Information

Myelination and time constant


In textbooks, it says that myelination doesn't really affect the time constant as tau=RC where R is the membrane resistance and C is the membrane capacitance. Myelin increases membrane resistance while decreasing membrane capacitance so there isn't really an overall effect on the time constant. However, during active regeneration of the action potential, the current only goes the path with least resistance i.e. the nodes of Ranvier, so wouldn't the R be the resistance of the node of Ranvier instead of the resistance of the entire membrane as stated in the textbooks? If this argument is correct then wouldn't myelination increases decreases time constant? Thanks!


No. When a signal is sent through a myelinated axon, the Nodes of Ranvier act as repeaters in electronics. The signal/action potential will disperse down the axon, the Node of Ranvier will pick up the signal, amplify it, and send it to the subsequent Node of Ranvier. I think you're thinking of the Nodes of Ranvier as cracks in the myelin/axon rather than as mini re-amplification sites.


THE EPIDEMIOLOGY OF DELAYED MYELINATION

A. Leviton , . E.C. Dooling , in The Developing Human Brain , 1983

MULTIVARIATE ANALYSIS ( Table 13-5 )

Myelinogenesis is a process that should begin months before delivery. A delay in myelination of at least several weeks was necessary for a newborn to qualify as a delayed myelinator. Since most of the newborns in this sample died during the first few postnatal days, and all died before the end of the seventh day, postnatal events were unlikely to have contributed appreciably to a delay in myelination. For this reason confounding postnatal events were excluded from the multivariate analysis.

Table 13-5 . Multivariate Analysis of Risk of Delayed Myelination

Risk Ratio
Risk FactorRisk GroupReferent GroupPoint Estimate95%Confidence Limits
Seizures, convulsions, epilepsy – all siblingsAnyNone *** 872.43,200
Cigarettes/day≥ 20&lt 20172.6110
Birthweight (gms)≤ 2,000≥ 2,501 * 6.91.826
2,001-2,500≥ 2,501 * 153.083
Gestational age (weeks)≥ 36≤ 357.02.124
Third trimester uterine bleedingYesNo * 4.41.315
Lowest hematocrit – gravida (%)&lt 35≥ 353.41.29.3
Infections of neonateAnyNone * 0.20.10.8

An unexpected finding was the magnitude of the increased risk of delayed myelination associated with cleft palate ( R R ^ = 690 .0, 95% confidence limits = 2 .8 and 170,000 ) . Because only two children had cleft palate, we were concerned that the association between cleft palate and delayed myelination might be a random phenomenon. This concern reflected our perception of the instability of small samples. For example, if one less child with cleft palate had delayed myelination, then the association between cleft palate and delayed myelination would disappear. This prompted us to perform a second multivariate analysis, excluding cleft palate ( Table 13-5 ).

When cleft palate is excluded, the risk factor with the largest point estimate of the risk ratio is “seizures in siblings” ( R R ^ = 87 ) ( Table 13-5 ).

The risk factor with the next largest risk ratio is “smoking at least one pack of cigarettes per day” ( R R ^ = 17 ) .

Both low birth weight categories (≦ 2,000 grams, and 2,001-2,500 grams with R R ^ s of 6.9 as estimated by the pathologist ( R R ^ = 7.0 ) are associated with an increased risk of delayed myelination. Both the mothers’ and the pathologists’ estimates of gestational age were entered into the multivariate analytic function. The fact that the pathologists’ estimate discriminated better than the mothers’ may indicate that if gestational age is really important, then a) the pathologists’ estimates are more valid than the mothers’ or b) the pathologists’ estimates convey additional information beyond just gestational age.

Women who had third trimester uterine bleeding were at increased risk of their infant having delayed myelination ( R R ^ = 4.4 ) as were women whose lowest hematocrit during pregnancy was less than 35% ( R R ^ = 3.4 ) .

Newborns were at decreased risk if they had a postnatal infection ( R R ^ = 0.2 ) .


How is myelin made?

Myelin is the protective lipid sheath wrapped around a nerve. It functions as an insulator, akin to the protective coating on a wire, speeding up electrical transmission of signals along a neuron. Myelin also plays a role in maintaining the health of neurons. Myelin function is dysregulated in many neurological disorders, including multiple sclerosis.

Oligodendrocytes are the myelin-producing cells of the central nervous system. The myelin sheath around a neuron is part of an oligodendrocyte&rsquos plasma membrane, and a single oligodendrocyte can myelinate as many as 50 neurons. During myelination, an oligodendrocyte stretches out tubes of membrane in search of a neuron. When it finds one, it sends the necessary building materials down the tubes and, still operating from a distance, assembles a myelin sheet around the neuron: Composition, number of wraps and total coverage all matter. A myelinated neuron that loses its coating cannot transmit electrical signals properly, leading to loss of muscle control and other neurological problems.

The myelin sheath is mostly made of lipids, including sphingolipids, which are critical to myelin&rsquos structure and function. The enzyme serine palymitoyltransferase, or SPT, produces the backbone of all sphingolipids, and the membrane-bound protein ORMDL monitors sphingolipid levels and regulates SPT activity. ORMDL&rsquos activity must be precise: Too little sphingolipid production impedes myelination, and too much can be toxic.

Binks Wattenberg, a professor of biochemistry and molecular biology at Virginia Commonwealth University, studies membrane biogenesis and now focuses on lipid biogenesis. &ldquoI am very curious about how the cell knows when to make sphingolipid and when to stop,&rdquo Wattenberg said. &ldquoI think ORMDL might be the key to answering that question.&rdquo

Wattenberg&rsquos next-door lab neighbor, Carmen Sato&ndashBigbee, a professor in the same department, studies myelination, with a focus on oligodendrocytes. The two joined forces to study the role of sphingolipid biosynthesis in myelination in developing brains. They report their recent results in the Journal of Lipid Research.

To uncover the dynamics of sphingolipid content and synthesis during myelination, Wattenberg and Sato&ndashBigbee&rsquos team worked with newborn rat brains, because peak myelination occurs directly after birth. Only one in five cells in the brain is an oligodendrocyte, so the team isolated these myelin-producing cells for their experiments.

The researchers found that a large portion of the sphingolipids present in oligodendrocytes during myelination have an atypically long backbone &mdash an 18-carbon chain instead of a 16-carbon chain. &ldquoThe 18-carbon chain backbone points to a change in lipid composition during myelination, which might explain the insulating properties of myelin,&rdquo Wattenberg said. &ldquoIn future work, we want to look at the role of each type of sphingolipid in myelination.&rdquo

The study also found that SPT activity increases for the first few days of myelination and then begins to decrease. ORMDL activity is not measurable, but the team deduced that ORMDL isoform expression varies over time. These findings pave the way for future experiments.

&ldquoThe control of sphingolipid biosynthesis is key to myelination, and understanding how this process works will enable us to alter it in future treatments,&rdquo Wattenberg said. &ldquoOur pie-in-the-sky goal is to understand sphingolipid biosynthesis so well that we can reprogram oligodendrocytes and reverse demyelination in degenerative myelination diseases like MS.&rdquo


Abstract

Remyelination involves reinvesting demyelinated axons with new myelin sheaths. In stark contrast to the situation that follows loss of neurons or axonal damage, remyelination in the CNS can be a highly effective regenerative process. It is mediated by a population of precursor cells called oligodendrocyte precursor cells (OPCs), which are widely distributed throughout the adult CNS. However, despite its efficiency in experimental models and in some clinical diseases, remyelination is often inadequate in demyelinating diseases such as multiple sclerosis (MS), the most common demyelinating disease and a cause of neurological disability in young adults. The failure of remyelination has profound consequences for the health of axons, the progressive and irreversible loss of which accounts for the progressive nature of these diseases. The mechanisms of remyelination therefore provide critical clues for regeneration biologists that help them to determine why remyelination fails in MS and in other demyelinating diseases and how it might be enhanced therapeutically.


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Written on Thursday 11 April 2013

Honoring the 20th ELA anniversary, Pr. Charles ffrench-Constant, recongnized expert on myelin research, chairman of the Scientific Committee of the ELA Foundation and director of the MRC Centre for Regenerative Medicine at the University of Edinburgh (Scotland), tells us about the research advances on myelin biology and repair.

How the field on myelin biology has evolved over the last 20 years?

Evolution has been considerable. Building on cell work in 80s, we now have a good understanding of molecular mechanisms that lead to generation of oligos from precursor cells. We also now understand the relationship between oligos and stem cells in adult brain. However we still have a poor understanding of the mechanisms of perhaps the most remarkable feature of myelin biology – how does a oligo form a large number of sheaths composed of as many as 50 membrane wraps and each exactly correct in size for the axon being myelinated.

Do we understand better the mechanisms leading to myelin damage?

In multiple sclerosis (MS), careful neuropathological studies have greatly improved the understanding of how the immune system can damage myelin. In leukodystrophy, progress has been slower and we still have important questions to address.

In what circumstances endogenous remyelination occurs in humans?

The neuropathology has revealed that remyelination can be extensive in MS, especially early in the course of the disease. We have known for some time that experimental lesions in animal models can repair very well – the finding that such repair was so extensive in humans was a surprise, but a very important discovery. In leukodystrophy, remyelination is much less studied but given that the disease usually results from an abnormality of the oligodendrocyte itself the amount of endogenous repair is likely to be small.

How can we promote endogenous remyelination in humans?

This is one of the major goals of current MS research, and we now have a handful of possible targets for drug development.

Is cell therapy a good option to repair myelin?

We need to be very clear what we mean by cell therapy and which diseases we are trying to treat.
If we are trying to put in new oligodendrocytes to replace damaged cells, then this will be a limited option for MS as the disease is so widespread and it would be difficult to get cells to spread far enough in the damaged adult brain. For leukodystrophy cell replacement therapy is much more promising as :

  • the disease is usually due to an intrinsic cell abnormality rather than an external immune attack
  • the childhood brain is smaller and “primed” for myelination as this normally occurs for at least 20 years after birth.

However we will need to use oligodendrocytes from another individual as the patients own cells will still have the genetic defect.

How do you see the transition from the lab bench to the bedside?

With increasing optimism – researchers are now focusing on this transition, as is ELA in its funding priorities, and the science is now poised for clinical application. We need a generation of young clinicians trained in leukodystrophies to deliver this promise, and here again ELA is leading efforts to find and train the best young medics.

Most of the research done on the myelin field focuses on MS. Can we expect more research focusing on leukodystrophies? If not, how can we improve it?

I am confident that we will see much more leukodystrophy research. It’s clear that treatments are now a realistic possibility and that advances in gene and cell therapy offer wonderful new possibilities. At the same time, our understanding of the basic science has progressed to the stage where clinical application is possible and we have developed sufficiently good technologies so the results of treatments can be accurately measured. By making sure that funding continues for this vital research, we can ensure that the possibilities are realized


Myelination and time constant - Biology

A- represents negatively charged proteins.

Inside a cell considerable calcium is sequestered. For example, from the endoplasmic reticulum of muscle cells Ca++ is released when the cell is stimulated.

Cells have ionic pumps that maintain concentration gradients. The ions themselves can "diffuse" in or out of the cell through specialized protein channels.

Reading:
George B. Benedek & Felix M. H. Villars, Physics With Illustrative Examples from Medicine and Biology, Vol 3: Electricity and Magnetism, Addison-Wesley, Reading, Massachusetts (1979).

Howard C. Berg, Random Walks in Biology, Princeton Univ. Press (1983). A statistical physics look at the diffusion-drift development that leads to the Nernst potential (p. 141). Berg is well-known for his "Life at low Reynolds number" essay: see p. 75 of the book.

Bertil Hille, Ion Channels of Excitable Membranes, Sinauer Associates, 814 pp., (2001)

Fick's First Law
Consider diffusion flux J along one dimension: where the last form is the gradient in 3D.
In these equations J is a flux [a vector, particles/(area-sec)] and concentration is C at point x. D is the diffusion coefficient, and has dimensions of cm^2/sec. Notice the minus sign! A positive concentration gradient leads to a negative direction for diffusion. See figure below. Imagine we're considering a concentration gradient for K+=potassium ions across a cell's membrane.

Flux as current: Fick's 1st Law tells us about a diffusive flux of particles, charged or uncharged. For example, glucose is an uncharged particle in solution, and is subject to Fick's Law just as well as charged K+ and Cl-, but glucose flux is NOT a current! A flux of anions (+) is a positive current in the same direction as the flux, while a flux of chloride cations (-) is a current in the opposite direction.

The diffusion of charged particles (in the case we're considering, of K+) will set up an E field which will oppose the diffusion flux, and in fact will set up a voltage difference across the membrane.

Consider also that charged ions going in and out of a cell are going in and out of a fairly confined space, and the charge accumulation or deficit can be enough to generate a significant E field across the membrane. Even outside the cell space is rather confined, with the spacing between cells again able to be measured in angstroms, leaving "outside" not the same as a resevoir.

Drift of charged particles in an E field. In a material, charged particles will "drift" with a velocity proportional to their mobility &mu, their charge, and the strength of the E field:

If the charged particles were in a plasma they would move under the influence of F=ma, accelerating, but here in a material the particle reach a "terminal velocity". (Demo with corn syrup, where a ball bearing falls faster under the influence of gravity than a marble of the same size.)
mobility &mu is a property of a particle in a material (in this case aqueous electrolyte, and has units cm/(sec N ) .

The flux due to drift in the E field will be proportional to the concentration of the ions.
(which can be morphed to Ohm's Law, and, again, compare to F = ma)

We can now combine the diffusion flux and the drift flux in a steady-state version of KCL (flux as current with explicit account taken of area normal to current flow).

We need to relate diffusion constant D to mobility &mu : Einstein found
(in metals), where gas constant R and Boltzmann constant k are related by
R = kN, where N is the number of molecules in question. (ref: Van Vlack, Elements of Materials Science 2nd Ed. Addison-Wesley, 1964. pp 105, 98). As a result (considering other material factors too), when temperature rises, diffusion coefficient D always increases while mobility &mu increases for non-metals and decreases for metals. Further information: in F. Reif, Statistical Physics McGraw-Hill, New York, (1967), page 337 shows that, in general, .

Rewriting the flux equation, we have

where you can see that temperature T is still involved but mobility &mu has cancelled out. Later, when different ionic species have their own mobility, various &mu's will survive in a multi-ion formula.

Definition of potential difference = voltage. Now we need to remind you that
Since this integral is "conservative" you can go along any path from gnd to point P (in our case, from outside to inside the cell, across the membrane). Therefore integrate the flux balance equation to end up computing voltage. We will be grounding the extracellular space, called OUT in the integral.

Remembering log(X) - log(Y) = log(X/Y), and computing that, at room temperature, kT/q = 25 mV we have
,the Nernst equation for a singly-charged positive ionic species at room temperature. Consider a ratio of internal to external potassium of 10:1, we find that V K = -58 mV, which turns out to be what is measured.

E field strength across the membrane: 70 mv divided by 10 Angstroms

= 100M V/meter. near the breakdown strength.

What happens to the Nernst potential if calcium instead of potassium is considered? Ans: Calcium is Ca++, a doubly charged ion, in solution. Therefore substitute 2q in the kT/q term of the Nernst equation. The Nernst voltage is reduced by a factor of 2! Think of it this way: in the same E field a Ca++ ion will experience twice the force as a K+ ion. Therefore half the field strength would be needed to exert the same force on Ca++.

What happens if chloride ion is considered in a Nernst potential calculation? There are two ways to think about that question: (1) Since chloride ion has the opposite sign of K, then all other things being equal, the sign of the answer for the Nernst potential should be opposite to that of potassium. (2) Since chloride has a higher concentration outside than inside the cell, then the sign of the answer should be the same as potassium.

Where does the sign change come in when considering negatively charged ions? Consider the diffusion flux. If chloride Cl- concentration is greater OUT than IN the direction of diffusion of chloride ions from OUT to IN (left to right below). But because chloride has a negative charge, the direction of the chloride diffusion current will be opposite, from right to left.

Therefore the diffusion current term in the flux balance will have the opposite sign for a negatively charged ion.

Now consider the drift flux due to the electric field of charge separation. In our K+ equation the term q was +e, where e is the magnitude of the charge on an electron. Now q becomes -e for the chloride ion. But the electric field changes direction too, because negative charges instead of positive charges have moved into position to block the further diffusion of chloride ions. You can write the drift equation as

and you see it will have the same sign as the potassium version. Thus the only effective sign change occurs in the diffusion flux term, and we will expect a sign change in the answer if we're considering Cl- to have the same concentration gradient as K+.

Given the concentration gradient of sodium Na+, what will be the sign of the "sodium equilibrium potential" (Nernst potential considering sodium alone)? Because sodium concentration is higher outside the cell than in, it's Nernst potential will be positive, and will follow the same Nernst equation logarithmic law as potassium.

Protein as pumps and channels for ions. We have been working with mobilities of ions as factors in their Nernst potentials. In fact, most ions move relatively freely inside and outside the cells it's at the membrane barrier that mobility becomes important. We understand that ionic imbalances are maintained by pumps, in the form of proteins in the cell membrane, in somewhat the same way that an air conditioner in a window helps maintain a temperature gradient from inside to outside the house. Proteins also form channels for specific ions, and the permeability (a more common term for ionic mobility in the membrane) of a channel can be modulated by synaptic activity or transmembrane voltage. It is possible to record, by means of a patch clamp electrode isolating a small section of membrane, the signals of individual channels. See below, from www.ccs.fau.edu/

Reading Bertil Hille, Ion Channels in Excitable Membranes, 3rd Ed., Sinauer Associates (2001).

The 1991 Nobel Prize in Physiology or Medicine went to Bert Sakmann and Erwin Neher for their work on patch clamping.

EXAMPLE: Say the concentration of K+ and Cl- is the same C(x) everywhere and that the mobility of K+ > mobility Cl-. What is an expression for the transmembrane voltage? ANS: You need to add up the fluxes of K+ and Cl-. We already have the K+ flux:

and the Cl- flux sum will be
because the Cl- diffusion flux is in the opposite direction, but the electrical flux is the same direction. Add the two contributions up, sum to zero, separate variables and see

now integrate from OUT to IN, as before, and obtain
, which gives the potassium-only answer from before if &mu Cl = 0.

Another problem: Now assume the concentrations differences of sodium and potassium are equal and opposite across a membrane. If the mobilities of Na and K are equal, then the transmembrane voltage will be zero. In fact the voltage is negative, in the direction of the K equilibrium potential. Assume the mobility of K > mobility of Na ions. What will be the voltage difference? Note that if C(x) is the potassium concentration function, then the Na concentration function is C(-x). Furthermore . How far can you get?
A better approach, when the concentration gradients of the ionic species differ: compute independently the Nernst potential of each ionic species. Then use superposition and compute the total of the respective ionic mobilities, for a weighted sum:

where N is the total number of ionic species, and Vj is the Nernst potential of the jth species.
Example. Assume the concentration gradients of Na and K are equal and opposite, +58 and -58mV. Suppose at rest the mobility of K is 3 times greater than the mobility of Na. Then Vinside = 0.75*(-58) + 0.25*58 = -29mV.

Increase in Na+ permeability during excitation: In the resting state of a nerve cell sodium mobility across the membrane is much lower than potassium, and the cell maintains a negative voltage. When a nerve or muscle cell is stimulated by synaptic transmission, the mobility (or channel conductance, or permeability) for sodium transiently increases to a value greater than that for potassium and the cell internal voltage "spikes" above zero volts for about a millisecond. (image below from http://www.bio.psu.edu/Courses/Fall2002/Biol142/neurons/neurons.html)

The following assumes passive conduction of voltage changes down an axon or dendrite. Consider a cylindrical tube as a model for a dendrite or axon process. The wall of the tube will be a high resistance membrane and the inside of the tube will be low resistance axoplasm.

Say the inside of the tube has resistance/length = r-in, as shown below.


How will r-in depend on cable diameter? Consider the material property we called rho in strain gauge development: here it will appear again, as specific axial resistance of "axoplasm", and its value is about 100 &Omega--cm. Therefore r-in = rho/area.

Conductance g-in = 1/r-in will increase as the square of the diameter.

Now consider the leakage current im going out of the membrane. Per "compartment" of length we have a picture like,

If you differentiate again and substitute the above equation in, you have

where the minus signs cancel out.

Now on to the time domain: Consider that there is capacitance in the membrane:

the membrane current is now to be expressed as:

How are r-in, r-m and c-m calculated from physical properties of the cell and its membrane? Recall from the strain gauge lecture that resistance R, in ohms, for a rod of length L and cross-section A is

where rho is the resistivity of the material of the rod. The units of resistivity are Ohm-cm. Resistance per unit length, by a "dimensional analysis," is therefore

where d is the diameter of the cable under consideration. Therefore knowing radius r allows calculation of resistance r-in.

It is known that resistivity of axoplasm is 100 Ohm-meter (From Neuron to Brain , p. 141)
Compare this to the resistivity of metal, like copper: about 10^-8 ohm-m!
Axoplasm is the same order as crystal silicon resistivity.

What's the resistance of a 1 cm long axon, diameter 10 microns? about 10^10 Ohms!

Next, consider membrane as a sheet of material specified by capacitance/cm^2 Cm, and by conductance/cm2 Gm. Conductance has units of mhos. Why use conductance? It's proportional to the area of the membrane under consideration. For a cable of diameter d, the circumference is pi·d. So pi·d*1cm is the area of a unit length (cm) of membrane. Capacitance per unit length c-m is then Cm·pi·d and conductance per unit length is Gm·pi·d. therefore

Both capacitance per unit length and resistance per unit length can therefore be calculated from material properties of membrane.
Membrane has 1 muF /cm^2 and a RESISTANCE of about 2000 Ohms/cm^2
These factors allow calculation of time constant and length constant of membrane.
A passive voltage change will decay toward zero with length constant lambda.

Solutions to the cable equation: See D. J. Aidley, The Physiology of Excitable Cells, page 50 ff and
B. Katz, Nerve, Muscle and Synapse, Oxford Univ Press (1970)

Myelination significantly increases Rm and therefore increases the length constant of a axon, typically from 10 to 2000 microns! The nodes of Ranvier are closer than one length constant. A myelin wrap can also reduce membrane capacitance (remember capacitors in series?) so the effective time constant of the membrane is about the same the result is faster propagation of an action potential in a myelinated axon.

Calculating the speed of conduction down a cable:

From ICHIJI TASAKI, "ON THE CONDUCTION VELOCITY OF NONMYELINATED NERVE FIBERS," Journal of Integrative Neuroscience, Vol. 3, No. 2 (2004) 115𤩬.

The cable equation is a PDE run matlab function pdex4.m to see a related example. For both time and position x, V can vary. a 3-D plot is needed.
C:MatlabR12ToolboxMatlabdemos


What Is the Myelin Sheath?

Definition & Facts

The myelin sheath is a cover made out of fats and proteins that wraps around the axons (projection) of nerve cells. It insulates neurons so they can send electrical signals faster and more efficiently. This supports brain health and nervous system function [1, 2].

Here are some quick facts about myelin:

  • About 80% fats/cholesterol and 20% proteins.
  • Considered an outgrowth or extension of a type of glial cell (oligodendrocyte &ndash CNS, Schwann cell &ndash PNS).
  • Continues to grow throughout adolescence and even into our early 20s.
  • Myelinated axons are white in appearance, hence the term &ldquowhite matter&rdquo of the brain.

Function

Myelin improves the conduction of action potentials, which are needed to send information down the axon to other neurons [3].

The myelin sheath increases the speed of impulses in neurons. It facilitates conduction in nerves while saving space and energy [1].

Myelin helps prevent the electrical current from leaving the axon. It allows for larger body sizes by maintaining efficient communication at long distances.

When babies are born, many of their nerves lack mature myelin sheaths. As a result, their movements are jerky, uncoordinated, and awkward. Scientists think that, as myelin sheaths develop, movements become smoother, more purposeful, and more coordinated [4, 5].

Research suggests that myelination might improve children&rsquos cognitive performance improves as they grow and develop [6].

Additionally, when a peripheral fiber is severed, the myelin sheath provides a track along which regrowth can occur [7].

When Does Myelination Stop?

Researchers think that myelination occurs most significantly during childhood, but some brain imaging studies suggest it may continue until 55 years of age and possibly even throughout life [8].

Oligodendrocytes vs. Schwann Cells

Oligodendrocytes and Schwann cells are types of cells that produce, maintain, and repair myelin [9].

Schwann cells normally produce myelin in peripheral nerves (outside the brain), but can enter the brain when needed [9].

On the other hand, oligodendrocytes are found solely in the brain. They are responsible for the formation of new myelin in both the injured and healthy adult brains [9].


Discussion

We show that oligodendrocytes expressing a single copy of a dominant-negative 㬡 integrin transgene myelinate small-diameter axons in optic nerve less efficiently than wild-type or control (dominant-negative 㬣) oligodendrocytes. This presents as a transient failure to myelinate small-diameter axons during development, demonstrated by a shift to the right of the dose–response curve created by plotting axon diameter versus the percentage myelinated as schematized in Fig. 9 . A similar phenotype was seen when the downstream signaling molecule FAK was deleted in myelinating glial cells. In mice expressing the dominant-negative 㬡 integrin transgene, we also observed that the thickness of the myelin sheath in those axons that do become myelinated is normal, as is the length of the myelin internodes as measured in myelinating co-cultures. We conclude, therefore, that inhibition of integrin signaling delays the initiation of myelination but has no effect on the signals required for the formation of the sheath once the axoglial interactions that lead to myelination have been established. It follows, therefore, that integrins contribute to the axoglial interactions that initiate myelination.

The expression of dn㬡 integrin shifts the axon diameter threshold for myelination toward larger diameters, shown here schematically by a shift to the right of the dose–response curve created by plotting the percentage of axons myelinated against the axon diameter. By contrast, the increased expression of Nrg1 in the PNS was previously shown to shift the myelination threshold toward lower axon sizes, i.e., to the left.

Given the evidence from myelinating co-culture experiments revealing an intrinsic defect in oligodendrocyte myelination, two broad mechanisms can be put forward to explain our observations—perturbation of the axoglial signaling that initiates myelination or a failure of oligodendrocyte differentiation ( Fig. 10 ). The first would be based on the observation that in the PNS an axonal signal proportional to the diameter and above a certain threshold is required to initiate myelination by the contacting glial cell (Taveggia et al., 2005 Nave and Salzer, 2006). If there is a reduction in the glial response to the axonal signal due to perturbation of integrins, then the prediction would be that the signal on the small axons will not be detected as above the required level and a reduced percentage of myelination of these small-diameter axons will be observed. In contrast, the large axons could either provide a signal that is in excess of the threshold level or express an additional signaling molecule that initiates myelination without any requirement for integrin function, and thus would not be affected by integrin perturbation. In the second mechanism, small-diameter axons could be preferentially affected by the reduction in 㬡 integrin signaling as a result of a compromised ability of the oligodendrocyte to change their morphology and generate multiple processes for myelination. It has been shown that toad oligodendrocytes myelinating small axons extend more processes than those myelinating large axons (Stensaas and Stensaas, 1968 Hildebrand et al., 1993). Additionally, oligodendrocytes in rat white matter areas constituted of small axons possess more branches than those in areas with both large and small axons (Matthews and Duncan, 1971). A reduction in 㬡 integrin signaling that compromises process outgrowth via the established role of this receptor in cytoskeletal reorganization would therefore be expected to reveal a phenotype in those tracts where more processes are required, i.e., those with small-diameter axons. In agreement with this, knockout mice for the regulator of the actin cytoskeleton WAVE1 exhibited a reduced number of myelinated axons in the optic nerve and corpus callosum, with WAVE1 knockout oligodendrocytes showing a reduced number of processes in cell culture (Kim et al., 2006).

Models for the failure in myelination of small-diameter axons resulting from perturbed 㬡 integrin signaling. (a) The oligodendrocyte initially extends processes to reach large and small axons and then initiates myelination. Large axons are myelinated earlier than small axons. (b) Model I: an axonal signal proportional to the diameter and above a certain threshold is required to initiate myelination by the contacting glial cell. Due to the expression of dn㬡 integrin there is a reduction in the glial signaling in response to this signal, such that the signal initiated by some small axons will now not be above the required (threshold) level for myelination. By contrast, the large axons provide a signal that is significantly in excess of the threshold level and/or additional signals, and thus will not be affected by perturbation of integrin signaling. (c) Model II: myelinating tracts rich in small-diameter axons require more oligodendrocyte processes than those tracts rich in large axons. Small axons are, therefore, particularly susceptible to any failure of the oligodendrocyte to generate multiple processes for myelination, as might occur after perturbation of integrin signaling, leading to an impaired ability of the cell to reorganize the cytoskeleton. See text for a discussion of the evidence for and against the two models.

Three observations argue against the latter mechanism—perturbation of cytoskeletal function as the cause of the phenotype in the dn㬡 mice. First, although the number of internodes myelinated by each dn㬡 oligodendrocyte in the myelinating cocultures is reduced, the length of internodes is unaltered, with the majority of internodes myelinated by wild-type, dn㬡, and dn㬣 oligodendrocytes distributed between 27 and 40 µm as recently reported for cortical oligodendrocytes in vivo (Murtie et al., 2007 Fig. S4 c). A compromised ability of the oligodendrocyte to extend processes would be expected both to reduce the number of internodes and to shorten internodal length, as this latter measurement must reflect process extension along the axon once contact has been established. Second, the similar g-ratios seen in the P17 optic nerve of wild-type and dn㬡 mice show that the cytoskeletal reorganization required for myelin sheath compaction is not affected by inhibition of integrin signaling. Third, the number of primary processes is not significantly different between wild-type, dn㬡, and dn㬣 oligodendrocytes in premyelinating co-cultures. Dn㬡 oligodendrocytes are, therefore, able to extend processes normally, as evidenced by their number of primary processes and internodal length, but then myelinate less efficiently with fewer internodes.

The conclusion that oligodendrocyte integrins form part of the recognition complex for axonal signals that determine whether or not myelination occurs is important, as the identity of such axonal signals in the CNS is unknown. In the PNS, the level of axonal type III Nrg1 has been shown to play a crucial role in determining both whether an axon is myelinated and in the regulation of myelin thickness (Michailov et al., 2004 Taveggia et al., 2005). We have shown that laminin-binding integrins amplify neuregulin signaling as part of the mechanisms that regulate target-dependent survival of newly formed oligodendrocytes in the CNS (Colognato et al., 2002). Our results here would therefore be consistent with a model in which axonal neuregulins in the CNS, recognized by oligodendrocyte ErbB receptors, generate a signal to initiate myelination that is further amplified by integrins. In the case of small-diameter axons, this amplification is essential for triggering myelination, whereas large-diameter axons generate sufficient signal without integrin amplification. However, the role of neuregulins in CNS myelination has not been resolved. Exogenous addition of Nrg1 promotes myelination in co-cultures of oligodendrocytes and DRG neurons (Wang et al., 2007) and myelination of type III Nrg1-deficient DRG neurons is significantly reduced in co-culture (Taveggia et al., 2008). Hypomyelination is seen in transgenic mice expressing a dominant-negative ErbB receptor in oligodendrocytes, and in mice haploinsufficient for type III Nrg1 (Roy et al., 2007 Taveggia et al., 2008). However, in contrast to the PNS, increased expression of this axonal signal does not promote myelination of very small, and normally unmyelinated, axons in co-cultures of superior cervical ganglion neurons and oligodendrocytes (Taveggia et al., 2008). Moreover, conditional mutants with ablation of Nrg1, ErbB3, or ErbB4 exhibit no cortical myelination abnormalities (Brinkmann et al., 2008), a result that rather compellingly argues against a necessary role for Nrg1 signaling in CNS myelination. Additional neuregulin isoforms may be important in the CNS but equally, and in contrast to the PNS, other axonal signals may be required for regulating myelination. These may also be amplified by integrins, as seen for a number of different growth factors in other cell types. The hypothesis that such multi-component signaling complexes initiate and regulate myelination by oligodendrocytes provides a mechanism to explain the �tch-up” by the mutant oligodendrocytes in the older animals, as compensatory interactions within the complex will facilitate restoration of normal axoglial interactions.

Interestingly, both in monkeys and humans the area of the CNS most prone to remyelination failure is the optic nerve (Lachapelle et al., 2005). A model in which amplification of a myelination signal is required for small- but not large-diameter axon myelination could explain both the vulnerability of the optic nerve and the regional selectivity of the phenotype seen in the dn㬡 mouse, as the optic nerve contains entirely small-diameter axons. Similar regional differences in myelination are observed in other mutants that perturb integrin signaling, such as the Fyn knockout and the laminin-2�icient (dy/dy) mouse. In these mice myelination defects are seen in optic nerve but not in spinal cord, where the density of larger axons is higher. Another possibility to explain regional heterogeneity, intrinsic differences in the oligodendrocytes arising from different regions of the developing CNS, seems less likely given that ablation of one such population is followed by replacement from a different region (Kessaris et al., 2006). In addition, transplantation of oligodendrocytes from optic nerve into spinal cord reveals that the transplanted cells can myelinate the full range of axon diameters in their new environment, even though these diameters are much greater than those present in their original location (Fanarraga et al., 1998).

Although the two previous studies examining the role of 㬡 integrins in regulating CNS myelination appear contradictory (Benninger et al., 2006 Lee et al., 2006), the current study allows their reconciliation. Here we have concluded that inhibition of integrins perturbs axoglial signaling and delays myelination of small-diameter axons, but has no effect on the subsequent formation of the myelin sheath. This conclusion is consistent with our previous report on the absence of g-ratio disturbances in the oligodendrocyte-specific conditional 㬡 knockout (Benninger et al., 2006), where the analysis focused on myelin sheath thickness in older animals. Hypomyelination after expression of 㬡㥌 (Lee et al., 2006) can be explained by a requirement for dystroglycan in sheath formation. As noted in the introduction, the 㬡㥌 integrin extracellular domain will form heterodimers with integrin-α subunits and thus compete for ligand with other oligodendroglial laminin receptors. One of these is dystroglycan (Colognato et al., 2007), for which the main binding site on laminin overlaps with that of integrins at the laminin G domains LG1-3 (Talts et al., 1999 Wizemann et al., 2003). We have shown that dystroglycan is a significant laminin receptor for CNS myelination in vitro, and transgenic studies reveal an essential role in PNS myelination (Saito et al., 2003 Occhi et al., 2005 Colognato et al., 2007). We propose, therefore, that the study of Lee et al. (2006) expressing 㬡㥌 reveals a requirement for dystroglycan in later stages of myelin sheath formation in addition to the role of integrins in the earlier stages of initiation. Our present and previous studies, by contrast, specifically target integrin signaling and would thus not reveal any later role of other laminin receptors.

The identification of the signals regulating the initiation of myelination may lead to the development of strategies to promote effective remyelination by oligodendrocytes within MS lesions arrested at the premyelinating stage and therefore unable to contribute to repair (Chang et al., 2002). Further studies examining the partners and ligands of the integrins expressed on oligodendrocytes represent promising new approaches toward understanding this goal.


Discussion

We have shown that a number of Ephs and ephrins are expressed in oligodendrocytes at different developmental stages, and that both forward and reverse signaling through these receptors have an impact on the ability of the oligodendrocytes to undergo morphological differentiation in culture and interact with neurons during the myelination process. Of particular interest, our results show distinct functions of forward signaling through the EphA and EphB receptors, and reverse signaling through ephrin-B. Forward signaling through EphA receptors reduced the ability of the oligodendrocytes to extend processes and form myelin sheets in culture. In addition, blocking of bidirectional signaling between ephrin-A1 and EphA receptors enhanced myelination in a coculture of oligodendrocytes and DRG neurons whereas stimulation of EphA receptors inhibited myelination. Combined, this suggest that EphA forward signaling into the oligodendrocyte causes process retraction, and thus prevents permanent axo-glia contact formation and initiation of myelination. We further showed that this effect of EphA is independent of the activational status of 㬡 integrins. By contrast, forward signaling through EphB only had an effect in the presence of the integrin ligand laminin, and the inhibitory effect of ephrin-B2 on myelin sheet formation could be reversed by overexpression of a constitutive active 㬡-integrin. This implies that in oligodendrocytes, ephrin-B2-induced forward signaling through EphB receptors is involved in controlling the activation of 㬡-integrins. In addition, we show that reverse signaling through ephrin-B, induced either by EphA4 or EphB1, stimulates myelin sheet formation, and finally the opposing effects of forward and reverse EphB-ephrin-B signaling were confirmed in the myelinating coculture system.

Together, this implies that the balance between forward and reverse Eph and ephrin signaling represents a possible mechanism of intercellular communication, controlling which axons are to be myelinated. We propose a working model in which negative regulatory guidance clues from ephrin-A and ephrin-B must be overcome by positive clues from EphA4 and EphB1, present on the axon surface, in order for the axons to be selected for myelination ( Figure 10 ).

Working model: Opposing signals from Ephs and ephrins regulate axo-glia interaction and myelination. (a) EphA forward signaling in the oligodendrocyte inhibits oligodendrocyte process extension and myelination in an integrin independent manner. (b) EphB1 forward signaling in oligodendrocytes modulates integrin activation and thereby regulates axo-glia interactions and myelination. (c) Ephrin-B reverse signaling in oligodendrocytes, induced by EphB1 or EphA4, enhances myelin sheet formation. We propose that the specific combination of Ephs and ephrins, presented on the axonal surface, will determine whether a given axon becomes myelinated.

The existence of such an Eph-ephrin-guided selection process bares resemblance to the process of axonal guidance and growth cone dynamics (Xu and Henkemeyer, 2012), where the control of integrin-mediated adhesion also is required for decision making (Gupton and Gertler, 2010 Hines etਊl., 2010). We have previously shown that integrin activation is involved in controlling both the morphological differentiation of the oligodendrocytes and timely translation of MBP mRNA (Laursen etਊl., 2011). The latter suggests that a tight regulation of integrin activation is required to coordinate the wrapping process with MBP expression. This may explain why an increase in the level of the EphB1 receptor is necessary to control integrin activation in mature oligodendrocytes. In line with our findings, forward signaling through EphB has previously been reported to control integrin-mediated cell adhesion in other cell types through a mechanism involving phosphorylation of tyrosine-66 of R-Ras, causing decreased R-Ras activity (Zou etਊl., 1999). Interestingly, in oligodendrocytes, R-Ras has also been shown to regulate 㬖㬡 integrin activation, which is required for process extension and myelin membrane formation in culture (Olsen and Ffrench-Constant, 2005). This implicates that the same signaling pathway is in use in the oligodendrocytes. The inhibitory effect of inactive R-Ras could be overcome by direct activation of the integrin with manganese ions (Olsen and Ffrench-Constant, 2005). Similarly, we show here that ephrin-B has no effect on cells expressing a constitutive active 㬡-integrin, and that the presence of laminin does not affect the ability of ephrin-B2 to induce EphB receptor phosphorylation, supporting the existence of a dynamically regulated system, where EphB signaling regulates the activation status of the β-1 integrin. Currently, the only factor known to induce integrin activation during the myelination process is laminin, present in the extracellular matrix surrounding the axons (Baron etਊl., 2005 Colognato etਊl., 2002 Olsen and Ffrench-Constant, 2005). This implicates that a balance between the amount of laminin in the extracellular matrix and ephrin-B2 on the axon surface controls the activation status of the integrin.

We also identified EphA4 and EphB1 as axonal adhesion molecules, which have a positive effect on the ability of the oligodendrocytes to extend myelin sheets. As a similar effect on myelin sheet formation was not observed with EphA2, this point to ephrin-B reverse signaling as a novel regulatory pathway during myelination. Interestingly, recent experiments have shown that EphA4 is present on hippocampal neurons at the time of myelination, and electron microscopy has further revealed clusters of EphA4 at contact points between axons and oligodendrocytes (Tremblay etਊl., 2009). This adds EphA4 and EphB1 to the list of very few axonal adhesion molecules, thought to be able to stimulate myelination. Previously, this list included only L1 (Laursen etਊl., 2009 Wake etਊl., 2011) and neuregulin (Brinkmann etਊl., 2008 Taveggia etਊl., 2008). A further understanding of how signals from these receptors integrate and overcome the negative regulatory signals will be essential for understanding the selection process. The observation that myelination is not completely inhibited in the absence of any one of these factors may suggest that the lack of one factor can be compensated for by upregulation of others.

We further identified ephrin-A1 as a novel negative regulatory molecule on the axonal surface, and we suggest that it may need to be downregulated for myelination to be initiated. Possible mechanisms behind such regulation could be cell surface shedding or reduced expression. Ephrin-A1-induced forward signaling was shown to prevent process extension from oligodendrocytes in an integrin-independent manner. In other systems, EphA forward signaling has been implicated in regulation of integrin activation (Bourgin etਊl., 2007 Miao etਊl., 2000) and has also been shown to directly regulate the activity of signaling molecules downstream of the integrins (Knoll and Drescher, 2004 Miao etਊl., 2000 Yamazaki etਊl., 2009). However, alternative downstream signaling pathways, controlling activation of the Rho family of GTPases, have also been implicated in EphA-mediated process retractions (Lisabeth etਊl., 2013). Even though we show that a constitutive active 㬡-integrin cannot reverse the inhibitory effect of ephrin-A1 on process extension, we cannot exclude that the EphA receptors may directly inhibit signaling molecules downstream of the 㬡-integrins. Interesting candidate molecules include ILK, FAK, and Src-family kinases, which are all known to be regulated by EphA receptors in other cell types (Knoll and Drescher, 2004 Miao etਊl., 2000 Yamazaki etਊl., 2009), and also known to be important for process extension in the oligodendrocytes (Camara etਊl., 2009 Chun etਊl., 2003 Colognato etਊl., 2004 Forrest etਊl., 2009 Liang etਊl., 2004 O’Meara etਊl., 2013).

Generally, the signaling pathways reported to be induced by forward and reverse signaling through the Eph-ephrin system are highly cell-type-specific. This is likely to reflect the specific expression profiles of receptor and ligands in adjacent cells at a given time point, and is further complicated by redundancy between family members and cis-interaction between ligands and receptors, reported to modulate the effect of trans-interactions (Carvalho etਊl., 2006 Kao and Kania, 2011). The observed upregulation of ephrin-A5 during oligodendrocyte differentiation may lead to inhibition of trans-signaling and thereby be an intrinsic mechanism by which the oligodendrocyte can overcome the inhibitory effect of EphA forward signaling.

In addition to the role of bidirectional Eph-ephrin signaling during axo-glia interactions and myelin sheath formation in the central nervous system we report here, others have suggested that the Eph/ephrin receptors may also play a role during migration of OPCs (Prestoz etਊl., 2004). It is interesting to note that Eph-ephrin signaling has also been reported to regulate Schwann cell migration (Afshari, etਊl., 2010). However, it is still an open question whether this signaling system also is involved in regulating axo-glia interactions in the peripheral nervous system.

Altogether our data suggest that Ephs and ephrins represent a novel family of adhesion molecules involved in controlling oligodendrocyte morphological differentiation and axo-glia interactions. This also points toward potentially novel ways, by which it will be possible to interfere with axo-glia interactions in order to enhance remyelination in demyelinating diseases, for example, multiple sclerosis. In particular, based on the enhanced myelination caused by soluble ephrin-A1 in cocultures, blocking of EphA forward signaling in the oligodendrocytes may be of specific interest. In support of the relevance and potential effect of such a strategy, ephrin-A1 and several EphA receptors were found to be upregulated in active lesions of multiple sclerosis patients (Sobel, 2005). Furthermore, recent data have shown that EphA4 knockout mice show a less severe clinical score after Myelin Oligodendrocyte Glycoprotein (MOG)-induced Experimental Autoimmune Encephalomyelitis (EAE) compared with wild-type animals (Munro etਊl., 2013).