Alexa Kiss: Molecular methods
in neurobiology (4 papers)-
Assignment for the Journal Club, Kabai's class, 2006
1) Peter Estibeiro and Jenny Godfray:
Antisense as a neuroscience tool and therapeutic agent
(TINS Vol.24 No.11, Nov. 2001)
Using antisense is a technology that enables researchers to reach into a specific cell type in the brain, and switch off the expression of a single gene product. But this technology is not reliable enough for using in therapy, because there are lots of technical and biological hurdles (and in my case, financial hurdles, as well L ). But this could be dominant in medical practice in a few years; considering, that RNA is not a toxic chemical, it’s a natural part of our organism.
The mechanism of antisense is simple –in theory J: the genomic DNA sequences are transcribed into mRNA, that are translated into proteins. An antisense molecule is a short, complementary nucleic acid that hybridizes specifically to its mRNA target, and prevents translation.
The most frequent technological hurdles are the following:
- Stability: unmodified nucleic acids have a half-life of only a few minutes in biological systems, because of the endogeneous nucleases. There are some attempts to prevent this progress; for example S-ODNs (phosphorothioate oligodeoxynucleotides), or deoxinucleotides with deoxythimidins added at the 3’-end. (Tuschl et al.)
- Toxicity: S-ODNs
bind heparin-binding proteins, and auto-phosphorylate EGF-receptor. They
concentrate in kidneys and liver, causing immune stimulation. They can’t
cross the blood-brain.barrier. And this is the third problem:
Delivery: different type cells take up S-ODNs with different efficiencies, these chemicals are mostly taken up by neurons. To improve cellular uptake, oligonucleotides can be complexed with cationic lipids ( e.g.:lipofectamine) or polyamine carriers. / The cheapest chemical, which we use for this purpose, is PEI (poly-ethylen-immine). This is a kind of detergent, and it can be found in some liquid soaps as well. So we say: „Be careful with handwash!” J /
/Another note: I read in this article that NP(N3’-P5’phosphoramidate) and MF (morpholino phosphodiamidate) oligonucleotides show nuclear localization. I don’t understand why it is an advantage. As far as I know, the translation takes place in the cytoplasma!/
- Sequence targeting: mRNA is not a simple „line”, it has complex higher oder structures. Over 90% of them are prevented by intra-molecular interactions from hybridizing to an antisense molecule.
The key to successful antisense design, is to identify accessible areas of a transcript. There are some computational algorithms to define the structure from the mRNA-sequence, by minimum free energy calculations. (Just type „RNA-fold”in the Google, and you will see, how many there are! J)
/ The program that I use is called „m-fold”. I attach a picture of EGFP (enhanced green fluorescein protein) mRNA-structure. ( Fig. 1)/
- Specificity: antisense-mediated gene-product knockdown should be very specific and selective. But there can be some non-specific biological effects. This can cause a lot of problems for example in neuroscience, because behavioural changes, and neuron-degeneration could occur.
Researchers use controls in order to avoid these events. For example there are mismatch siRNA-s, or they change a base in the original sequence, and examine the effects.
The efficiency of silencing can be detected by Western blot, too.
- Immune response: it is known, that oligonucleotides longer than 30 base pairs, induce IFN1 (interferon) response. And the immune system responds strongly to nucleic acids containing unmethylated CpG dinucleotides, which can be found in bacterial genome. But, in the brain (because of the blood- brain barrier), there are no immune cells, and the immune response is not very strong.
The using of antisense in neuroscience will have to solve the problem of crossing the blood-brain barrier. Conjugation of antisense reagents to small molecules can be the solution.
Antisense is probably the most powerful functional genomics tool to understand the genome, and it may become a widely used therapeutic agent in the future.
Figure 1: The mRNA-structure of EGFP
( Watch out for loops! J)
2) N. Joan Abbott:
Dynamics of CNS barriers: Evolution, Differentiation and Modulation
Cellular and Molecular Neurobiology, Vol. 25, No. 1, February 2005
Until the early 1970s, studies on the barriers in the nervous system were done in situ and in vivo. Ferenc Joó was the first one to isolate cerebral capillaries, and made studies in vitro.
The central nervous system (CNS) is protected by barrier layers at three sites:
- The blood-brain barrier
- Blood-cerebrospinal fluid barrier (choroid plexus epithelium)
- Arachnoid epithelium of the meninges
There are „additional barrriers”, too:
- the „inner” blood-retinal barrier, formed by the retinal capillary endothelium
- the „outer” blood-retinal barrier,formed by the retinal pigment epithelium
- the inner blood-nerve barrier (BNB), endoneurium capillaries
- outer BNB, perineurium
- CVO (circumventricular organs), tight junctions between ependymal cells
The blood-brain barrier is maintaned by the endothelial cells, end-feet of pericapillary astrocytes, and (probably) pericytes.
Evolution of CNS barrier layers:
It’s interesting to examine non-mammalian groups of animals. Elasmobranch fish (sharks, rays) have a BBB, but formed not by the endothelium, but by perivascular glial end-feet.
Invertebrate groups tends not to have a BBB (or people didn’t research them enough J); but some of them have (for example: insects, crustacea, cephalophods). Their barrier is glial. That’s why it is said, that a glial barrier is a primitive condition, and during evolution the barrier has shifted to the endothelium.
Why do we need a blood-brain barrier?
The mammalian BBB has some properties that regulate the brain function. It constitutes the basis of a physical barrier, protecting the ISF from fluctuations that occur in plasma (due to respiration, digestion/absorption, exercise), reducing „noise” in neural communication, and providing the environment for „parasynaptic transmission”.
Studies among invertebrates suggest, that the main factor in the evolution of BBB was the need for ionic homeostasis around central synapses.
The sequence of changes in CNS barriers during fetal development can give clues to understand earlier evolutionary stages:
a.) Neural plate
b.) Closure of neural tube
c.) Separation of neural tube from remaining ectoderm
d.) Leaky blood vessels, CSF high in protein
e.) Vascularization, CSF low in protein, arachnoid appears
The subventricular zone is the major site of neurogenesis during development, which progress continues here, in the olfactory bulb, and in the subgranular zone of the dentate gyrus even in the adult(!).
The evolutionary shift from glial to endothelial barriers may have several advantages: glial cells have a number of specialized functions in regulating the ISF composition and supporting neurons.
Modulation of the blood-brain barrier
Cell-cell influences in the brain endothelium can be separated into induction (long-term, via regulation of gene transcription), and modulation (short-term, mediated by protein modification). Both progresses influence the physiology of the BBB in normal physiology and in pathology.
BBB modulation plays an important role in CNS inflammation. Short-term barrier opening might have benefits, including influx of plasma components such as antibodies and growth factors. Permeability increaeses by histamine and ROS (reactive oxygen species). There are researches targeting the blood-brain barrier, in order to drug delivery.
Cell culture models have permitted dissection of cellular and molecular events. Primary cultures are often used ( I like them, too J), with the condition that they still have some features of the original, in vivo barrier, while immortalized cell lines (they can be produced by using telomerases) do not express the „full” in vivo phenotype.
Several mechanism are known, which are able to BBB modulation. A lots of agents act via membrane receptors and G-proteins to acivate PLC (Phospholipase C), and elevation of [Ca].
Brain endothelial cells can act in „autocrine” mode, releasing factors such as NO and ATP that modulate their own function. A large number of agents are released paracrine mode from astrocytes, and from nerve terminals and activated microglia.
3) Craig C. Mello & Darryl Conte Jr:
Revealing the world of RNA interference
(Nature, Vol.431. Sept, 2004)
The term „RNA world” was first used to describe an early evolutionary stage, when RNA may have been the base of life on Earth. Now, there is another RNA world within our cells- that is RNA silencing or RNA interference. It is a sequence-specific mechanism, which helps to defend cells against foreign nucleic acids. RNA signals can be transmitted horizontally between cells and vertically through the germ line.
Why is dsRNA a trigger for RNAi? First, dsRNA was thought to be a nonspecific silencing agent, that causes the complete suppression of protein translation in mammalian cells. Second, it is energetically stable and incapable of further base pairing. There must be a cellular mechanism, which unwinds dsRNA. The „antisense-mediated silencing” was first examined in Caenorhabditis elegans, in 1995. The silencing effect could be passed through the germ line, by sperm or the egg for several generations. Another surprising observation was, that the silencing effect spread from tissue to tissue within the injected animal.
In another experiment, animals were soaked in dsRNA or fed with bacterially expressed dsRNA. These methods helped a lot in identification of many C. elegant, Drosophila, plant (where this phenomenon is known as post-transcriptional gene silencing) and fungus genes required for RNAi.
Another agent can trigger RNA-silencing besides dsRNA .Even transgenes trigger silencing. There is a gene family, called RdRPs (RNA-dependent RNA polymerases). In this type of silencing, the RdRP recognises transgene products as „aberrant”. This system hasn’t been found in mammals, yet.
In plants, which were infected by RNA viroids, there is a de novo cytosine methylation of genomic DNA. It happened to those genomic sequences, which were homologous to the viroid sequences. This process is called RNA- directed DNA methylation or RdDM.
Now it is already clear, that different organisms have evolved different mechanisms, at least variations on a common theme. It is useful to examine gene regulation using small interfering RNA. And it is important to know, how dsRNA spreads in germline and in neighbouring cells. These questions could be answered within the next ten years.
4) Leonid Gitlin and Raul Andino:
Nucleic Acid-Based Immune System:
the Antiviral Potential of Mammalian RNA silencing
(Journal of Virology, July 2003)
RNA-silencing is a highly conserved cellular mechanism that can regulate gene expression in response to double-stranded RNA, which may be transported by viruses. This process was described first in plants; where the overexpression of transgenes cosuppressed the homologous endogenous genes. This suppression or the resistance to RNA viruses arises from a sequence-specific RNA degradation system.
It is generally accepted that RNA-silencing plays an imprtant role in antiviral defense in plants, because RNA viruses replicate through dsRNA intermediates, which may cause strong gene silencing. The mechanism of silencing works in invertebrates as well. In one experiment, mosquitoes or their cells were preinfected with Sindbis virus carrying dengue virus genome fragments, and it inhibited dengue virus replication.
It is a logical assumption, that the RNA-silencing exists in mammals, too. But the early experiments denied this assumption, because the mammalian cells responded to the presence of dsRNA with a nonspecific shutdown translation. And mammals have a very developed immune system which protects them effectively. This system could have functionally replaced the RNA-silencing system. But the mammalian viruses initiate RNAi response! SiRNA has been shown to inhibit production of HIV, Rous sarcoma virus, poliovirus, and human papillomavirus. And the former experiment worked in mammalian cells, too, when it was repeated with shorter (21-25 nt long) RNA, wich did not elicit the interferon response.
In the future, it would be important to understand the role of RNA silencing –if it has any- in antiviral defense. If it has, the scientists will have the opportunity to use RNAi in therapy. They have to solve some problems until then: the persistence of the inhibitory effect, the targeting of specific cell types (with viral vectors), and preventing the viral escape by mutations.