are used to deliver genes that provide instructions for generating molecules such as antibodies or fluorescent proteins that help researchers study, identify, and track certain cells.
Largely because of the blood-brain barrier problem, scientists have had only limited success delivering AAVs and their genetic cargo to the central nervous system. In general, they have relied on surgical injections, which deliver high concentrations of the virus at the injection site but little to the outlying areas. Such injections are also quite invasive. "One has to drill a hole through skull, then pierce tissue with a needle to the injection site," explains Viviana Gradinaru (BS ཁ), assistant professor of biology and biological engineering at Caltech, Heritage Principal Investigator, and senior author on the paper. "The deeper the injection, the higher the risk of hemorrhage. With systemic injection, using the bloodstream, none of that damage happens, and the delivery is more uniform."
In addition, Gradinaru notes, "many disorders are not tightly localized. Neurodegenerative disorders like Huntington's disease affect very large brain areas. Also, many complex behaviors are mediated by distributed interacting networks. Our ability to target those networks is key in terms of our efforts to understand what those pathways are doing and how to improve them when they are not working well."
In 2009, a group led by Brian Kaspar of Ohio State University published a paper, also in Nature Biotechnology, showing that an AAV strain called AAV9 injected into the bloodstream could make its way into the brain&mdashbut it was only efficient when used in neonatal, or infant, mice.
"The big challenge was how do we achieve the same efficiency in an adult," says Gradinaru.
Although one might like to design an AAV that is up to the task, the number of variables that dictate the behavior of any given virus, as well as the intricacies of the brain and its barrier, make that extremely challenging. Instead, the researchers developed a high-throughput selection assay, CREATE (Cre REcombinase-based AAV Targeted Evolution), that allowed them to test millions of viruses in vivo simultaneously and to identify those that were best at entering the brain and delivering genes to a specific class of brain cells known as astrocytes.
They started with the AAV9 virus and modified a gene fragment that codes for a small loop on the surface of the capsid&mdashthe protein shell of the virus that envelops all of the virus' genetic material. Using a common amplification technique, known as polymerase chain reaction (PCR), they created millions of viral variants. Each variant carried within it the genetic instructions to produce more capsids like itself.
Then they used their novel selection process to determine which variants most effectively delivered genes to astrocytes in the brain. Importantly, the new process relies on strategically positioning the gene encoding the capsid variants on the DNA strand between two short sequences of DNA, known as lox sites. These sites are recognized by an enzyme called Cre recombinase, which binds to them and inverts the genetic sequence between them. By injecting the modified viruses into transgenic mice that only express Cre recombinase in astrocytes, the researchers knew that any sequences flagged by the lox site inversion had successfully transferred their genetic cargo to the target cell type&mdashhere, astrocytes.
After one week, the researchers isolated DNA from brain and spinal cord tissue, and amplified the flagged sequences, thereby recovering only the variants that had entered astrocytes.
Next, they took those sequences and inserted them back into the modified viral genome to create a new library that could be injected into the same type of transgenic mice. After only two such rounds of injection and amplification, a handful of variants emerged as those that were best at crossing the blood-brain barrier and entering astrocytes.
"We went from millions of viruses to a handful of testable, potentially useful hits that we could go through systematically and see which ones emerged with desirable properties," says Gradinaru.
Through this selection process, the researchers identified a variant dubbed AAV-PHP.B as a top performer. They gave the virus its acronym in honor of the late Caltech biologist Paul H. Patterson because Deverman began this work in Patterson's group. "Paul had a commitment to understanding brain disorders, and he saw the value in pushing tool development," says Gradinaru, who also worked in Patterson's lab as an undergraduate student.
To test AAV-PHP.B, the researchers used it to deliver a gene that codes for a protein that glows green, making it easy to visualize which cells were expressing it. They injected the AAV-PHP.B or AAV9 (as a control) into different adult mice and after three weeks used the amount of green fluorescence to assess the efficacy with which the viruses entered the brain, the spinal cord, and the retina.
"We could see that AAV-PHP.B was expressed throughout the adult central nervous system with high efficiency in most cell types," says Gradinaru. Indeed, compared to AAV9, AAV-PHP.B delivers genes to the brain and spinal cord at least 40 times more efficiently.
"What provides most of AAV-PHP.B's benefit is its increased ability to get through the vasculature into the brain," says Deverman. "Once there, many AAVs, including AAV9 are quite good at delivering genes to neurons and glia."
Gradinaru notes that since AAV-PHP.B is delivered through the bloodstream, it reaches other parts of the body. "Although in this study we were focused on the brain, we were also able to use whole-body tissue clearing to look at its biodistribution throughout the body," she says.
Whole-body tissue clearing by PARS CLARITY, a technique developed previously in the Gradinaru lab to make normally opaque mammalian tissues transparent, allows organs to be examined without the laborious task of making thin slide-mounted sections. Thus, tissue clearing allows researchers to more quickly screen the viral vectors for those that best target the cells and organs of interest.
"In this case, the priority was to express the gene in the brain, but we can see by using whole-body clearing that you can actually have expression in many other organs and even in the peripheral nerves," explains Gradinaru. "By making tissues transparent and looking through them, we can obtain more information about these viruses and identify targets that we might overlook otherwise."
The biologists conducted follow-up studies up to a year after the initial injections and found that the protein continued to be expressed efficiently. Such long-term expression is important for gene therapy studies in humans.
In collaboration with colleagues from Stanford University, Deverman and Gradinaru also showed that AAV-PHP.B is better than AAV9 at delivering genes to human neurons and glia.
The researchers hope to begin testing AAV-PHP.B's ability to deliver potentially therapeutic genes in disease models. They are also working to further evolve the virus to make even better performing variants and to produce variants that target certain cell types with more specificity.
Deverman says that the CREATE system could indeed be applied to develop AAVs capable of delivering genes specifically to many different cell types. "There are hundreds of different Cre transgenic lines available," he says. "Researchers have put Cre recombinase under the control of gene regulatory elements so that it is only made in certain cell types. That means that regardless of whether your objective is to target liver cells or a particular type of neuron, you can almost always find a mouse that has Cre recombinase expressed in those cells."
"The CREATE system gave us a good hit early on, but we are excited about the future potential of using this approach to generate viruses that have very good cell-type specificity in different organisms, especially the less genetically tractable ones," says Gradinaru. "This is just the first step. We can take these tools and concepts in many exciting directions to further enhance this work, and we&mdashwith the Beckman Institute and collaborators&mdashare ready to pursue those possibilities."
The Beckman Institute at Caltech recently opened a resource center called CLOVER (CLARITY, Optogenetics, and Vector Engineering Research Center) to support such research efforts involving tissue clearing and imaging, optogenetic studies, and custom gene-delivery vehicle development. Deverman is the center's director, and Gradinaru is the principal investigator.
Additional Caltech authors on the paper, "Cre-dependent selection yields AAV variants for widespread gene transfer to the adult brain," are Sripriya Ravindra Kumar, Ken Y. Chan, Abhik Banerjee, Wei-Li Wu, and Bin Yang, as well as former Caltech students Piers L. Pravdo and Bryan P. Simpson. Nina Huber and Sergiu P. Pasca of Stanford University School of Medicine are also coauthors. The work was supported by funding from the Hereditary Disease Foundation and the Caltech-City of Hope Biomedical Initiative, a National Institutes of Health (NIH) Director's New Innovator Award, the NIH's National Institute of Aging and National Institute of Mental Health, the Beckman Institute, and the Gordon and Betty Moore Foundation.