Celastrol Enhances Atoh1 Expression in Inner Ear Stem Cells and Promotes Their Differentiation

that's what it seems like to me. I think we need to figure out how to induce atoh1 expression somehow.

im looking into if anything can in fact go through the eardrum.

 

Celastor Can induce this, as you know. Side effects are not too severe as in LY411575 and injecting thru the eardrum can cause -30dB all range.

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I think ATOH1 is the most important.
"Notch intracellular domain (NICD) is cleaved by gamma secretase (GS) and activates Hes1. Hes1 blocks Muc2 activation via blockage of ATOH1 and the cell retains a progenitor state. In the cell where Notch is inactive, ATOH1 activates MUC2 and the cell adopts a goblet cell fate."

Good article:
Notch Signaling and Atoh1 Expression During Hair Cell Regeneration in the Mouse Utricle
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2902641/

"Using immunohistochemistry, we observed that Jagged1 was located in the supporting cells of the normal utricle and remained in those cells after a lesion. Atoh1 was not detected in the normal tissue but appeared after the trauma. qRTPCR data showed a post trauma decrease for Hes5 and an increase in Atoh1 after the lesion, with no significant change in Notch1, Jagged1 and Hes1.

Our results present a new ototoxic regimen for a complete elimination of utricular hair cells. We document changes in expression level of Atoh1 and Hes genes and no marked change in Jagged1. We find Atoh1 and myosin VIIa-positive cells early after the lesion suggesting that the process of spontaneous regeneration may start several days after the insult. Our data also suggest that Atoh1 up-regulation leading to hair cell regeneration may be induced by more than one signaling pathway."




We have shown that the response of cells in the utricle to an ototoxic trauma involve changes of Notch signaling molecules and Atoh1 expression. Using qRTPCR we detected down-regulation of Hes5, up-regulation of Atoh1 and no changes in levels of Notch1, Jagged1 and Hes1. Jagged1 is expressed in the supporting cells of normal utricle and remains in supporting cells after the lesion. Atoh1 immuno-fluorescence, undetectable in the normal mouse utricle, appears after trauma in nuclei with or without myosin VIIa labeling. Our results suggest that the spontaneous regeneration may arise earlier than previously thought and Atoh1 up-regulation may be initiated by more than one signaling pathway.

Inhibition of Notch signaling gives rise to ectopic hair cells in the developing and damaged organ of Corti (Hori et al., 2007; Yamamoto et al., 2006). In non-mammalian inner ears, inhibition of Notch signaling causes excessive regeneration of hair cells following a lesion, and over-expression of activated Notch in supporting cells inhibits hair cell regeneration (Daudet et al., 2009; Ma et al., 2008). These data raise the possibility that a therapy that would repress Notch signaling may also be applied in the traumatized mammalian vestibular epithelium, in order to enhance hair cell regeneration. Notch inhibition could be accomplished, for instance, by using a γ-secretase inhibitor, or repressing the expression of critical Notch ligands or receptors using specific antagonists.

Virus mediated over-expression of Atoh1 in the damaged mammalian vestibular epithelium is a promising method for enhancing regeneration of hair cells (Staecker et al., 2007). However, non-invasive pathways using small molecules with no side effects may be necessary for clinical applicability. In addition, elimination of Hes5, the repressor of Atoh1, using siRNA at a suitable time point after a lesion may potentially increase expression of Atoh1, which could lead to a larger numbers of new hair cells.

Pathways other than Notch signaling may additionally be involved in regulating proliferation and other aspects of tissue response to trauma in the mammalian vestibular epithelium.

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This is way beyond me. I am technically educated.

 

@JohnAdams look, what i've found. Your curcumin.
Natural compounds targeting major cell signaling pathways
https://jhoonline.biomedcentral.com/articles/10.1186/s13045-016-0373-z


and this: Effects of curcumin on protein expression in the liver of HFD rats



https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5780160/

 

@JohnAdams Look, this is interesting. This is comparition of DAPT treatment (r-secretase inhibitor in the Notch signal pathway) and Atoh1 overexpression can induce supernumerary hair cells.

Effects of DAPT and Atoh1 Overexpression on Hair Cell Production and Hair Bundle Orientation in Cultured Organ of Corti from Neonatal Rats
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3197578/

"Our results demonstrated that DAPT treatment and overexpression of the Atoh1 gene induce the formation of extra hair cells in an additive but not synergistic manner. Furthermore, the increase in the number of OHCs during the treatment depended on their location and culture time, and we observed that DAPT treatment changed the orientation of stereociliary bundles dramatically. However, Atoh1 over-expression did not markedly change the polarization of the stereociliary bundles. These results suggest that Atoh1 and DAPT act differently in the development of the stereocilia bundles on hair cells, even though they both can induce the production of extra hair cells."


Disturbance of Notch signaling pathway may affects the arrangement of hair cells and their hair bundles
Although both Atoh1 overexpression and DAPT treatment were able to induce extra hair cells in cultured Organ of Corti from newborn animals, their effects on the orientations of the hair cells stereocilia bundles were different. In our results, we noticed that DAPT treatment resulted in the loss of the polarity of the stereocilia on most of the hair cells. The arrangement of the stereocilia was drastically changed that their orientation lost its normal pattern, which means that the opening direction of stereocilia bundles on almost all of the hair cells were affected. Our results are in accordance with those of Kiernan et al [27] in that the polarity of the hair bundles was also changed drastically in the Dll1hyp/− Jag2−/− mice cochleae. Doetzlhofer et al [32] have also treated neonatal Organ of Corti with DAPT and showed that the arrangement of hair cells has considerably changed. All these results demonstrated that the disturbance of Notch pathway in the developing Organ of Corti would affect the arrangement of hair cells as well as the polarity of their hair bundles. As it is well known that planar cell polarity (PCP) pathway determined the arrangement of cells and the orientation of hair bundles in mammalian auditory sensory organ [33], we postulate that the normal function of Notch pathway may be necessary for the action of PCP pathway in the development of inner ear sensory epithelia. When the Notch signal was blocked by r-secretase inhibitor, the new hair cells trans-differentiated from supporting cells might contributes to the disarrangement of hair bundles because they also brought up new hair bundles but not in the same row with the original hair cells. When we compared the hair bundle shape in the Atoh1 overexpression groups with those treated by DAPT, we noticed that the orientation of the stereocilia did not change so drastic and were kept relatively more regular, which may be attributed to the less increase of new hair cells inducted by Atoh1 overexpression. We don't know the exact mechanism behind these changes. Further studies on related genes' expression may reveal how these two factors work in arranging the hair cells and their stereociliary bundles."

Look at this fucked up hair cells with g-secretase.




A-no treatment
B- Atoh1 over-expression
C- g-secretase inhibitor in the Notch signal pathway
D- Atoh1 over-expression + g-secretase inhibitor WTF!! I think It wouldn't be so drammatic sound reception. Surly some of us have more disortion that this would be.

ATOH1 expression - very nice.

Do U still want to get LY411575?

 

I took 8 grams of curcumin per day for 3 weeks. no significant permanent reduction of tinnitus. im back on it now. I have hope etc.....

 

I think that Atoh1 is expressed only in Cochlea and exactly in Organ of Corti (there are some exceptions (cancers, etc) and we should induce it expression by some way. To do i we have to neutralise side effects of Celastrol in high doses (hepatoxicity by other herbs for example Silybum marianum but there are others side effects but also other herbs) and eliminate repressors of Atoh1 (its activity is completely antagonized by the negative regulator of neurogenesis HES1) - what is actually curcumin doing and it should change something.


This need deeper investigation.

@JohnAdams look at this. So fucked up.

We describe here four chick hes genes that are expressed during neurogenesis: three hes5-like genes (hes5-1, hes5-2 and hes5-3) and one hes6-like (hes6-2). We show that hes6-2 represses transcription of the hes5 genes, thus functioning as a negative regulator of Notch signaling. Conversely, hes6-2 may be repressed by hes5 activity. In cells committing to differentiation, we find that hes6-2 is up-regulated by proneural genes and contributes to the proneural program of neuronal commitment by preventing Notch activity in these cells. In neural progenitors, Notch signaling produces an initial burst of hes5 activity, which represses hes6-2. However, as hes5 transcription declines due to negative auto-regulation, hes6-2 may become active and inhibit the remaining hes5 activity to end Notch signaling.)

Is there any geneticis here? We need help.

I think this is important too:
Selection of cell fate in the organ of Corti involves the integration of Hes/Hey signaling at the Atoh1 promoter.
https://www.ncbi.nlm.nih.gov/pubmed/26932672

 

How do you know how much Celastrol is in Thunder God Root?

 

I don't know, but it's not a problem.
https://www.alibaba.com/product-detail/99-Celastrol-tripterygium-wilfordii-extract_60330216220.html

I would rather ask how much Celastrol do we need to properly saturate the body with it to start expression of Atoh1, and avoid severe side effects.

To use this homemade method, we need to eliminate other strong repressors of Atoh1, I think.

For example Prox1 - Quantification showed that only 2% of Prox1+OHCs were immunoreactive for Atoh1, while this value was 100% for GFP+OHCs in AdGFP-infected cultures. I am not a geneticist. I don't know what is even this gene function in adult human. I'm rather interested in herbs and many times i saw already their work. And i like to read.....

 

The Notch Inhibitors Isolated from Nerium indicum.
https://www.ncbi.nlm.nih.gov/pubmed/29693393

Notch signaling plays a crucial role in differentiation and cell maintenance, but once aberrantly activated, it contributes to cancer progression. Notch inhibitors were isolated from plant extracts and tested using an originally constructed cell-based assay system. We isolated eight compounds from Nerium indicum that showed inhibition of the Notch signaling pathway. HES1 and HES5 are target genes of the Notch signaling pathway, and oleandrin (1) decreased the protein levels of HES1 and HES5 in assay cells. Oleandrin (1) showed potent cytotoxicity against HPB-ALL cells and decreased HES1 and the Notch intracellular domain in these cells. The main mechanism of action of 1 appears to be inhibition of Notch signaling by acceleration of Notch intracellular domain degradation.
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That plant is somewhat toxic (as everything) f.ex. - overdosing -
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3089829/

But I think it is relatively safe:

"Non-lethal dose of 70% ethanol extract of the Nerium oleander dry leaves (1000 mg/kg body weight) was subcutaneously injected into male and female mice once a week for 9 weeks (total 10 doses). One day after the last injection, final body weight gain (relative percentage to the initial body weight) had a tendency, in both males and females, towards depression suggesting a metabolic insult at other sites than those involved in myocardial function. Multiple exposure of the mice to the specified dose failed to express a significant influence on blood parameters (WBC, RBC, Hb, HCT, PLT) as well as myocardium. On the other hand, a lethal dose (4000 mg/kg body weight) was capable of inducing progressive changes in myocardial electrical activity ending up in cardiac arrest."
https://www.ncbi.nlm.nih.gov/pubmed/12597564​

Dosing: powdered oleander leaves - Pulvis Fol. Oleandri 0.05 g 2-3 times a day (100-150 mg per day). The powdered leaf can be mixed with warm beeswax, form suppositories and administered rectally (50 mg leaf powder per suppository). Intractum Oleandri: 3 times a day for 10-20 drops. Tincture Oleandri 3 times a day for 10-20 drops
http://rozanski.li/591/folium-oleandri-lisc-oleandra/​

 

I think maybe a better candidate for experimentation is egcg.

 

@JohnAdams
I use EGCg for some months. Don't see any effects. It indeed impacts Notch1, Hes1, Hes5 (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4619967/) but I think it is necessary to overexpress Atoh1.

And what about Hey1. There is contradictory research about curcumin and Hey1.

 

just FYI, ATOH is the generic term for this gene. In humans it is specifically called HATH.

 

Co-activation of GATA3 or POU4F3 and ATOH1 promoted conversion of SCs to HCs in adult mice. Activation of POU4F3 alone also converted mature SCs to HCs in vivo.
https://www.ncbi.nlm.nih.gov/m/pubmed/19403287/

These results may lead to the use of berberine to target the induction of specific genes such as GATA-2 and GATA-3
https://www.ncbi.nlm.nih.gov/m/pubmed/19403287/

I use berberine for some months too for lyme. I dont see any effects but who knows how i would feel if i stopped.

Or Math1.

 

In 2018 they discovered celastrol influence on Atoh1. How many herbs and interactions are undiscovered? How many plants are not described?

If I had OAE and ABR 32kHz device I would personally feed some rats with amoglicosydes and then Celastrol and look how it is affecting their sense of hearing.

(I remember Ly411575 intraperitoneal injection induced regeneration of hearing, so not only intratympanic - but it's the case of Notch inhibition, not Atoh1 forced expression, however it's strongly connected, and mutually interacting with each other).

Atoh1 is expressed only in cochlea so if we found a level of body saturation with Celastrol that is inducing Atoh1 expression, the rest would happen automatically, I think.

 

I think that level of Notch1 inhibition can have adverse effects in other body systems. If you read the supplemental information SB2 in this paper it explains it.
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3573859/#SD1

 

yeah, ew.

http://www.jbc.org/content/282/44/32264.long
oral ly411575 made them get tumors, skin lesions, hair loss etc.

 

I think Math1 is mice.

 

I think Celastrol is Notch inhibitor too.

Effects of Celastrol on growth inhibition of U937 leukemia cells through the regulation of the Notch1/NF-kappaB signaling pathway in vitro.
https://www.ncbi.nlm.nih.gov/pubmed/20346213

"Overexpression of Notch1 was found in U937 cells, while Celastrol could downregulate it at both the protein and mRNA level in a dose-dependent manner, and expression of NF-kappaB decreased in nuclei and increased in the cytoplasm"


1-3mg/kg dose is not considered to be rather dangerous, but the results!
1-3mg/kg downregulate NFkB in vivo - so Notch1 also, I think, so maybe it will express some Atoh1 too.

 

@JohnAdams what do you think? Why researchers don't try to apply it intratympanically if they had success in vitro with Atoh1? Even if it won't work orally (but we see it works intraperitoneally in tumors - post above), it costs less than Ly411575 (20USD/kg) and main question... Why nobody is interested in this on this forum, moreover it's funny for some people.

 

Well, they may very well be experimenting right this moment but just havent published their results or they have and the are still in the peer review process. The thing about apathy in this community regarding this type of independant research is beyond explanation for me. I'm just glad you're interested enough to even discuss these things. I really do appreciate your interest. I'd think 100% of us would be participating in these discussions. I've got some thunder god root coming my way. I'm going to use it with egcg. I'm almost positive curcumin is not going to achieve this at all. Even then, what is the maximum potential here? Are we just simply out of luck?

 

"To analyze whether ongoing histone acetylation is required for progressive Atoh1 expression in the developing cochlea, E13.0 Atoh1-GFP cochlear ducts were treated with the histone acetyl-transferase inhibitor (HATi) curcumin (Balasubramanyam et al., 2004). Curcumin (30 µM) was efficient in preventing Atoh1 expression (Fig. S3)."
http://dev.biologists.org/content/142/20/3529

 

This is interesting. How does it look in Celastrol's case?

Response of the flat cochlear epithelium to forced expression of Atoh1.
"We determined that seven days after unilateral elimination of hair cells with neomycin, differentiated supporting cells are absent, replaced by a flat epithelium. Nerve processes were also missing from the auditory epithelium. (...) We then inoculated an adenovirus vector with Atoh1 insert into the scala media of the deafened cochlea. The inoculation resulted in upregulation of Atoh1 in the flat epithelium. However, two months after the inoculation, Atoh1-treated ears did not exhibit clear signs of hair cell regeneration. Combined with previous data on induction of supporting cell to hair cell transdifferentiation by forced expression of Atoh1, these results suggest that the presence of differentiated supporting cells in the organ of Corti is necessary for transdifferentiation to occur."
https://www.ncbi.nlm.nih.gov/pubmed/18430530

 

Yeah I drew this graph a few weeks ago.

 

Also remember this is direct application of these drugs, not oral systemic application.

 

That graph makes it seem like at the .5 dose that atoh1 was enhanced 50% over the control group looks very very dose dependent. Maybe this is in fact the answer. Too bad there is no way to test the concentration of curcumin in the endolymph, but .5micro (molar?) Seems very low. So yes, maybe a small dose of curcumin and celastrol is worth a try.

 

@JohnAdams,

Did you buy God Thunder root already?

 

@JohnAdams if there exists one plant that can simply block Atoh, there have to be a plant that simply expresses Atoh. If there is one, there have to be others.

I think I've got something new. I will post it tonight.

 

Affirmative.

 

Question becomes: will expressing atoh1 cancer us up?

 

Msx1 and Msx2 act as essential activators of Atoh1 expression in the murine spinal cord.
http://dev.biologists.org/content/141/8/1726.long

"These experiments indicate that Msx1 directly binds the Atoh1 3′ enhancer in vivo."

"However, even when weakly temporally initiated by Msx1 and Msx2, Atoh1 would by itself sustain its own expression by binding its enhancer E-box. "

"This further confirms that Msx1 and Msx2 control Atoh1 expression by promoting the activity of its 3′ enhancer."

A Chinese Herbal Decoction, Danggui Buxue Tang, Stimulates Proliferation, Differentiation and Gene Expression of Cultured Osteosarcoma Cells: Genomic Approach to Reveal Specific Gene Activation
https://www.researchgate.net/public...c_Approach_to_Reveal_Specific_Gene_Activation

Table 1: Genes related to bone development are up regulated by DBT

Msh (Drosophila) homeo box homolog 1 MSX1 AA464197 1.66 1.45 1.28
Msh (Drosophila) homeo box homolog 2 MSX2 AA195636 1.86 1.14 0.82

Moreover:
Transforming growth factor, β1TGFβ1 R36467 1.56 1.94 2.00
Transforming growth factor, β2TGFβ2 AA233738 2.60 3.40 1.77

And:
Insulin-like growth factor 1 IGF1 AA456321 2.47 — —
Fibroblast growth factor receptor 1 FGFR1 R54846 1.36 1.17 1.25
Fibroblast growth factor receptor 3 FGFR3 AA419620 1.65 0.97 0.82

Moreover:

Danggui Buxue Tang, a simple Chinese formula containing Astragali Radix and Angelicae Sinensis Radix(...)
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5568261/

"Results
The application of DBT in cultured neuroblastoma cells showed the efficacies in: (1) up-regulation of nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF) and glial cell line-derived neurotrophic factor (GDNF); (2) activation of transcriptional activities of promoters coding for NGF, BDNF, GDNF"

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Astragali radix - the root of Astragalus membranaceus
https://www.magicznyogrod.pl/traganek_nalewka_1_1_50ml.html'


Angelicae Sinensis Radix - the root of Angalicae Sinensis
OTHER NAME(S):
Angelica China, Angelica sinensis,
https://evita.sklep.pl/pl/dong-quai...KwVFqGE0kJSi3E-HxuN9bVdxeuvlxMScaAuhzEALw_wcB