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Mitochondrial origins of deafness?
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Mitochondrial origins of deafness?



Can some forms of deafness be attributed to mitochondrial deficiencies? These are my notes based on literature research

Can some forms of deafness be attributed to mitochondrial deficiencies? These are my notes based on literature research



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    Mitochondrial origins of deafness? Mitochondrial origins of deafness? Document Transcript

    • - Bending of cilium in model kidney cells leads to influx of calcium (Praetorius 2003). What is the route of entry of the calcium ions ? - Proximal tubule kidney cells exhibit primary cilia (Praetorius 2001) - It takes a few seconds, after bending of the cilium, before intracellular calcium concentration starts to increase (Praetorius 2003). Is there a g-protein coupled receptor involved? It is hypothesized by Nauli (2003) that bending of the cilium induces minimal calcium influx, which then later leads to release of intracellular calcium, cicr, Calcium induced calcium release. - Gd3+ completely blocks the influx of calcium (induced by movement of the cilium) (Praetorius 2003). Does this imply that mechano-electrical transduction in these kidney cells is inhibited? Gadolinium is known to block MET and it is known to block some TRP channels. - Single channel recordings from renal primary cilia show that the single channel conductivity is around 80 pS (Raychowdhury 2005) This conductivity is close to what is accepted for the MET single channel conductance. Unfortunately the Raychowdhury paper does not show if the channels in the primary cilium are permeable to Ca2+ . - Apparently when polyaspartic acid is co administered with either gentamicin or amikacin protects against early and late signs of aminoglycoside nephrotoxicity (Mingeot-LeClerq 1999). Does polyaspartic acid also prevent AG induced degeneration of HCs in cultures? - Mitochondrial density apparently is higher in the basal turn of the cochlea and within a HC it is higher in the infra-nuclear region (Fischel-Ghodsian 2004). Has this been demonstrated though? Apparently the source of this information is from a 2003 ARO abstract by Kopke et al.
    • Aminoglycosides can cause ototoxicity to varying degrees depending on the specific AG. What is the ranking order of aminoglycosides with respect to causing ototoxicity. Is this list the same as for nephrotoxicity? Effects of AGs on cultured OHCs: Neomycin>gentamycin>dihydrostreptomycin>amikacin>neamine>spectinomy cin (Kotecha 1994) - Aminoglycosides enter the hair cells through the MET channels (Marcotti 2005) Are there AGs which are too big to enter through the MET channels? Could non reactive chemical groups be attached to existing AGs which will prevent them from entering though the MET channels but will still be effective against bacterial infections? - Aminoglycosides also lead to degeneration of vestibular hair cells (Sha 2005). Has it been shown that aminoglycosides enter the VHCs through the MET channels? - Aminoglycosides lead to destruction of initially OHCs and then IHCs (N.Fischel-Ghodsian 2004) Why this difference? Does this reflect the fact that IHCs have less stereocilia (48) then OHCs (81) ? ( Stauffer 2007) - Aminoglycosides can also induce neuromuscular paralysis, block of calcium channels at the neuromuscular synapse (Sha 2005) What is the mechanism? Is it related to ototoxicity and nephrotoxicity? AGs affect Ca2+ channels, HCs, the neuromuscular junction, kidney cells and mitochondria.
    • - Apparently streptomycin is almost exclusively vestibulotoxic whereas dihydrostreptomycin is almost exclusively cochleotoxic (Sha 2005). Why is this? Because of size differences? Is there a difference in structure of the cochleal HC MET channel and the VHC MET channel? - Hair cell loss due to aminoglycosides starts in the basal coil and progresses apically (Sha 2005). This is not attributable to a concentration gradient because cochlea in culture (i.e. with a homogenous concentration of AGs) display the same behaviour. Therefore the pattern of sensitivity must be based on some inherent properties of the hair cells in different regions of sensory epithelia. Does this reflect a difference in mitochondria (number, size, etc) between basal and apical HCs? Basal OHCs have larger transducer currents than apical OHCs and OHCs have larger transducer currents than IHCs (Marcotti 2005). So if the HC degeneration is dependent upon the entry of AGs through the METs than the difference in conductivity of the METs in basal and apical OHCs can account for the pattern of sensitivity. -Aminoglycosides induce ROS and anti-oxidants can protect against AG induced hearing loss (Sha 2005)
    • Does the formation of ROS occur through the mitochondria? Do AGs target mitochondria which then produce ROS and go into apoptosis? Or do the AGs induce ROS formation which then triggers the mitochondria to go into apoptosis? How about culturing cochleae incubated with AGs and determining the mitochondrial ∆Ψ per day (per hour) ? - It appears that basal OHCs are much more sensitive to ROS than apical ones. Why? - Carriers of the 1555 mutation of mitochondrial ribosomal RNA are more susceptible to AG induced hearing loss. This mutation is present in all mitochondria of a person affected. (Forge 2000) How come only the HCs are affected? Do individuals who carry this mutation also show AG induced nephrotoxicity? - Aminoglycosides are the preferred treatment against bacterial infections in developing countries. Studies from southern China claim that 2/3 of all deaf- mutism in that area was due to administration of aminoglycosides to children. Two populations of patients, suffering from AIDS or cystic fibrosis are also facing the prospect of developing ototoxicity due to aminoglycoside treatment (Forge 2000). - The glycoprotein megalin has been hypothesized to transport aminoglycosides. In OHCs (so far) the presence of megalin has not been determined (Forge 2000). Is megalin present in IHCs and OHCs ? According to Mingeot-Leclercq (1999) megalin IS present in inner ear epithelia. However in the paper referenced by M-L the word megalin doesn’t even occur, i.e. the M-L paper is shit. - Aminoglycosides have a high affinity for polyphosphoinositides. Phosphoinositides are hypothesized to function as electron donors and contribute to ROS formation. Could it be the case that aminoglycosides induce ROS formation, which subsequently triggers mitochondria to induce apoptosis? - Glutathione in vivo, protects against aminoglycosides induced ototoxicity (Forge 2000).
    • Glutathione is cellular antioxidant, does this mean that the level of glutathione in HCs is too low to effectively deal with aminoglycoside induced ROS formation? According to Usami (1996) the levels of GSH in OHCs and IHCs are very low. - It is hypothesized that aminoglycosides can chelate iron. This complex can then subsequently mediate between oxygen and an electron donor (Forge 2000) To which iron atom do the AGs bind? Would they be part of any of the electron transport chain proteins? Also, would incubating with iron chelators prevent hair cell degeneration in cultures? - OHCs subjected to aminoglycosides display apoptosis. It seems to be the case that first row OHCs are more severely affected than the other rows (see Figure 4 in Forge, 2000). Why? Also, if aminoglycosides induce apoptosis, can this be prevented by incubation with CSA? Apparently caspase inhibitors, such as BAF and z-VAD-fmk, can prevent gentamicin induced HC degeneration (Forge and Li 2000). - Spermine is reported to induce release of cyt c by disrupting the mitochondrial OMM. Spermine inhibits PTP and does not lead to swelling (Mather 2001) - It is reported that aminoglycosides stimulate electrogenic uptake of Ca2+ by mitochondria but do not inhibit PTP (Rustenbeck 1998) - The release of SIMP (soluble mitochondrial intermembrane proteins) induced by aminoglycosides is inhibited by CsA to various extents depending on the AG used. CsA highly effective with streptomycin, gentamicin induced SIMP release partially inhibited by CsA and neomycin induced SIMP release only slightly inhibited by CsA (Mather 2001). So, aminoglycosides target mitochondria in different ways, is this reflected in the severity of ototoxicity induced by those aminoglycosides? - Spermine causes damage to OHCs comparable to DHS and amikacin (Kotecha 1994). Does spermine lead to mitochondrial damage in OHCs which subsequently leads to OHC degeneration? Also, does spermine enter the OHCs through the MET? - Neomycin causes more morphological damage basally than apically in cochlear cultures (Kotecha 1994). - Spermine causes HC damage, whereas other cells in the organ of Corti are
    • not affected (Kotecha 1994). Does this mean that spermine specifically targets HCs entering through the MET? - Apparently the death receptor pathway does not play a key role during aminoglycoside induced hair cell death (Cheng 2005). - In contrast (?) to what Cheng reports, Cunningham (2002) reports that treatment with neomycin leads to Caspase 9 activity primarily with only slight Caspase 8 activity. Is one of them incorrect? Or does neomycin specifically lead to Caspase 9 activity? - incubation with z-VAD-fmk (general caspase inhibitor) protects hair cells against neomycin induced damage (Cunningham 2002). - incubation with Caspase 9 inhibitors protects against neomycin induced cell damage. Caspase 8 inhibitors have no protective effect (Cunningham 2002). Also, inhibition of Caspase 9 prevents activation of Caspase 3. Does this show conclusively that aminoglycosides specifically induce apoptosis through the mitochondrial pathway? Does neomycin specifically induce caspase 9? - There are EM studies showing changes in mitochondrial structure in response to aminoglycoside treatment (Cunningham 2002). These are all publications from obscure journals, try to get via interlibrary request or e-mail authors. - Cloramphenicol can be used to specifically inhibit mitochondrial biogenesis. It binds to the 50S subunit of mitochondrial ribosomes and inhibits transcription of proteins encoded by the mitochondrial genome. It was shown that the presence of chloramphenicol led to an increase in degree of HC loss in chick cochleae exposed to gentamicin (Hyde 1995). Does chloramphenicol exacerbate the effects of aminoglycosides on cochleal cultures exposed to aminoglycosides? It would be interesting to culture mouse cochleae and incubate them with various inhibitors of mitochondria for several days and patch HCs during this period. To distinguish long term effects from acute effects. - Congenital thyroid dysfunctions are associated with hearing loss. Upon treatment with thyroid extract patients showed improvement of hearing,
    • it has been shown that thyroid hormone controls mitochondrial function by regulating the production of nuclear- and mitochondrial encoded mitochondrial proteins (Hyde 1995). - Iron deficiency sensitizes animals to acoustic trauma (Hyde 1995). Can this be attributed to mitochondrial dysfunction? Does the iron deficiency affect those respiratory complexes which require iron atoms? - The paper of Ding and Salvi (2005) mentions specifically that AGs enter the mitochondria. Where is the experimental proof for it? - Figures 3A,C&D and 4B&E and 7A&B in the Ding and Salvi (2005) paper show TEM pics of guinea pig OHCs. The mitos look small. The diameter being smaller than 1 micron. Unfortunately it is difficult (if not impossible) to ascertain the age of the animals used as these papers are either in Chinese or never published. - It has been reported that labelling with gentamicin is restricted to the organ of Corti. Other cochlear tissues such as the stria vascularis, Reissner’s membrane and the spiral ganglion did not demonstrate any labeling (de Groot 1990). If AGs would enter the cell via endocytosis how come there is only labeling for gentamicin in HCs and not in the other cell types? Is this issue ever explained in any of the papers that advocate endocytosis? Incidentally, de Groot et al. never claim that AGs enter HCs through endocytosis; they only hypothesize that this might be the case. Also, I don’t find the paper by de Groot very convincing, Ding (2005) is not very critical however. - It has been reported that immature hair cells take up less gentamicin than mature hair cells, also mature cells die faster than immature cells upon exposure to AGs (Dai 2006). Could this be due to the fact that transducer currents in adult animals are larger than in immature ones? No, transduction current amplitude stays constant during maturation of the animal, see Figure 4 (Kennedy 2003). Is the establishment of the endocochlear potential involved? Increased driving force for AGs to go into the HCs?
    • - It has been reported that within the kidney gentamicin targets proximal tubule cells strongly. Labeling of distal tubules and glomerulus is negligible (Dai 2006). In vitro both proximal and distal tubule cells take up AGs. Are there MET channels in proximal tubule cells? Dai et al suggest that the lack of labeling in distal tubule cells in vivo could be due to: -GTTR uptake by proximal cells reduces availability to distal cells -Distal tubule cells are able to rapidly clear GTTR from the cytoplasm. -The electrophysiological conditions in the nephron lumen changes between the proximal and distal sectors and may not favour uptake of GTTR by distal tubule cells. - Aminoglycosides bind strongly to phosphoinositides (PIP2) which are constitutive components of all membranes (Dai 2006). - It is reported that gentamicin uptake (by cultured kidney cells used as model cells for HCs) is not dependent on endocytosis (Myrdal 2005). Is there in fact real experimental evidence which shows that AGs can enter HCs through endocytosis? Homozygous mutant varitint mice (both TRPML3 alleles mutated) do not load FM1-43 or gentamicin. Is there an interaction between TRPML3 channels and TRPV1 receptors? Is the MET channel a supercomplex of various TRP subunits? What about endocytosis of AGs in varitint HCs? - It has been reported that membrane depolarisation of cultured kidney cells reduced the uptake of AGs. Does this mean that entry of AGs into HCs is electrophoretic? Does this also mean that when the endocochlear potential is established the rate of entry of AGs is increased? Can this explain why in vivo HCs in adult animals seem to degenerate at a higher rate than HCs in immature animals? - TRPV4 is expressed in epithelial cells of kidney tubules (Cuajungco 2007). Is TRPV4 involved in mechanotransduction in kidney tubule cells? - It has been reported that in the presence of Gd3+ and La3+ the uptake of gentamicin in kidney cell line cells was decreased (Myrdal 2005). Does this indicate that gentamicin enter these cells through MET channels? Do these cations block TRP channels? Most TRPC channels are inhibited by lanthanides (Gd3+ , La3+ ) however, TRPC4 is activated by micromolar concentrations of extracellular La3+ .
    • Also, TRPC5 is activated by micromolar concentrations of extracellular La3+ or Gd3+ (Pedersen 2005). Find out about Lanthanide sensitivity of other TRP channels. - It has been reported that in HCs TRPML3 shares a similar expression profile with TRPA1 (Pedersen 2005). Could it be the case that the MET channel is formed of a multimer of both TRPML3 and TRPA1 subunits? Perhaps the TRPML3 mutation leads to two effects. It affects the functionality of the MET and it leads to a constitutively open TRP channel in the basolateral membrane. Suppose the MET is a multimer consisting of both TRPML3 and TRPA units (and possibly other components). A mutation in TRPML3 would lead to a complete lack of function of METs in homozygous varitint HCs and it would lead to a decreased population of functional METs in heterozygous varitint HCs. Then how do we explain the limited transduction which we DID see in homozygous varitint HCs? Maybe, a mutated TRPML3 subunit decreases, but does not abolish functionality of the MET channel, i.e. homozygous varitint HCs do have working METs, be it with decreased functionality. The reason we haven’t been able to record IT from the majority of the HCs measured is due to the very poor condition these cells are in. The leak could be explained by having monomeric TRPML3 channels in the basolateral membrane. In homozygotes, all these channels would be constitutively open. In heterozygotes there would be predominantly tetramers of mutated and wild type TRPML3 subunits, with only a small fraction of TRPML3 channels which consist solely of homozygous TRPML3 units. The leak caused by these channels would be undetectable. - It has been recorded that TRPP2 localises to both motile and primary cilia. It is also hypothesized that TRPP2 functions as a mechanosensor in the nonmotile, primary cilia (Pedersen 2005). Could it be the case that mechanosensation in kidney cells is dependent on TRPP2? Is there expression of other TRP channels in kidney tubule cells? - It has been reported that administration of both iron chelators and ROS scavengers lends complete protection against gentamicin induced HC damage (Song 1997). It is also reported that iron chelators protect against GM induced nephrotoxicity. Does this indicate that there are multiple sites and mechanisms of action underlying aminoglycoside induced HC damage? Also, the Song paper mentions that mannitol may scavenge hydroxyl radicals. I didn’t know mannitol was a ROS scavenger? Song et al further hypothesize that gentamicin forms a complex with an iron complex. Get the Priuska paper.
    • - It has been reported that exposure to streptomicin leads to release of cytochrome c in vestibular guinea pig hair cells, although the experimental evidence given isn’t very strong (Nakagawa 1999). - It has been reported that aminoglycoside HC degeneration is concentration dependent, see figure 2 in Cheng 2002. - It has been reported that gentamicin exposure leads to significant activation of caspase-9 which indicates that mitochondria are involved in aminoglycoside induced hair cell degeneration. It was also found that caspase-8 was activated by gentamicin, but anti-body staining did not show any staining for caspase-8 paradoxically enough (Cheng 2002). This means that aminoglycosides induce HC degeneration via both the mitochondrial pathway and the death receptor pathway. - It has been reported that in Va mutants staining for SDH activity was decreased when compared to control mice (Cable 1998). This would imply that the hair cell degeneration seen in varitint waddler mice is to an extent dependent on the mitochondria. As these cells load up with calcium this might trigger apoptosis. Perhaps we should stain varitint HCs for Caspase-9 and determine mitochondrial membrane potential. - It has been reported that the destruction of OHCs by cisplatin occurs in a base-to-apex gradient (Forge 2000). Does this mean that cisplatin enters the HCs through the METs? Guy told me that Schacht and co did experiments on isolated OHCs which did not respond to neomycin. These isolated OHCs do not transduce, hence there is no uptake of AGs in these cells. On the basis of that result it was postulated that AGs form an interaction with an iron complex which generates cytotoxic agents. Find the reference for this, Priuska 1995? It is reported that non HCs in the cochlea are not affected by AGs. Find a reference for this. This is probably due to the fact that these cells do not transduce. Since it is postulated by people who do not believe that AGs enter the HCs through the METs that AGs enter cells via endocytosis, how come non HCs are not affected? Isn’t it a coincidence that the only other cells which are affected by AGs in mammals are kidney cells which, just like HCs, also transduce? - It has been reported that during noise exposure OHCs are more readily damaged than IHCs. It is unlikely that the difference in sensitivity of the HCs for noise is attributable to a difference in conductivity of the METs. So what could be the cause?
    • Is there also a base-to-apex gradient of OHC destruction upon noise exposure? If not this would go against Schacht’s hypothesis. - It has been reported that after glomerular filtration a small but sizable proportion (5%) of aminoglycosides is retained in the epithelial cells lining the S1 and S2 segments of the proximal tubules (Mingeot-LeClercq 1999). - It has been reported that gentamicin forms complexes with mitochondrial Fe2+ (Mingeot-LeClercq 1999). This could mean the aminoglycosides complex with iron complexes on the outside of the IMM, i.e. without entering the matrix. Get: Aminoglycoside ototoxicity: prevention in sight? Schacht J (1998) To confirm whether gentamicin indeed complexes with mitochondrial Fe2+ . The Song paper (1997) is also referenced as a source, however there is no mention of interactions between aminoglycosides and mitochondrial Fe2+ at all. - It has been reported that blocking of caspase-8 does not prevent neomicin induced activation of caspase-3, whereas caspase-9 inhibitors prevented caspase-3 activation and apoptosis (Corbacella 2004). This means that neomycin induced HC death via the mitochondria. Read Cheng and Cunningham papers again. - It has been reported that minocycline protects against gentamicin induced HC loss by preventing MPTP and the release of cyt c.[ref] Based on the work of Mather and Rottenberg this would mean that minocycline would not be effective in protecting against aminoglycosides, such as neomycin as that aminoglycoside does not appear to induced MPTP. Determine if minocycline indeed has been tried with other AGs. - It has been reported that OHCs in culture show a base to apex viability gradient, i.e. basal OHCs die sooner than apical OHCs in the absence of AGs (Sha 2001). What are the inherent differences between basal and apical OHCs (apart from conductivity) that could account for these differences? - It has been reported that glutathione levels in basal OHCs are lower than
    • apical OHCs by Sha (2001) however, the method used for quantifying these levels is unclear. Also, based on the numbers there don’t appear to be any differences. It is reported that there is a significant difference, however, it is not mentioned which statistical test is applied (probably a T-test) and more seriously, it is not specified whether variation is expressed as SD or SEM. When testing for statistical significance we found that with using SD there is a significant difference but with SEM there isn’t. Rather cheeky to omit this information. Is the base – apex pattern of viability as seen in OHCs also present in IHCs? - It has been reported that the OHCs in cochleal cultures incubated with radical scavengers show improved viability. Although the rest of that specific seems rather weak, this result appears genuine (Sha 2001). Do HCs have higher levels of free-radical species than other cells? If so, why? Release of cytochrome c induces activation of caspase 9, see figure 1 in Green (1998). Does ROS formation lead to apoptosis or does apoptosis lead to ROS generation? Or both? The release of cyto c before or in the absence of a drop in DCm in some cells suggests that different regulatory events control permeability of the inner and outer mitochondrial membranes. A rapid opening and closing of the PT pore at its reversible low conductance state may allow a repeated, respiration-driven reestablishment of DCm (39) so that outer membrane disruption and cyto c release can occur before DCm collapse (Green 1998). - It has been found that AGs target mitochondria [ref], but in Hashino (1997) Immunogold labeling shows labeling of lysosomes and not to mitochondria. How come mitochondria were not labeled at all? Perhaps first lysosomes are targeted and then with a time delay the mitochondria? -It has been reported that HEK cells expressing mutated TRPML3 (both A419P and A419P/I362T) label positive for annexin V (an indicator for early apoptosis. Also, these same cells load with Ca2+ (Grimm 2007). This suggests a role for mitochondria. -It has been reported that astrocytes in culture develop mitochondria with strange shapes. The same study showed that in the presence of AGs the
    • occurrence of ‘strange’ mitochondria increased (Robert 2007). I have no idea what this means and whether the work done in fact is any good, but it does show some effect of AGs on mitos, so it may be relevant. -It has been reported that AGs enter HCs via receptor mediated endocytosis (Hashino 1997). Has the identity of these illustrious receptors been confirmed yet? Also, if AGs enter via receptor mediated endocytosis how come cells with mutated myosins do not accumulate AGs? How would myosin molecules be instrumental in receptor mediated endocytosis? Cisplatin is used for chemotherapy. - It has been reported that cisplatin therapy results in loss of high frequency hearing and deafness. Cisplatin primarily damages OHCs. Also, there apparently is cross talk between the two pro-caspase pathways, i.e. activation of caspase-8 can lead to activation of caspase-9. Cisplatin leads to caspase-8 activity before caspase-9 activity (Devarajan 2002). Does this mean that cisplatin induced HC degeneration starts in the basal coil? What is the effect (if any) of cisplatin on IHCs? In the Devarajan paper it is reported that PMT was determined, however, this was done on the basis of measuring mitochondrial membrane potential. So, how does this specifically implicate PMT? Do varitint mutant HCs show morphological signs of apoptosis? - It has been found in proximal tubule cells that gentamicin localises to both mitochondria and lysosomes. Initially AGs accumulate in the brush border (Beauchamp 1991). Does the brush border contain the kidney epithelial cells containing the mechano electrical transducing cilia? - Apparently, TRPML and TRPP channels are similar, much more so than any of the other TRP channels (Venkatachalam 2006). Could this indicate that kidney diseases are related to ototoxicity through dysfunction of TRP channels? - Apparently one of the hallmark features of programmed cell death (PCD) is membrane blebbing (Matsui 2002). Does exposure to AGs to HCs lead to blebbing? And more specifically, do varitint HCs show blebbing?
    • Richardson (1997) shows blebbing as a response to AG exposure. What about varitints though? - It was found by Matsui (2002) that upon exposure to neomycin chick utricle HCs went into apoptosis, the mitochondria however appeared to be intact. This was on the basis of morphology, it would be interesting to determine the membrane potential of these mitos. - Apparently adult mice, rats and gerbils show little, if any, drug induced Toxicity under conditions that would produce severe auditory and vestibular deficits in guinea pigs or chinchillas. Apparently, adult mice are not susceptible to aminoglycoside induced ototoxicity. In cochlear explants however there appear to be no differences between adult mouse, gerbil and guinea pig OHCs with respect to their response to AGs (Wu 2001). - 2,3-Dihydrobenzoate (an antioxidant) protects against kanamycin induced ototoxicity (Wu 2001). - It has been reported that the end of the ‘sensitive period’ for AGs in the young rat coincides with the maturation of glutathione-S-transferases, enzymes which use glutathione as a substrate in drug detoxification (Wu 2001). Can glutathione protect against AG induced HC degeneration in cultures? - It has been reported that the order of resistance to kanamycin correlates with the pigmentation of mouse strain used. It his hypothesized that this is due to enhanced antioxidant capability of melanin-containing cochlea (Wu 2001). Would homozygous varitint mice benefit from antioxidants? If apocytochrome c is encoded by the nucleus it has to be transported across the OMM. How does that work? And is this transport process involved in the release of holocytochrome c ? - It has been reported that caspase-8, apart from directly stimulating downstream caspases can also induce release of cytochrome c from mitochondria. Apparently caspase-8 interacts with mitochondria indirectly using Bid as an intermediate (Luo 1998 and Li 1998). Does this mean that mitochondria are always involved in apoptosis? - It has been reported that AGs enter HCs via receptor mediated endocytosis on the basis of colocalisation of immunogold labeled KM and ferritin (Hashino 1995). It has also been reported for cultured kidney cells that AGs enter via endocytosis (Ford 1994).
    • Is the presence of ferritin a good measure for determining that? I have the impression that the Hashino 1995 paper isn’t all that good. - It is reported that AGs are nonmetabolizable (Ford 1994). Is this in fact true? Find out about AG metabolism. - It has been reported that Bid (and tBid) can induce release of cytochrome c from mitochondria in the presence of CsA, i.e. PTP is not involved in this process Zhai 2000). - Minocycline inhibits cytochrome c release, it crosses the BBB and is a non- toxic drug (Zhu 2002). Would minocycline be able to protect against AG hair cell degeneration in HC cultures? Given the non-toxicity of this drug, it may be a candidate drug to be co-administered with AGs. - It has been reported in cultured kidney cells that the presence of gentamicin leads to reduction of mitochondrial membrane potential. In the same study it is shown that AGs can be trafficked via retrograde transport through both the golgi complex and ER to subsequently be released into the cytosol and interact with other organelles, such as mitochondria (Sandoval 2004). Would it be safe to assume that AGs enter cells by both endocytosis and MET channels? Do non transducing HCs (such as Myo 7A and varitints) not load with AGs at all? What if you leave the cultures for 4 to 6 hours? - It has been found that TRPP channels in kidney cells (PKD1 and PKD2) flux calcium upon stimulation with fluid flow, suggesting the presence of mechano electrical transduction (Lin 2005). - It has been found that HEK cells expressing TRPV1 channels readily take up FM1-43 when these channels are opened. Ruthenium red , a TRPV1 antagonist, blocked the uptake of FM1-43 into the HEK cells (Meyers 2003). It is well known that FM1-43 can enter HCs through the MET. The results obtained with HEK cells suggest that the MET might be a TRP channel. Also, does Ruthenium red block HC mechanotransduction? How does apo cytochrome c cross the OMM? - Apparently FM1-43 labels ER and mitochondria (Meyers 2003). - It was found that FM1-43 enters various cells (Merkel cells, neurites innervating Merkel cells, nociceptors and enteric neurons, in various structures (hair follicles, skeletal muscles, cornea), all of these structures have mechano-electrical transduction properties (Meyers 2003).
    • What is the effect of aminoglycosides on these structures? Does FM1-43 enter kidney cells? - Meyers (2003) suggest that in mature mice FM1-43 loses access to the endolymph. This might explain why mature mice, in vivo, are not affected (that much) by aminoglycosides. - HCs from homozygous Myo7ash1 accumulate high levels of gentamicin, whereas HCs from homozygous Myo7a6J do not accumulate gentamicin (Richardson 1997). Are there any differences in mechanoelectrical transduction between these two types? Apparently, the shaker1 mutants have relatively normal transduction, including a resting transducer current, which is absent in the myo7a6j type. There are some nice pictures of mitos in OHCs in the Richardson (1997) paper, see Figure 1. Probably AGs can enter cells in general via endocytosis. However, in cells which display mechanotransduction there is an extra port of entry which allows for flooding of the cell with AGs in a relatively short time. Short term effect of AGs apoptosis and long term effects, both apoptosis and necrosis in HCs? Do AGs lead to ROS formation directly? Do AGs generate ROS via the mitochondria? Can ROS stimulate caspase-8 and caspase-9? Do AGs directly stimulate caspase-8 and caspase-9? - Combining KM with ethacrynic acid leads to massive apoptosis (Ding 2005). Do KM and EA work synergistically? What does EA do ? EA generates free radicals (Ding 2005). - According to Ding (2005) AGs generate ROS/ How? - According to Ding (2005) inhibiting calcium activated proteases prevents AG induced HC loss. How about calcium release by damaged mitochondria (induced by AGs) ?
    • - Hair cells take up FM1-43 but surrounding cells show little or no labeling. (Gale 2001). This indicates that HCs have uptake mechanisms that other cells are lacking, i.e. MET channels serve as routes of entry many large polycations. - FM1-43 labeling is much stronger in the basal coil than in the apical coil (Gale 2001). How does that compare with AG loading? - It can be seen that loading of FM1-43 in control HCs is immediate whereas in homozygous Myo7a6j HCs no immediate loading is seen (Gale 2001). Is there any loading if you let the culture incubate with FM1-43 (or AGs) for several hours? - FM1-43 competes with AGs for entry into the HCs (Gale 2001). Can FM1-43 prevent entry of AGs into kidney cells? - OHCs exposed to neomycin (1 hour) show blebbing. Whereas OHCs pre- treated with calcium chelators are protected against AGs (Gale 2001). The blebbing suggests that these cells go into apoptosis. The exposure to neomycin was 1 hour, during that interval, when did the blebbing start? Also, if there is any endocytosis of AGs then this process takes longer than 1 hour since the OHCs pre-treated with calcium chelators did not show any blebbing. It could of course be the case that AGs were in fact getting into the hair cells through endocytosis. Again it would be useful to know when exactly the blebbing started in the experiment with just neomycin present. Suppose AGs do enter HCs also via endocytosis, how slow/fast is this process and how long does it take to reach a steady state AG concentration within the HC? - Continued exposure to FM1-43 leads to some blebbing of OHCs (Gale 2001). Does this tie in with the fact that FM1-43 also targets mitochondria? See Meyers (2003). Temperature dependence of FM1-43 or AG entry into HCs may be due to temperature dependence of the opening of MET channels which may be dependent on myosin ATPase
    • - EM pictures of rat and guinea pig vestibular HCs show clearly that mitochondria are very small, less than a micron along the long axis, look at figure 2 in (Vautrin 2006). - Apparently ocsyn and mitochondria co-localise in vestibular HCs (Vautrin 2006). To me the fluorescence images look oversaturated, not sure about the quality of this work. What is the connection between lysosomes and AGs? Do AGs get taken up by them? The Ding paper shows in figure 4B that tritium labeled neomycin co-localises with mitochondria, the text claims that AG labeling is IN the mitochondria. - Exogenous SOD protects against AG induced HC degeneration, however, exogenous SOD also elicits an immune response (Ding 2005). - AG induced influx of calcium is hypothesized to occur through L-type calcium channels (Ding 2005). Does Calcium influx induce PMT in mitochondria? Could it be that AGs induce apoptosis via parallel mechanisms? Both calcium influx and ROS formation? - TRP channels interact with PDZ-domain containing scaffold proteins (Pedersen 2005). This suggests a role for TRP channels in the process of mechanotransduction since PDZ-domain proteins are known to be important components as part of the adaptation-motor complex. - In oocytes TRPC1 was identified as the mechanosensitive cation channel, which transduces membrane stretch into cation currents (Pedersen 2005). This suggests a role for TRP channels in mechanotransduction. Whereas most TRPC channels are inhibited by lanthanides (which suggests a role for TRPC channels in mechanotransduction) TRPC4 and TRPC5 is activated by micromolar concentrations of extracellular La3+ (Pedersen 2005). - One of the physiological functions of TRPV4 is thought to be mechano- sensing (including endothelial cell responses to shear stress) (Pedersen 2005). This again may imply that a TRP channel is involved in mechanotransduction.
    • - TRPN1 is an important mechano-transduction channel in C. elegans, Drosophila and zebra fish, but no detected in mammals (Pedersen 2005). - IHC mitochondria appear to be small, maximum length, along the long axis being about 1 micron, see figures 1-3 in Spicer (2007). - It has been shown, in both IHCs and OHCs, that mitochondria make contact with cisternae. It is assumed that these cisternae have ATPases. I don’t see the relevance at all, but maybe AGs find their way into these cisternae and can target mitochondria more directly? - It has been found that AGs can inhibit TRPV1 in the order: Neomycin ≅ Streptomycin > Gentamicin (Raisinghani 2005) How does this compare with the potency of AGs inducing HC damage? Effects of AGs on cultured OHCs: Neomycin>gentamycin>dihydrostreptomycin>amikacin>neamine>spectinomy cin (Kotecha 1994) - TRPV1 regulators mediate gentamicin penetration of cultured kidney cells (Myrdal 2005). If AG’s inhibit TRPV1 channels and if TRPV1 regulators mediate the entry of gentamicin into kidney cells could it then be that the MET channel is a TRPV1 channel? Are there specific TRPV1 inhibitors? It is worth trying to measure transduction of hair cells in the presence of these inhibitors. How does capsaicin influence mechanoelectrical transduction? Corey doesn’t think the MET channel is a TRPV channel but he doesn’t comment on TRPV1 at all. It is know that TRPV4 can be found in HCs, what about TRPV1 though? - It has been reported that TRPV1 is present in OHCs (guinea pig) (Zheng 2005). The evidence given in the Zheng paper is mostly indirect. The only immuno assay shown is not all that convincing. - Formation of heteromeric TRP channels has been observed. The only heteromer, so far, which combines TRP subunits from different subfamilies is a combination of PKD2/TRPC1 (Schaefer 2005). Is there any evidence that TRPML3 subunits may combine with TRPA or TRPV subunits?
    • It has been demonstrated that TRPML subunits can form heteromers of all combinations of TRPML1, TRPML2 and TRPML3 (Venkatachalam 2006). - Both TRPN1 and TRPA1 contain many ankyrin repeats in their N-termini. It has been hypothesized that these ankyrin repeats could function as a gating spring (Christensen 2007). - Ruthenium red blocks TRP channels and also of mitochondrial calcium inlfux (Christensen 2007). Is there a relationship between TRP channels and mitochondria? - In the kidney nephron the following TRP channels have been found: TRPC1, TRPC3, TRPC6, TRPP2, TRPV4, TRPV5, TRPV6 and TRPM6. (Hsu 2007). - PKD1-like subgroup of the TRPP family is somewhat unusual because it has 11 TM domains. It is considered a TRP channel because it assembles with PKD2-like proteins to form functional complexes (Hsu 2007). That sounds completely wrong to me. - TRPV4 is highly expressed in kidney cells (Hsu 2007). - It has been reported that noise exposure induces Hensen’s bodies. Hensen’s bodies are circular formations composed of ER and encased by Mitochondria (Fredelius 2001). Are Hensen’s bodies specific for HCs? Hensen's bodies - assemblies of vesicles in upper region of cochlear outer hair cells and related to ion transport - It has been reported that noise exposure leads to a decrease in SDH s staining density in IHCs and OHCs (Canlon 2001). When incubated with FM1-43, if left to incubate for a long time, it does enter the Myo7a6j OHCs, i.e. endocytosis does occur, very slow (unpublished results Kros group). - It has been reported that FM1-43 staining distributes homogeneously within the IHCs whereas in the OHCs the staining appears to coincide with Hensen’s bodies (Meyer 2001). Experiments in this paper were done on isolated OHCs which do not transduce.
    • - OHC loss far exceeds ICH loss when the cochlea is exposed to AGs (Ding 2005). Can this be explained by the fact that OHCs have more MET channels and hence load up with AGs more quickly? Could it be the case that OHC Hensen bodies, which are hypothesized to be in contact with mitochondria, accumulate AGs? In this way AGs would very effectively target mitos in OHCs. - It has been reported that OHCs contain an apical network of so called ‘canalicular reticulum’. Apparently, similar structures have been found in kidney proximal tubule cells (Spicer 1998). - EM micrographs from OHCs show that they are loaded with mitochondria, See the figures in Spicer (1998). It is also very clear that mitochondria in OHCs are not homogeneously distributed. See Figure 2 where mitochondria are lined up along the lateral wall. Do OHCs (and IHCs) have more mitochondria than other cell types? - It has been reported that mitochondria from HCs are not distinguishable from liver mitochondria (Spector 1974) That study however compared labeling for dehydrogenase activities in isolated cochleae with labeling of isolated liver mitochondria. I don’t know if you can actually conclude from the results in the Spector paper that HC mitochondria are similar to liver mitochondria. The proper way of comparing would be to isolate mitochondria from HCs directly and biochemically characterise them. - Signals such as Ca2+ , nitric oxide and ROS may induce release of proapoptotic proteins from mitochondria independent of proapoptotic Bcl-2 proteins (Mather 2001). - It has been reported that lysosomes are the first site of accumulation of AGs in kidney proximal tubules, in vivo as well as in cultured cells (Servais 2005). - Depending on concentration of AG present cells go into apoptosis or Necrosis. It was found that cultured kidney cells went into apoptosis when incubated with gentamicin at concentrations up to 3 mM whereas at higher concentrations the cells started showing signs of necrosis (Servais 2005). - In LLC-PK1 cells (kidney cell line) incubation with gentamicin leads to the following chain of events: after 2h gentamicin appears to be released from lysosomes, after 10h loss of mitochondrial membrane potential, after 12h release of cytochrome c and activation of caspase-9, after 16 to 24h later caspase-3 activity and appearance of fragmented nuclei (Servais 2005).
    • - TRPV4 is found in hair cells (Corey 2006) Do TRPML channels associate with PDZ proteins? - TRPA channels are permeable to FM1-43 (Lin 2005). Are TRPA channels found in hair cells? - All of the epithelia of the mammalian kidney (except for the intercalated cells of the collecting duct) express a single primary cilium on their apical (lumenal) surface (Praetorius 2001). So not just proximal tubule cells then? - Bending of the cilium of MDCK cells in the presence of Gd3+ does not lead to Ca2+ influx (Praetorius 2001). - In the vestibular organ AGs affect type I hair cells before type II hair cells (Forge 2000). Do type 1 and type 2 hair cells have different MET conductances? - It has been reported that aminoglycosides stimulate calcium uptake in Mitochondria (Rustenbeck 1998). - Streptomycin charge = +3 (maximum charges) neomycin charge = +6 tobramycin charge = +5 gentamicin charge = +5 At pH = 7 Tobramycin charge = + 3.39 ± 0.04 Neomycin charge = + 4.09 ± 0.24 (Rustenbeck 1998) - aminoglycosides lead to decreased velocity of calcium uptake in mitochondria but an increased accumulation of calcium (Rustenbeck 1998). - Oxygen consumption rate under state 4 conditions in liver mitochondria was inhibited to 49.1 ± 4.7 % of control the rate (9.2 ± 0.4 nmol O2 min-1 * mg of protein-1 ) by 250 µM gentamicin (Rustenbeck 1998). - Apparently spermine and aminoglycosides can compete with each other for binding sites on mitochondria (Rustenbeck 1998). - polyamines affect calcium transport into mitochondria (Salvi 2004)
    • Does that mean polyamines interact with the Ca2+ uniporter? Has the effect of aminoglycosides been investigated properly? (aside from the Rustenbeck work?) Calcium influx can induce MPT, so perhaps aminoglycosides can induce MPT via calcium influx? - Calcium influx into mitochondria is electrophoretic and dependent on the mitochondrial membrane potential (Salvi 2004). - Ruthenium red is a polycation and inhibits the Ca2+ uniporter (Salvi 2004) - Apparently there are binding sites for polycations on the IMM (Salvi 2004) - Somehow Dehne et al. conclude that aminoglycosides induce ROS formation which leads to MPT which induces apoptosis (Dehne 2002). Although this is an interesting scenario I don’t see how it can be concluded on the basis of the data in their paper. - It was shown that hair cells have lower concentrations of glutathione than neighbouring cells (Usami 1996) How good is their research? - Apparently gentamicin stimulates state 4 respiration and inhibits state 3 and uncoupled respiration in renal cortical mitochondria (Weinberg 1980). This is different from what was reported by Rustenbeck - It is suggested that gentamicin interacts with mitochondria at the IMM (Weinberg 1980) - It has been reported that dehydrogenase activity is greater in IHCs than in OHCs by Vosteen (get ref) whereas it was found by Koide et al (get ref) that OHCs were either equally or more metabolically active than IHCs. According to Ding (1997) SDH activities in IHCs and OHCs are comparable. There is clearly confusion about HC metabolism and also, some of these reports are very old, using outdated techniques by current standards. So HC metabolism should be investigated again. - it has been reported that aminoglycosides antagonise the effects of natural polyamines like spermine. Spermine inhibits PM. Perhaps aminoglycosides compete with spermine for binding sites. Also it was found that aminoglycosides induced a depolarization of the IMM (Rustenbeck 1998). - It has been reported that gentamicin stimulates respiration and reduction of cytochrome c (Sha 1998)
    • I’m not sure about the methods, but if the findings are true, they suggest that gentamicin might uncouple mitochondria (given the increased vO2). Or because of increased cytochrome c reduction there is more O2 reduction. Cytochrome P450 is found in the IMM - It has been reported that gentamicin is metabolised into some cytotoxin in the cytosol of liver cells. Incubation of gentamicin with other fractions, such as the mitochondria do not metabolise gentamicin (Crann 1992) - The mitochondrial mutation A3243G is associated with both hearing loss and diabetes mellitus (Fischel-Ghodsian) - The A1555G mutation on its own can lead to deafness (Fischel-Ghodsian) It would be interesting to study HCs from a mouse with a mitochondrial mutation Johnson et al. (2001) are the first to report on a mouse model in which a mitochondrial DNA mutation affecting a clinical phenotype. - Bax can translocate from the cytoplasm to the mitochondria and promote the formation of pores in the mitochondrial membrane (Cheng 2005). What is the nature of the interaction of other Bcl-2 members with mitochondria? - Bcl-2 proteins act upstream of caspase activation (Cheng 2005). How do AGs (if they do) stimulate Bcl-2 protein activity? Does Caspase-9 induce release of cytochrome c or not? - Activated nuclear p53 can directly translocate to and damage mitochondria (Cheng 2005). This is a separate apoptosis pathway from the caspase pathway? - It has been shown that mitochondria-associated oxidants are involved in pathways regulating cytochrome c translocation and caspase activation (Cheng 2005). - Phosphorylation of JNK and c-jun have been demonstrated in inner ear hair cells when treated with neomycin and cisplatin (Cheng 2005). What is the role of JNK and c-jun in apoptosis? - a technique has been established where mitochondria in situ can be imaged
    • using label-free optical imaging (Lasne 2007) Perhaps we could try this approach with Mischa’s setup? - It has been reported that incubation with CsA partially protects against gentamicin induced HC damage (Dehne 2002). - It has been reported that aminoglycosides lead to reduced mitochondrial respiration in the inner ear and in the kidney (Dehne 2002). This suggests that aminoglycosides affect mitochondria. Would respiration in isolated cochleal cells be affected by aminoglycosides? Compare with liver cells? - It has been reported that with age mitochondrial respiration decreases (van Remmen 2001). - Hair cells mutant for the inositol lipid phosphatase Ptprq were found to be hypersensitive to aminoglycoside exposure (Jane Bryant thesis). Why? What does Ptprq do? How do increased PIP2 levels lead to increased aminoglycoside sensitivity? More binding of AGs? - In chick basilar papillae aminoglycoside induced HC damage spreads from proximal to distal, i.e. it starts with the high frequency range HCs similar to what happens in the mammalian cochlea (Bryant thesis). Do proximal HCs have larger transducer currents than distal HCs? - Apparently, chick embryonic basilar papillae demonstrate a pronounced insensitivity to neomycin (Bryant thesis). Lack of transduction? However, FM1-43 does load at the same ages. - It was found that serum from individuals susceptible to AG induced hearing loss can be cytotoxic to isolated OHCs. It is hypothesized that these individuals can metabolise AGs into a cytotoxic compound (Guthrie 2008). - According to Dulon (1989) isolated OHCs (by means of trituration) are not affected by the presence of 5 mM gentamicin when incubated for 6 hours. When you look at the paper by Kotecha (1994) it is very clear that in cochleal cultures in the presence of 1 mM gentamicin for 1 hour the OHCs are severely damaged. This suggests that transduction is negatively affected in the isolated OHCs in Dulon’s study.
    • Is there a correlation between ototoxicity of various aminoglycosides and their effects on mitochondria? i.e. are mitochondria more severely affected by strong ototoxic AGs (such as neomycin) than weaker ones, such as spectinomycin? - Ethacrynic acid can deplete mitochondrial and cytosolic GSH (Clerici 1996) - Apparently regeneration of mammalian vestibular hair cells has been reported (Schacht 2000). - Apparently OHCs are affected by aminoglycosides in a particular order, first the first row then the second row then the third row OHCs (Schacht 2000). - Polyphosphoinositides have a high binding affinity for aminoglycosides (Schacht 2000). - Gentamicin gets taken up quicker by OHCs in the presence of background noise as opposed to in animals maintained under noise-attenuated conditions (Schacht 2000). Channels were open because of the noise? - It was found that gentamicin enhances hydrogen peroxide production in a dose dependent fashion in isolated kidney and liver mitochondria (Walker 1987). - Heat shocked utricle hair cells (utricles kept at 43 degrees Celsius for 30 min) inhibits neomycin induced hair cell death (Taleb 2008). The results in the paper are not….shocking. - Individuals with the 1555 mutation in mitochondrial ribosomal RNA are extremely sensitive to aminoglycosides. A single injection may induce deafness. Interestingly enough the vestibular system in these people seems not to be affected (Sinswat 2000). - Iron chelators deferoxamine and DBH reduce gentamicin induced ototoxicity and damage to the kidney (Song 1996). Suggesting a common mechanism? - According to Gale et al. within the apical coil there is a gradient of FM1-43 loading during development of cochlea in culture. What aspect of development influences the uptake of FM1-43? (Gale 2001) - The rate of FM1-43 loading in BC HCs is much faster than in AC HCs. FM1-43 loading is inhibited by incubation with EGTA, which breaks tip-links.
    • Myo7a mutant hair cells do not load with FM1-43, unless stimulated mechanically . (Gale 2001) - A correlation is found between aminoglycoside ototoxicity and inhibition of protein synthesis of mitochondrial ribosomes (Hobbie 2008). - It has been reported that aminoglycosides distribute to different regions of the inner ear, on the basis of their molecular structure (Perletti 2008). How is this possible? It would be very interesting to see the effects of a range of AGs on vestibular cultures and compare them to the results obtained by the Kotecha 1994 study. - It has been reported that concanavalin A prevents uptake of gentamicin in rat cochlear explants (Zheng 1999). Guy doesn’t believe this study was very good. - Aminoglycoside induced hair cell death has been characterized as apoptotic (Cunningham 2002) - It has been reported that exposure to aminoglycosides leads to ultrastructural changes in hair cell mitochondria (Cunningham 2002). - Exposure to gentamicin in vitro leads to increased levels of free intracellular calcium in hair cells (Hirose 1999). Where does this calcium come from? Nifedipine (inhibitor of low voltage-gated calcium channels, L-type) inhibits the increase of intracellular calcium induced by gentamicin (Hirose 1999). This suggests the calcium has an extracellular origin. Does the calcium influx lead to apoptosis? - It has been shown in cultured avian sensory epithelia that exposure to gentamicin induces increased levels of ROS (Hirose 1999). - It has been reported that streptomycin and gentamicin are primarily vestibulotoxic whereas amikacin, neomycin, dihydrostreptomycin and kanamicin are primarily cochleotoxic. Also, it was reported that gentamicin has less ototoxic and vestibulotoxic
    • effects in newborns than in older children or adults. It is reported that gentamicin does not cause apoptosis via the death receptor pathway. Kanamicin however seems to involve the death receptor pathway (Selimoglu 2007). This suggests that different aminoglycosides induce different apoptotic pathways. - It is reported that megalin is expressed in cochleal sensory cells and in kidney proximal tubule cells (Selimoglu). Is this true? Check references. Could this be some connecting mechanism which accounts for ototoxicity and nephrotoxicity?