Lecture #9 Flashcards
what is myelin and what is its function?
a protrusion of the membrane of Schwann cells which wraps around the axon insulating it allowing for faster conduction of nerve impulses
which cells of the nervous system are not coated in myelin?
nodes of ranvier
where to Schwann cells come from?
neural crest cells
what two types of Schwann cells can be differentiated from neural crest cells after birth?
myelinating Schwann cells that wrap one single axon multiple times, or non-myelinating schwann cells where more than one small axon is circled by just one Schwann cell
what occurs for Schwann cells to differentiate?
signals coming from the axon to the Schwann cels activate a cascade of signaling molecules such as those in the phospho-AKT and phospho-ERK pathways as well as a series of tfs that act as positive regulators of myelinization that when activated allow the myelination process to progress
under what conditions can Schwann cells de-differentiate and go back to the immature type?
in cases of nerve injury
describe the difference between Schwann cells in the CNS vs PNS:
Schwann cells in the PNS have the ability to become de-myelinating and then revert back to myelinatiing cells (plastic) whereas the CNS cannot
what is Charcot Marie tooth syndrome?
a genetic disease that arises when you have problems in systems that give rise to myelination
describe CMT1:
based on demyelination (bound defects, contraction of tendons, loss of muscle)
describe CMT2:
axonal type based electrophysiology and morphology
what is the biggest problem faced with CMT?
there are more than 1000 mutations found in over 100 genes that can cause hereditary neuropathies, so there are patients with very similar phenotyoes but the origin of the disease can be very different
what is the most common form of CMT and what does it entail?
CMT1A: there is a loss of finer nd in some axons you can see onion bulbs
what are onion bulbs?
protrusions that proliferate and attempt to re-myelinate the axon
what is Dejerine-Sottas Syndrome (DSS)?
sever disease in children where the nerve and muscles never develop properly and have a notable presence of large onion bulbsk
since there are many different mutations on different genes involved in this disease, scientists decided to focus on a broad approach that targets a mechanism - what did the focus on?
myelin protein zero (P0)
describe myelin protein zero:
a single passage transmembrane protein which has an extracellular domain the folds like an immunoglobulin domain → the most present protein in myelin of peripheral nerves (200 different mutations for CMT)
describe the function of P0 in peripheral myelin:
most abundant protein and forms tetramers which can allow for the packing of myelin
which specific amino acid did they decide to focus on and why?
S63 → there was more than one mutation in this aa and different mutations produced different phenotypes
what does the deletion of S63 cause?
classical CMT1 de-myelinating disease
what does a substitution of the S63 with S63C cause?
DSS - much more severe
they generated mice models with both the deletion and the substitution, what type of mutation did they discover?
both mutations gave rise to a different phenotype so both mutations cause a gain of function
describe the normal path of a myelin protein:
goes to the ER then to the golgi, and then most likely folded next to the myelin sheet
what would theorhetically happen if the protein was not folded as usual?
autophagy is activated or ERAD, but if there is an excess of the protein the UPR is activated
what are the three pathways of the UPR?
IRE1, PERK, ATF6
describe the IRE1 pathway:
encodes for a potent transcription factor (XBP1)that translocates to the nucleus and activates genes encoding for chaperones or associated to ER degradation → activates genes that try to fold proteins and remove them from the ER
describe the PERK pathway:
dimerization and autophosphorylation of PERK, which causes the phosphorylation of the the translation initiation factor eIF2⍺ which attenuates protein translation
what is another name for the PERK pathway?
integrated stress response → PERK targets eIF2⍺ not only in cases of misfolded proteins but also in viral infections and other challenges to the cell
describe the ATF6 pathway:
translocates to the Golgi where it is cleaved into the active form and then inactivates a series of genes partially overlapping with the IRE1 pathway
describe the adaptivity of the UPR:
on one side you have misfolded proteins in the ER and then you activate eIF2⍺ to reduce protein translation because you don’t want have too many misfolded proteins, but also activate genes which try to fold the proteins
what happens if the UPR goes on for too long?
it becomes maladaptive and causes cell death
how does a chronic UPR cause cell death?
through ATF4 there is the activation of another tf called CHOP which leads to the activation of genes involved in cell death, namely GADD34
what is another name for GADD34?
PPP1r151
what did scientists discover when studying the downstream effects of GADD34?
it reactivates translation in the context of the UPR
when GADD34 was ko in mice what was seen?
they ran on the rotor similar to the WT mice and nerve conduction velocity was partially recovered
why did they see an improvement in mice when GADD34 was removed?
if translation is not reactivated, there should be less protein in the ER and therefore a reduction to ER stress
what does stress derived from the mutant protein result in?
leads to eIF2⍺ being phosphorylated and then translation is attenuated → however since P0 is expressed, ATF4 is activated together with CHOP so the Gadd34 gene is expressed, leading to the dephospho rylation of eIF2⍺ and as a result protein synthesis restarts producing more stress on the cell
what was seen in S63del mice models when eIF2⍺ was removed?
there is an increase in demyelinated fibers with a very odd looking cytoplasm and signs of stress
in S63del mice it was expected that IRE1 ad ATF6 would be increased when eIF2⍺ was removed - what was seen instead?
there is a strong increase in gene which are negative regulators of myelination such as c-Jun → it was the lack of phosphorylation on eIF2⍺ telling the cell to stop myelinating
how is c-Jun activated?
activated through p-ERK which is hugely increased in mutant mice without eIF2⍺ → acts through phospho-ERK and phospho-AKT pathways
in β-cells what normally happens under stress?
there is the activation of the 3 UPR pathways leading to the phosphorylation of eIF2⍺, blocking protein translation and leading to the attenuation of ER stress
removal of the possibility of eIF2⍺ phosphorylation leads to the blockage of the other two pathways (IRE1 and ATF6) leading to a general increase in stress leading to apoptosis
what happens in Schwann cells after the removal of eIF2⍺ that is different than in β cells?
removal of eIF2⍺ does not lead to the blockage of the other two pathways which instead are continued - we do not have oxidative stress or an increase in the stress response → there is the activation of the p-ERK pathway that will increase c-Jun activation and block Schwann cell differentiation and myelination
this is what Schwann cells de-differentiate in the case of injury
what is the function of Guanabenz?
used in disease like hypertention - has a strong affinity for the ⍺2-adrenergic receptor and an inhibitory of Gadd34
what was the issue with the mice treated with Guanabenz?
it was too strong - the mice were very sleepy ad unhappy
how was Guanabenz modified in order to reduce its strength and what was its new name?
Sephin 1: they removed the part with the ⍺2-adrenergic receptor activity but maintained the Gadd34 inhibition
what was seen in mice who were given Sephin 1?
there was in increase in the phosphorylation of eIF2⍺ because Gadd34 was inhibited
myelination improved significantly which was also accompanied with the reduction of stress response genes
was Sephin 1 able to enter the BBB?
yes - accumulated in the sciatic nerve
what was observed when mice were treated with Sephin1 twice a day for 6 months?
there was an improvement in morphology, motor functional reduction of the stress repose and a reduction in the formation of onion bulbs and fibers → the nerve conduction velocity almost returned to normal
describe the R98C mutation:
patients with this mutation are classified as DSS since they develop almost no myelin
what is seen when R98C patients are treated with Sepin1?
there is an improvement in myelination particularly in thickness of the myelin → mice are stronger in terms of grip strength and have better holding capacity as well as nerve conduction
describe the genetic phenotype of CMT1A:
there is a rearrangement causing the duplication of a 1.4Mb segment on chromosome 17 containing the PMP22 gene → this leads to the production of three copies of the protein and therefore overexpression
what is unique about the mutation in CMT?
if there is a duplication on one allele, then there will be a deletion on another segment
what neuropathy is caused when there is a deletion in CMT1A?
HNPP (hereditary neuropathy with pressure palsies) - only manifests when there is pressure to the nerve
in general this deletion does not cause a clinical manifestation
what disease occurs when there is a point mutation on PMP22?
a different neuropathy called CMT1E
describe what is seen in both CMT1A and CMT1E:
there is the activation of the stress response mechanism → in both cases the protein is unfolded so the mechanism is similar to what was seen in P0
when there is too much PMP22 protein produced, it is immediately degraded, therefore high levels means that the system is overwhelmed and the stress response is activated
is there a difference seen in the healing ability of male and female mice?
yes - females largely improved in their motor capacity and males improved a little, but were still incapable of running on the treadmill
also seen that the nerve conduction velocity is improved in females and remains the same in males
what is G ratio?
measure of myelin thickness → ratio between the diameter of the axon and the diameter of the axon plus myelin
what happens to the axons upon treatment to females?
the morphology is improved and we see a rebalance in protein expression → in these mice there is more PMP22 because of its Overexpression but there is also hypomyelination - so if the protein id taken from the nerve and a WB is performed, theres less myelin protein in general, especially in P0 - but if we consider the ration between P0 and PMP22, PMP22 is relatively over expressed compared to P0
when we treated theses mice the ratio is partially re-established→ confirmed by the presence of BiP
how does aging effect the nerve health?
the overall UPR tends to fade with age, so the more the cells age, the more inefficient the quality control becomes → even if Gadd34 is removed and less mutant protein is produced there will still be na accumulation of mutants proteins disrupting the nerve
describe the pathway of IRE1:
IRE1 is dimerized and leads to the splicing of Xbp1, which becomes a potent tf the enters the nucleus and induces the transcription of genes involved in chaperone,s lipid synthesis, and ER associated degradation
what is the role of Xbp1 in a misfolded protein disease?
it is unclear → some papers show that it can be adaptive, and in other cases it appears to be maladaptive, or in the case of prion disease its complete removal has no effect
what is the effect on Schwann cells if Xbp1 (or ATF6 etc) is removed vs if it removed in WT mice?
Schwann cells don’t care if it is removed, they just keep myelinating as normal - in many other cells such as B cells, no Ab will be produced if Xbp1 is removed
what is the effect on S63del mice Schwann cells if Xbp1 is removed?
the phenotype worsens → it is needed to protect the cells at least partially from the accumulation of mutant protein and from toxicity
the same thing is seen in the R98C cells
upon gene analysis, what was seen when Xbp1 was removed?
in most cases, many genes are upregulated in response to unfolded proteins → in this case those genes are no longer activated suggesting that they may be targets of Xbp1 - it was seen that th over-all dyregulation of genes was much higher
what occurs in Schwann cells when Xbp1 is removed in regards to gene regulation?
everything gets dysregulated - they are unable to deal with the presence of the mutant proteins and the UPR is decreased because all of the genes that target Xbp1 are missing
the response is not activated because the genes necessary are not there
what is ERAD?
ER associated degradation - mechanism in which the cels dislocate protein that need to be degraded
what is seen in regards to ERAD genes in the S63del mice?
genes are increased because ERAD genes need to be increased in order to remove the mutant protein → in the case that the proteasome is inhibited and ERAD is blocked, the S63del protein starts to accumulate suggesting that it is degraded through this pathway whereas the other two proteins are not affected
also shown that the S63del protein is retained in the ER, so it co-localizes P0 with calnexin
in the WT or other mutant the S63C is able to reach the cell membrane
what is seen if one of the genes that are typically increased in the S63del mice (Derlin2) is blocked?
ERAD is blocked and the nerve is completely disrupted
what effect does GlcNac have on the cells?
part of the glycosylation cycle and products - known to increase protein qc mechanism as well as autophagy, so when cells are treated with GlyNac there is an improvement in myelination
what was seen in mice where Xbp1 was over-expressed?
there is an improvement in morphology and there is a much more efficient Er associated degradation system → G ratio decreased = myelin gets thicker and therefore nerve conduction velocity is partially corrected
what is seen in R98C cells if Xbp1 is removed vs if it is increased?
removed: stress markers (BiP, GRP94, P-eIF2) are increased suggesting that cells undergo even more stress
over-expressed: P-eIF2 starts to go back to normal levels since the mutant protein has been removed and the stress is being reduced, then P-eIF2 will be phosphorylated because it is no longer needed
describe the overall role of Xbp1:
a protective role → when increased either genetically or pharmacologically ERAD increases which leads to a reduction in the accumulation of the mutant protein in the ER, reducing stress
what is seen when ATF6 is removed?
similar to what happens with Xbp1 → the nerve conduction velocity decreases and the F-wave increases; BiP which is the target of ATF6 is no longer activated and the phenotype worsens (not as drastically as Xbp1)
how do the activation of both UPR pathways effect CMT1B?
they play a protective role → molecules that are able to activate the protein quality control, the ERAD, or the proteasome could be an alternative / additional treatment to Sephin 1
what happens if Sox2 or Id2 are removed in a normal mouse?
the schwann cells do myelinated more or start to myelinate earlier - these factors keep myelination stopped until birth and then they decrease and myelination starts
what happens when Sox3 or Id2 are removed in S63del mice?
the phenotype gets hugely worse → they think that this happens because if you remove the “brake” myelination starts too early so the axon has no time to become a single myelinated axon but are myelinated together
summary:
in normal Schwann cells P0 goes to myelin - in mutants like S63del cells the protein gets stuck in the ER and the UPR is activated which activates factors like c-Jun or other factors like Sox2 or ID2 → this limits the amount of myelin, so the mutant protein that is being produced, limiting the entry of the mutant protein in the ER and keeping the cell in equilibrium
But if Sox2 or ID2 are removed, the cells will just keep producing P0, and the stress will increase too much for the UPR to handle → there is a certain limit to which it is possible to activate the UPR, so its not possible to fix everything and at that point the response becomes maladaptive and the cell is blocked in its differentiation
at this point the system no longer works and the myelin is not formed and the cell is extremely stressed
