Lecture #9

Question 1

what is myelin and what is its function?

Answer 1

a protrusion of the membrane of Schwann cells which wraps around the axon insulating it allowing for faster conduction of nerve impulses

Question 2

which cells of the nervous system are not coated in myelin?

Answer 2

nodes of ranvier

Question 3

where to Schwann cells come from?

Answer 3

neural crest cells

Question 4

what two types of Schwann cells can be differentiated from neural crest cells after birth?

Answer 4

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

Question 5

what occurs for Schwann cells to differentiate?

Answer 5

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

Question 6

under what conditions can Schwann cells de-differentiate and go back to the immature type?

Answer 6

in cases of nerve injury

Question 7

describe the difference between Schwann cells in the CNS vs PNS:

Answer 7

Schwann cells in the PNS have the ability to become de-myelinating and then revert back to myelinatiing cells (plastic) whereas the CNS cannot

Question 8

what is Charcot Marie tooth syndrome?

Answer 8

a genetic disease that arises when you have problems in systems that give rise to myelination

Question 9

describe CMT1:

Answer 9

based on demyelination (bound defects, contraction of tendons, loss of muscle)

Question 10

describe CMT2:

Answer 10

axonal type based electrophysiology and morphology

Question 11

what is the biggest problem faced with CMT?

Answer 11

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

Question 12

what is the most common form of CMT and what does it entail?

Answer 12

CMT1A: there is a loss of finer nd in some axons you can see onion bulbs

Question 13

what are onion bulbs?

Answer 13

protrusions that proliferate and attempt to re-myelinate the axon

Question 14

what is Dejerine-Sottas Syndrome (DSS)?

Answer 14

sever disease in children where the nerve and muscles never develop properly and have a notable presence of large onion bulbsk

Question 15

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?

Answer 15

myelin protein zero (P0)

Question 16

describe myelin protein zero:

Answer 16

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)

Question 17

describe the function of P0 in peripheral myelin:

Answer 17

most abundant protein and forms tetramers which can allow for the packing of myelin

Question 18

which specific amino acid did they decide to focus on and why?

Answer 18

S63 → there was more than one mutation in this aa and different mutations produced different phenotypes

Question 19

what does the deletion of S63 cause?

Answer 19

classical CMT1 de-myelinating disease

Question 20

what does a substitution of the S63 with S63C cause?

Answer 20

DSS - much more severe

Question 21

they generated mice models with both the deletion and the substitution, what type of mutation did they discover?

Answer 21

both mutations gave rise to a different phenotype so both mutations cause a gain of function

Question 22

describe the normal path of a myelin protein:

Answer 22

goes to the ER then to the golgi, and then most likely folded next to the myelin sheet

Question 23

what would theorhetically happen if the protein was not folded as usual?

Answer 23

autophagy is activated or ERAD, but if there is an excess of the protein the UPR is activated

Question 24

what are the three pathways of the UPR?

Answer 24

IRE1, PERK, ATF6

Question 25

describe the IRE1 pathway:

Answer 25

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

Question 26

describe the PERK pathway:

Answer 26

dimerization and autophosphorylation of PERK, which causes the phosphorylation of the the translation initiation factor eIF2⍺ which attenuates protein translation

Question 27

what is another name for the PERK pathway?

Answer 27

integrated stress response → PERK targets eIF2⍺ not only in cases of misfolded proteins but also in viral infections and other challenges to the cell

Question 28

describe the ATF6 pathway:

Answer 28

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

Question 29

describe the adaptivity of the UPR:

Answer 29

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

Question 30

what happens if the UPR goes on for too long?

Answer 30

it becomes maladaptive and causes cell death

Question 31

how does a chronic UPR cause cell death?

Answer 31

through ATF4 there is the activation of another tf called CHOP which leads to the activation of genes involved in cell death, namely GADD34

Question 32

what is another name for GADD34?

Answer 32

PPP1r151

Question 33

what did scientists discover when studying the downstream effects of GADD34?

Answer 33

it reactivates translation in the context of the UPR

Question 34

when GADD34 was ko in mice what was seen?

Answer 34

they ran on the rotor similar to the WT mice and nerve conduction velocity was partially recovered

Question 35

why did they see an improvement in mice when GADD34 was removed?

Answer 35

if translation is not reactivated, there should be less protein in the ER and therefore a reduction to ER stress

Question 36

what does stress derived from the mutant protein result in?

Answer 36

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

Question 37

what was seen in S63del mice models when eIF2⍺ was removed?

Answer 37

there is an increase in demyelinated fibers with a very odd looking cytoplasm and signs of stress

Question 38

in S63del mice it was expected that IRE1 ad ATF6 would be increased when eIF2⍺ was removed - what was seen instead?

Answer 38

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

Question 39

how is c-Jun activated?

Answer 39

activated through p-ERK which is hugely increased in mutant mice without eIF2⍺ → acts through phospho-ERK and phospho-AKT pathways

Question 40

in β-cells what normally happens under stress?

Answer 40

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

Question 41

what happens in Schwann cells after the removal of eIF2⍺ that is different than in β cells?

Answer 41

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

Question 42

what is the function of Guanabenz?

Answer 42

used in disease like hypertention - has a strong affinity for the ⍺2-adrenergic receptor and an inhibitory of Gadd34

Question 43

what was the issue with the mice treated with Guanabenz?

Answer 43

it was too strong - the mice were very sleepy ad unhappy

Question 44

how was Guanabenz modified in order to reduce its strength and what was its new name?

Answer 44

Sephin 1: they removed the part with the ⍺2-adrenergic receptor activity but maintained the Gadd34 inhibition

Question 45

what was seen in mice who were given Sephin 1?

Answer 45

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

Question 46

was Sephin 1 able to enter the BBB?

Answer 46

yes - accumulated in the sciatic nerve

Question 47

what was observed when mice were treated with Sephin1 twice a day for 6 months?

Answer 47

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

Question 48

describe the R98C mutation:

Answer 48

patients with this mutation are classified as DSS since they develop almost no myelin

Question 49

what is seen when R98C patients are treated with Sepin1?

Answer 49

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

Question 50

describe the genetic phenotype of CMT1A:

Answer 50

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

Question 51

what is unique about the mutation in CMT?

Answer 51

if there is a duplication on one allele, then there will be a deletion on another segment

Question 52

what neuropathy is caused when there is a deletion in CMT1A?

Answer 52

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

Question 53

what disease occurs when there is a point mutation on PMP22?

Answer 53

a different neuropathy called CMT1E

Question 54

describe what is seen in both CMT1A and CMT1E:

Answer 54

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

Question 55

is there a difference seen in the healing ability of male and female mice?

Answer 55

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

Question 56

what is G ratio?

Answer 56

measure of myelin thickness → ratio between the diameter of the axon and the diameter of the axon plus myelin

Question 57

what happens to the axons upon treatment to females?

Answer 57

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

Question 58

how does aging effect the nerve health?

Answer 58

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

Question 59

describe the pathway of IRE1:

Answer 59

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

Question 60

what is the role of Xbp1 in a misfolded protein disease?

Answer 60

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

Question 61

what is the effect on Schwann cells if Xbp1 (or ATF6 etc) is removed vs if it removed in WT mice?

Answer 61

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

Question 62

what is the effect on S63del mice Schwann cells if Xbp1 is removed?

Answer 62

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

Question 63

upon gene analysis, what was seen when Xbp1 was removed?

Answer 63

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

Question 64

what occurs in Schwann cells when Xbp1 is removed in regards to gene regulation?

Answer 64

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

Question 65

what is ERAD?

Answer 65

ER associated degradation - mechanism in which the cels dislocate protein that need to be degraded

Question 66

what is seen in regards to ERAD genes in the S63del mice?

Answer 66

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

Question 67

what is seen if one of the genes that are typically increased in the S63del mice (Derlin2) is blocked?

Answer 67

ERAD is blocked and the nerve is completely disrupted

Question 68

what effect does GlcNac have on the cells?

Answer 68

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

Question 69

what was seen in mice where Xbp1 was over-expressed?

Answer 69

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

Question 70

what is seen in R98C cells if Xbp1 is removed vs if it is increased?

Answer 70

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

Question 71

describe the overall role of Xbp1:

Answer 71

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

Question 72

what is seen when ATF6 is removed?

Answer 72

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)

Question 73

how do the activation of both UPR pathways effect CMT1B?

Answer 73

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

Question 74

what happens if Sox2 or Id2 are removed in a normal mouse?

Answer 74

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

Question 75

what happens when Sox3 or Id2 are removed in S63del mice?

Answer 75

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

Question 76

summary:

Answer 76

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