Nahid G. Robertson

The etiology of primary open angle glaucoma, a leading cause of age-related blindness, remains poorly defined, although elevated intraocular pressure (IOP) contributes to the disease progression. To better understand the mechanisms causing elevated IOP from aqueous humor circulation, we pursued proteomic analyses of trabecular meshwork (TM) from glaucoma and age-matched control donors. These analyses demonstrated that Cochlin, a protein associated with deafness disorder DFNA9, is present in glaucomatous but absent in normal TM. Cochlin was also detected in TM from the glaucomatous DBA/2J mouse preceding elevated IOP but found to be absent in three other mouse lines that do not develop elevated IOP. Histochemical analyses revealed co-deposits of Cochlin and mucopolysaccharide in human TM around Schlemm's canal, similar to that observed in the cochlea in DFNA9 deafness. Purified Cochlin was found to aggregate after sheer stress and to induce the aggregation of TM cells in vitro. Age-dependent in vivo increases in Cochlin were observed in glaucomatous TM, concomitant with a decrease in type II collagen, suggesting that Cochlin may disrupt the TM architecture and render components like collagen more susceptible to degradation and collapse. Overall, these observations suggest that Cochlin contributes to elevated IOP in primary open angle glaucoma through altered interactions within the TM extracellular matrix, resulting in cell aggregation, mucopolysaccharide deposition, and significant obstruction of the aqueous humor circulation. The etiology of primary open angle glaucoma, a leading cause of age-related blindness, remains poorly defined, although elevated intraocular pressure (IOP) contributes to the disease progression. To better understand the mechanisms causing elevated IOP from aqueous humor circulation, we pursued proteomic analyses of trabecular meshwork (TM) from glaucoma and age-matched control donors. These analyses demonstrated that Cochlin, a protein associated with deafness disorder DFNA9, is present in glaucomatous but absent in normal TM. Cochlin was also detected in TM from the glaucomatous DBA/2J mouse preceding elevated IOP but found to be absent in three other mouse lines that do not develop elevated IOP. Histochemical analyses revealed co-deposits of Cochlin and mucopolysaccharide in human TM around Schlemm's canal, similar to that observed in the cochlea in DFNA9 deafness. Purified Cochlin was found to aggregate after sheer stress and to induce the aggregation of TM cells in vitro. Age-dependent in vivo increases in Cochlin were observed in glaucomatous TM, concomitant with a decrease in type II collagen, suggesting that Cochlin may disrupt the TM architecture and render components like collagen more susceptible to degradation and collapse. Overall, these observations suggest that Cochlin contributes to elevated IOP in primary open angle glaucoma through altered interactions within the TM extracellular matrix, resulting in cell aggregation, mucopolysaccharide deposition, and significant obstruction of the aqueous humor circulation. Glaucoma encompasses a group of blinding diseases classified generally as primary, for which there is no known etiology, or as secondary, in which a previous illness or injury is contributory. Primary open angle glaucoma (POAG) 1The abbreviations used are: POAG, primary open angle glaucoma; IOP, intraocular pressure; TM, trabecular meshwork; ECM, extracellular abbreviations used are: POAG, primary open angle glaucoma; IOP, intraocular pressure; TM, trabecular meshwork; ECM, extracellular is the of the and in but not glaucoma is to in intraocular pressure (IOP) with to the The of the is but the for increases with and a a of and of in glaucoma for and of to glaucoma disease mechanisms IOP glaucoma as a of aqueous humor humor is the in the of the and through the to the through the trabecular meshwork (TM) Schlemm's and the Glaucoma and to in the TM, which a extracellular of collagen with cells The mechanisms of not the cells in the TM a mucopolysaccharide that to in for of the TM of control of in the TM to disrupt the and in in IOP other in the cochlea associated with the and and disorder DFNA9, which pressure for the disease aqueous or aqueous but do not a is the of for and the of a of TM and of the aqueous through the to the To better understand the mechanisms in glaucoma and in the of aqueous we a to the protein of glaucomatous TM with that of normal TM. we present that Cochlin, a protein associated with the disorder DFNA9, is absent in normal TM but increases with in glaucomatous TM in with from normal and and of were used in and were through the were within of and TM was control were from with no no of glaucoma, and human TM used for were from the of the and were from donors. TM were from in the with of TM, but with of be TM cells for cell were from the associated with used for the and were from human within of and in from and were from The and in the TM were with the and of the from and was in and was for and protein was the of were to and the were used for proteomic analyses or for analyses protein were and in with and were a and a from the and and the and protein a and human Cochlin mouse and to and to were used with analyses to the analyses to Cochlin in were with within of and with was and in in with of and with of for were with and with a to a for was from to a for control and glaucomatous TM were with used for and To and control and glaucomatous were the were with to Cochlin and with a to and a of were and to a of the were from associated with used for were for in and extracellular TM primary cell were in and the aggregation cells were in and for in The cells were with the was and the cells were with of Cochlin or in cells or with of from cells after To the observed cell aggregation, Cochlin was for with of or and the was used to the primary TM cells in with was and cells were for as were with and the II with and a Cochlin and were in cells with and the and The were from the of cells to protein with Purified Cochlin was to sheer stress the protein through a in and and was with from and normal TM were to the were and the were and Overall, were of which were detected in glaucomatous TM but with Cochlin, a protein of was the in glaucomatous TM. Cochlin was detected in of the and analyses in of glaucomatous TM but not in the normal control TM of or these from human TM, we Cochlin in of glaucoma, the DBA/2J mouse IOP of with to the and analyses of TM from DBA/2J and from age-matched or control which do not develop IOP, revealed Cochlin in TM from the DBA/2J of DBA/2J mouse TM the of detected Cochlin of and demonstrated and of in DBA/2J from mouse TM was to with Cochlin in TM from and DBA/2J is with Cochlin is in TM from DBA/2J and a control mouse detected with in TM and in the TM of To the of Cochlin with TM we pursued of Cochlin in the of the human with or analyses of glaucomatous and normal the of Cochlin in glaucomatous but not normal TM and the of Cochlin in glaucomatous TM in and Cochlin was within glaucomatous TM to the of Schlemm's and revealed the of or in the of the Cochlin but not in control Cochlin and the of the glaucomatous around Schlemm's canal, and in but was around the of the The in the cochlea that in DFNA9 also Cochlin and or of Cochlin in human with is is with normal TM from a and glaucomatous TM from a was used for Cochlin and that Schlemm's and and and of is normal TM from a glaucomatous TM from a around Schlemm's and and in of TM Cochlin protein and The is after the which a The with in other in a of and with collagen as and and in to and aggregation of and To a for Cochlin in cell we aggregation human primary TM cells in and human Cochlin of the TM cells was observed with the of Cochlin to the but not with control protein or with the and Cochlin with to to the primary TM cell cell aggregation, with control not not the aggregation of Cochlin sheer stress was also observed suggesting that may be in the aggregation These in observations with extracellular for Cochlin interactions with components as of TM cells Primary TM cell were and as Cochlin in of in of from of Cochlin and of Cochlin and and after of Cochlin and after sheer in the Cochlin and of the of Cochlin in glaucomatous TM and a with of Cochlin and type II collagen were analyses of glaucomatous and normal TM from of in Cochlin was detected in glaucomatous TM, with a concomitant decrease in type II significant with was in the type II collagen of normal TM. These suggest that the of Cochlin may to the of interactions in glaucomatous TM, resulting in the or degradation of the in TM increases and type II collagen in glaucomatous analyses of normal and glaucomatous TM protein for Cochlin, type II collagen, and detected with and of analyses as in with to type II for the of proteomic detected Cochlin in the TM of glaucomatous but not normal human of the was a or a Cochlin was found to be associated with glaucomatous human TM and analyses of and normal TM donors. analyses also demonstrated the of Cochlin in TM from the DBA/2J glaucomatous mouse but not from three other mouse that do not develop elevated IOP. analyses other in human TM the of detected in glaucomatous TM remains to be in normal human TM, of the in the present demonstrated that protein and in not and the of not the of protein of with the be with proteomic analyses were TM the was from normal TM donors. The of the of protein which is the of of in normal TM in to of in of TM but in of normal TM to the present of Cochlin were observed in the of a in normal TM. detected the in normal and glaucomatous TM and to the protein in of the of TM suggesting that Cochlin was absent or in in normal is the of the and is in with the and disorder DFNA9 also in disease of and and in These and in and the of Cochlin the of the of the but is also in in the and of Cochlin within the not The protein is with human Cochlin and with the mouse and Cochlin is a protein with three in human with of and Cochlin and were detected in glaucomatous the we used the Cochlin in the of in the and but the which the that Cochlin a in the architecture of the cochlea to components of the with DFNA9, there is a decrease in and of that the and with POAG, Cochlin in the TM increases with with Cochlin around Schlemm's canal, which interactions with and other may to in TM induce aggregation in and may cell in the type and Cochlin in cells and aggregation we found that sheer stress induce the in aggregation of Cochlin, through interactions to the of Cochlin in glaucoma; may the may in the of glaucomatous TM for a and through with interactions and other components to collagen degradation and the may in collagen degradation in in collagen known to in a of absent in TM, Cochlin in with may disrupt and to collagen degradation in glaucomatous TM. The in Cochlin and decrease in type II collagen that we observed in glaucomatous TM with altered collagen be the Cochlin may to collagen from other TM the more susceptible to and The Cochlin observed in glaucomatous TM aqueous a and the to Cochlin cause IOP or IOP Cochlin and Purified Cochlin to TM primary cells in the aggregation of the the of remains to be is that Cochlin is associated with cell aggregation and deposition, resulting in and significant obstruction of the aqueous is that Cochlin is in the DBA/2J mouse TM after and the of elevated IOP of Overall, the that Cochlin may be in the in glaucoma that to IOP. the of in TM from for for for Cochlin or for Glaucoma encompasses a group of blinding diseases classified generally as primary, for which there is no known etiology, or as secondary, in which a previous illness or injury is contributory. Primary open angle glaucoma (POAG) 1The abbreviations used are: POAG, primary open angle glaucoma; IOP, intraocular pressure; TM, trabecular meshwork; ECM, extracellular abbreviations used are: POAG, primary open angle glaucoma; IOP, intraocular pressure; TM, trabecular meshwork; ECM, extracellular is the of the and in but not glaucoma is to in intraocular pressure (IOP) with to the The of the is but the for increases with and a a of and of in glaucoma for and of to glaucoma disease mechanisms IOP glaucoma as a of aqueous humor humor is the in the of the and through the to the through the trabecular meshwork (TM) Schlemm's and the Glaucoma and to in the TM, which a extracellular of collagen with cells The mechanisms of not the cells in the TM a mucopolysaccharide that to in for of the TM of control of in the TM to disrupt the and in in IOP other in the cochlea associated with the and and disorder DFNA9, which pressure for the disease aqueous or aqueous but do not a is the of for and the of a of TM and of the aqueous through the to the To better understand the mechanisms in glaucoma and in the of aqueous we a to the protein of glaucomatous TM with that of normal TM. we present that Cochlin, a protein associated with the disorder DFNA9, is absent in normal TM but increases with in glaucomatous TM in with from normal and and of were used in and were through the were within of and TM was control were from with no no of glaucoma, and human TM used for were from the of the and were from donors. TM were from in the with of TM, but with of be TM cells for cell were from the associated with used for the and were from human within of and in from and were from The and in the TM were with the and of the from and was in and was for and protein was the of were to and the were used for proteomic analyses or for analyses protein were and in with and were a and a from the and and the and protein a and human Cochlin mouse and to and to were used with analyses to the analyses to Cochlin in were with within of and with was and in in with of and with of for were with and with a to a for was from to a for control and glaucomatous TM were with used for and To and control and glaucomatous were the were with to Cochlin and with a to and a of were and to a of the were from associated with used for were for in and extracellular TM primary cell were in and the aggregation cells were in and for in The cells were with the was and the cells were with of Cochlin or in cells or with of from cells after To the observed cell aggregation, Cochlin was for with of or and the was used to the primary TM cells in with was and cells were for as were with and the II with and a Cochlin and were in cells with and the and The were from the of cells to protein with Purified Cochlin was to sheer stress the protein through a in and and was from normal and and of were used in and were through the were within of and TM was control were from with no no of glaucoma, and human TM used for were from the of the and were from donors. TM were from in the with of TM, but with of be TM cells for cell were from the associated with used for the and were from human within of and in from and were from The and in the TM were with the and of the from and was in and was for and protein was the of were to and the were used for proteomic analyses or for analyses protein were and in with and were a and a from the and and the and protein a and human Cochlin mouse and to and to were used with analyses to the Histochemical analyses to Cochlin in were with within of and with was and in in with of and with of for were with and with a to a for was from to a for control and glaucomatous TM were with used for and To and control and glaucomatous were the were with to Cochlin and with a to and a of were and to a of the were from associated with used for were for in and extracellular TM primary cell were in and the aggregation cells were in and for in The cells were with the was and the cells were with of Cochlin or in cells or with of from cells after To the observed cell aggregation, Cochlin was for with of or and the was used to the primary TM cells in with was and cells were for as were with and the II with and a Cochlin and were in cells with and the and The were from the of cells to protein with Purified Cochlin was to sheer stress the protein through a in and and was with from and normal TM were to the were and the were and Overall, were of which were detected in glaucomatous TM but with Cochlin, a protein of was the in glaucomatous TM. Cochlin was detected in of the and analyses in of glaucomatous TM but not in the normal control TM of or these from human TM, we Cochlin in of glaucoma, the DBA/2J mouse IOP of with to the and analyses of TM from DBA/2J and from age-matched or control which do not develop IOP, revealed Cochlin in TM from the DBA/2J of DBA/2J mouse TM the of detected Cochlin of and demonstrated and of with in TM and in the TM of To the of Cochlin with TM we pursued of Cochlin in the of the human with or analyses of glaucomatous and normal the of Cochlin in glaucomatous but not normal TM and the of Cochlin in glaucomatous TM in and Cochlin was within glaucomatous TM to the of Schlemm's and revealed the of or in the of the Cochlin but not in control Cochlin and the of the glaucomatous around Schlemm's canal, and in but was around the of the The in the cochlea that in DFNA9 also Cochlin and or of Cochlin in human with is is with normal TM from a and glaucomatous TM from a was used for Cochlin and that Schlemm's and and and of is normal TM from a glaucomatous TM from a around Schlemm's and and in of TM Cochlin protein and The is after the which a The with in other in a of and with collagen as and and in to and aggregation of and To a for Cochlin in cell we aggregation human primary TM cells in and human Cochlin of the TM cells was observed with the of Cochlin to the but not with control protein or with the and Cochlin with to to the primary TM cell cell aggregation, with control not not the aggregation of Cochlin sheer stress was also observed suggesting that may be in the aggregation These in observations with extracellular for Cochlin interactions with components as of TM cells Primary TM cell were and as Cochlin in of in of from of Cochlin and of Cochlin and and after of Cochlin and after sheer in the Cochlin and of the of Cochlin in glaucomatous TM and a with of Cochlin and type II collagen were analyses of glaucomatous and normal TM from of in Cochlin was detected in glaucomatous TM, with a concomitant decrease in type II significant with was in the type II collagen of normal TM. These suggest that the of Cochlin may to the of interactions in glaucomatous TM, resulting in the or degradation of the in TM increases and type II collagen in glaucomatous analyses of normal and glaucomatous TM protein for Cochlin, type II collagen, and detected with and of analyses as in with to type II for the of Cochlin with from and normal TM were to the were and the were and Overall, were of which were detected in glaucomatous TM but with Cochlin, a protein of was the in glaucomatous TM. Cochlin was detected in of the and analyses in of glaucomatous TM but not in the normal control TM of or these from human TM, we Cochlin in of glaucoma, the DBA/2J mouse IOP of with to the and analyses of TM from DBA/2J and from age-matched or control which do not develop IOP, revealed Cochlin in TM from the DBA/2J of DBA/2J mouse TM the of detected Cochlin of and demonstrated and of Cochlin with in TM and in the TM of To the of Cochlin with TM we pursued of Cochlin in the of the human with or analyses of glaucomatous and normal the of Cochlin in glaucomatous but not normal TM and the of Cochlin in glaucomatous TM in and Cochlin was within glaucomatous TM to the of Schlemm's and revealed the of or in the of the Cochlin but not in control Cochlin and the of the glaucomatous around Schlemm's canal, and in but was around the of the The in the cochlea that in DFNA9 also Cochlin and or Cochlin in of TM Cochlin protein and The is after the which a The with in other in a of and with collagen as and and in to and aggregation of and To a for Cochlin in cell we aggregation human primary TM cells in and human Cochlin of the TM cells was observed with the of Cochlin to the but not with control protein or with the and Cochlin with to to the primary TM cell cell aggregation, with control not not the aggregation of Cochlin sheer stress was also observed suggesting that may be in the aggregation These in observations with extracellular for Cochlin interactions with components as Age-dependent in the Cochlin and of the of Cochlin in glaucomatous TM and a with of Cochlin and type II collagen were analyses of glaucomatous and normal TM from of in Cochlin was detected in glaucomatous TM, with a concomitant decrease in type II significant with was in the type II collagen of normal TM. These suggest that the of Cochlin may to the of interactions in glaucomatous TM, resulting in the or degradation of the in TM proteomic detected Cochlin in the TM of glaucomatous but not normal human of the was a or a Cochlin was found to be associated with glaucomatous human TM and analyses of and normal TM donors. analyses also demonstrated the of Cochlin in TM from the DBA/2J glaucomatous mouse but not from three other mouse that do not develop elevated IOP. analyses other in human TM the of detected in glaucomatous TM remains to be in normal human TM, of the in the present demonstrated that protein and in not and the of not the of protein of with the be with proteomic analyses were TM the was from normal TM donors. The of the of protein which is the of of in normal TM in to of in of TM but in of normal TM to the present of Cochlin were observed in the of a in normal TM. detected the in normal and glaucomatous TM and to the protein in of the of TM suggesting that Cochlin was absent or in in normal is the of the and is in with the and disorder DFNA9 also in disease of and and in These and in and the of Cochlin the of the of the but is also in in the and of Cochlin within the not The protein is with human Cochlin and with the mouse and Cochlin is a protein with three in human with of and Cochlin and were detected in glaucomatous the we used the Cochlin in the of in the and but the which the that Cochlin a in the architecture of the cochlea to components of the with DFNA9, there is a decrease in and of that the and with POAG, Cochlin in the TM increases with with Cochlin around Schlemm's canal, which interactions with and other may to in TM induce aggregation in and may cell in the type and Cochlin in cells and aggregation we found that sheer stress induce the in aggregation of Cochlin, through interactions to the of Cochlin in glaucoma; may the may in the of glaucomatous TM for a and through with interactions and other components to collagen degradation and the may in collagen degradation in in collagen known to in a of absent in TM, Cochlin in with may disrupt and to collagen degradation in glaucomatous TM. The in Cochlin and decrease in type II collagen that we observed in glaucomatous TM with altered collagen be the Cochlin may to collagen from other TM the more susceptible to and The Cochlin observed in glaucomatous TM aqueous a and the to Cochlin cause IOP or IOP Cochlin and Purified Cochlin to TM primary cells in the aggregation of the the of remains to be is that Cochlin is associated with cell aggregation and deposition, resulting in and significant obstruction of the aqueous is that Cochlin is in the DBA/2J mouse TM after and the of elevated IOP of Overall, the that Cochlin may be in the in glaucoma that to IOP. the of in TM from for for for Cochlin or for proteomic detected Cochlin in the TM of glaucomatous but not normal human of the was a or a Cochlin was found to be associated with glaucomatous human TM and analyses of and normal TM donors. analyses also demonstrated the of Cochlin in TM from the DBA/2J glaucomatous mouse but not from three other mouse that do not develop elevated IOP. analyses other in human TM the of detected in glaucomatous TM remains to be in normal human TM, of the in the present demonstrated that protein and in not and the of not the of protein of with the be with proteomic analyses were TM the was from normal TM donors. The of the of protein which is the of of in normal TM in to of in of TM but in of normal TM to the present of Cochlin were observed in the of a in normal TM. detected the in normal and glaucomatous TM and to the protein in of the of TM suggesting that Cochlin was absent or in in normal TM. Cochlin is the of the and is in with the and disorder DFNA9 also in disease of and and in These and in and the of Cochlin the of the of the but is also in in the and of Cochlin within the not The protein is with human Cochlin and with the mouse and Cochlin is a protein with three in human with of and Cochlin and were detected in glaucomatous the we used the Cochlin in the of in the and but the which the that Cochlin a in the architecture of the cochlea to components of the with DFNA9, there is a decrease in and of that the and with POAG, Cochlin in the TM increases with with Cochlin around Schlemm's canal, which interactions with and other Cochlin may to in TM induce aggregation in and may cell in the type and Cochlin in cells and aggregation we found that sheer stress induce the in aggregation of Cochlin, through interactions to the of Cochlin in glaucoma; may the Cochlin may in the of glaucomatous TM for a and through with interactions and other components to collagen degradation and the may in collagen degradation in in collagen known to in a of absent in TM, Cochlin in with may disrupt and to collagen degradation in glaucomatous TM. The in Cochlin and decrease in type II collagen that we observed in glaucomatous TM with altered collagen be the Cochlin may to collagen from other TM the more susceptible to and The Cochlin observed in glaucomatous TM aqueous a and the to IOP. Cochlin cause IOP or IOP Cochlin and Purified Cochlin to TM primary cells in the aggregation of the the of remains to be is that Cochlin is associated with cell aggregation and deposition, resulting in and significant obstruction of the aqueous is that Cochlin is in the DBA/2J mouse TM after and the of elevated IOP of Overall, the that Cochlin may be in the in glaucoma that to IOP. the of in TM from for for for Cochlin or for and for and the for for and for and and for control

We report the genetic analysis of one large Belgian and two small Dutch families with autosomal dominant non-syndromic progressive sensorineural hearing loss associated with vestibular dysfunction. Linkage studies in the Belgian family mapped the disease to the DFNA9 locus on chromosome 14. Mutation analysis of the COCH gene, which is responsible for DFNA9, revealed a missense mutation changing a highly conserved residue. One of the patients, who had an earlier age of onset in comparison with most of the affected family members, was shown to be homozygous for the mutation. After the mutation was found in the Belgian family, we discovered that the same missense mutation was also present in two Dutch families with similar cochleo-vestibular symptoms. In all three families with hearing loss and imbalance problems, >25% of the patients showed additional symptoms, including episodes of vertigo, tinnitus, aural fullness and hearing loss. Clinically, these symptoms are consistent with the criteria for Menière's disease. The importance of genetic factors in Menière's disease has been suggested on many occasions, but this study is the first report of a mutation in a gene leading to the symptoms of Menière's disease in a significant portion of the carriers. The COCH gene may be one of the genetic factors contributing to Menière's disease and the possibility of a COCH mutation should be considered in patients with Menière's disease symptoms.