Home ❯ Research ❯ Funded Studies
Current Grant
09/23 – 08/24
In vivo treatment of MMex38 mice with 4-PBA: We plan to conclude our longitudinal drug study to determine if the clinically approved drug sodium 4-phenybutryic acid (4-PBA) is can ameliorate the dystrophic condition caused by the DYSF loss-of-function missense mutation L1360P in MMex38 animals. All animals (32 in total, 16 male, 16 female, age matched in equal numbers on drug or water control) in this study, will be trained in an Inclined Balance Beam (IBB) assay and periodically assessed for their ability to traverse beams of various width sizes (24mm, 12mm, 10mm, 8mm). We plan to assay the animals monthly from 12 to 14 months of age comparing MMex38 animals given 4-PBA ad libitum in their drinking water to untreated controls. Thereafter, as animals complete IBB assays, hind limb muscle groups, including Quadriceps (Q), Gluteus (GL), Gastrocnemius (GC), Tibialis Anterior (TA), Psoas (P), and Extensor Digitorum Longus (EDL) muscles will be isolated and processed for histological analysis. Previous analysis comparing muscles from wild type and MMex38 animals indicated significant weight differences in a number of muscle groups at 14-months of age, we will therefore determine if 4-PBA has salutary effects on muscle weights compared to untreated animals. Similarly, we will process both fixed and fresh frozen muscles using various stains to characterize the relative levels of dystrophic muscle phenotypic markers in either 4-PBA treated and untreated mice, histological analysis includes H&E staining, Mason’s Trichrome staining, Congo Red staining, and Dysferlin immunohistochemistry. To determine if long term treatment with 4-PBA maintains functional activity of myofibers will be isolated from explanted EDL muscles from both treated and untreated animals and assayed for membrane sealing kinetics following laser wounding.
Development of Monocyte Assay to access Dysferlin expression in murine PBMCs: Dysferlin is normally expressed in human CD14+ monocytes found in blood, which can be purified from peripheral blood mononuclear cells (PBMCs) by density gradient centrifugation and immunoaffinity binding to a-CD14-coated beads. Expression of human DYSF in CD14+ cells is determined by Western, and the results used to aid in dysferlinopathy diagnosis where DYSF is typically reduced by more than 90%. In order to assess the efficacy of 4-PBA and other putative therapeutics in preclinical mouse models (and potentially in patients) where blood draw amounts are smaller, we plan to isolate fixed PBMCs from mouse blood samples from MMex38 and wild type control mice either untreated or treated with 4-PBA using a series of PBMC isolation/fixation/permeabilization buffers (BD Bioscience). Following permeabilization, DYSF expression among monocytes will be determined by flow cytometry using antibody which binds the mouse monocyte surface marker Ly6C and the a-dysferlin Hamlet antibody to quantify dysferlin expression. Ultimately, the status of DYSF expression in monocytes from blood samples taken from mice in our longitudinal 4-PBA drug study will be performed and correlated with the immunohistochemical analysis of DYSF expression in muscle explants.
Modelling 4-PBA responsive patient missense mutations (PMMs) in myoblasts/myotubes: Apart from L1341P and R555W, we have identified a number of other DYSFPMMs that increase plasma membrane (PM) localization in response to 4-PBA treatment in HEK cells (Tominaga et. al 2021, iScience). We plan to express these DYSFPMMs in DYSF deficient GREG myoblasts, and interrogate if 4-PBA increases their PM expression using a flow cytometry-based assay protocol similar to that used for HEK cells. DYSFPMMs showing highest PM expression with 4-PBA treatment will be prioritized for differentiation into myotubes, and determination of functional restoration of membrane repair following laser wounding in the presence or absence of 4-PBA. This information will help determine the patient population that may benefit from 4-PBA treatment, and also aid our understanding the functionality of DYSFPMMs at the PM.
Project Results
Previous Grant Period
09/22 – 08/23
MMex38 mice harbor the murine dysferlin mutation DYSFL1360P synonymous to the dysferlinopathy patient missense mutation DYSFL1341P. We previously found that 4-PBA could increase plasma membrane localization and restore membrane repair function to of DYSFL1341P in vitro and could also do the same after a 2-day treatment of MMex38 mice in vivo (Tominaga et. al. 2021, iScience). During this grant period we began a study to determine if the clinically approved drug, sodium phenylbutyrate (4-PBA), is efficacious in preventing or reducing the severity of dystrophic phenotypes in the MMex38 mouse model of dysferlinopathy.
We first undertook an aging study of MMex38 mice in order to establish a baseline for histological and physiological changes that occur over time in this model in order to be able to establish endpoints for a longitudinal study of 4-PBA. To these ends, we established an aging colony of MMex38 (MUT) and wildtype (WT) control animals ranging from 3 months of age to up to 2 years of age, and isolated hind limb muscles from at least three male mice from each age group; including Quadriceps (Q), Gluteus (GL), Gastrocnemius (GC), Tibialis Anterior (TA), Psoas (P), and Extensor Digitorum Longus (EDL) muscles. We found statistically significant reductions in the weights of P, GL, and Q muscles from MUT animals at or over 12 months, and in GC muscles in MUT mice at or over 16 months. TA muscle show weight reduction at or over 20 months of age, while no differences were noted in EDL. We prepared fixed and fresh frozen tissue samples from all muscle groups for histological analysis, including H&E staining, Mason’s Trichrome staining, Congo Red staining, and Dysferlin immunohistochemistry in order to quantify dystrophic histological markers common to dysferlinopathy such as reduced myofiber size, increased numbers of central nuclei caused by regenerating myofibers, increased fat and collagen content/distribution indicative of fat infiltration and fiber replacement, and fibrosis. Most of these samples have been processed and analysis underway to determine differences between the various WT and MUT muscle groups.
To access the physiological effects on hind limb muscles, we developed an in-house Inclined Balance Beam (IBB) assay that scores the amount of time it takes a mouse to traverse a specified distance on inclined beams of various widths (24mm, 12mm, 10mm, 8mm). We found statistically significant differences in cross times between WT and MUT animals at 13 and 14 months of age on 12mm, 10mm, and 8mm width beams. We therefore plan to use the balance beam assay as a measure of muscle function in our longitudinal 4-PBA drug study.
During this granting period we established a longitudinal 4-PBA drug treatment trial. Thirty-two 2-month-old individually caged MMEx38 animals (16 male and female), were placed on a drug treatment protocol where half receive water containing 4-PBA (2mg/mL) and the remaining are given untreated water ad libitum, with water changed weekly. Animals were placed on protocol as they aged in; full enrollment occurred over three months. All animals were found to consume equivalent amounts of water, and showed equivalent increases in body weight over time; no adverse health events were found. Animals have been/are trained for IBB at 10 months of age, and assayed on the IBB each month thereafter. During our next grant period, we plan to end the longitudinal 4-PBA drug study after IBB assay are performed at 14 months of age, whereupon the various muscle groups listed above will be isolated, weighed, and processed for muscle histology.
Current Grant
09/23 – 08/24
In vivo treatment of MMex38 mice with 4-PBA: We plan to conclude our longitudinal drug study to determine if the clinically approved drug sodium 4-phenybutryic acid (4-PBA) is can ameliorate the dystrophic condition caused by the DYSF loss-of-function missense mutation L1360P in MMex38 animals. All animals (32 in total, 16 male, 16 female, age matched in equal numbers on drug or water control) in this study, will be trained in an Inclined Balance Beam (IBB) assay and periodically assessed for their ability to traverse beams of various width sizes (24mm, 12mm, 10mm, 8mm). We plan to assay the animals monthly from 12 to 14 months of age comparing MMex38 animals given 4-PBA ad libitum in their drinking water to untreated controls. Thereafter, as animals complete IBB assays, hind limb muscle groups, including Quadriceps (Q), Gluteus (GL), Gastrocnemius (GC), Tibialis Anterior (TA), Psoas (P), and Extensor Digitorum Longus (EDL) muscles will be isolated and processed for histological analysis. Previous analysis comparing muscles from wild type and MMex38 animals indicated significant weight differences in a number of muscle groups at 14-months of age, we will therefore determine if 4-PBA has salutary effects on muscle weights compared to untreated animals. Similarly, we will process both fixed and fresh frozen muscles using various stains to characterize the relative levels of dystrophic muscle phenotypic markers in either 4-PBA treated and untreated mice, histological analysis includes H&E staining, Mason’s Trichrome staining, Congo Red staining, and Dysferlin immunohistochemistry. To determine if long term treatment with 4-PBA maintains functional activity of myofibers will be isolated from explanted EDL muscles from both treated and untreated animals and assayed for membrane sealing kinetics following laser wounding.
Development of Monocyte Assay to access Dysferlin expression in murine PBMCs: Dysferlin is normally expressed in human CD14+ monocytes found in blood, which can be purified from peripheral blood mononuclear cells (PBMCs) by density gradient centrifugation and immunoaffinity binding to a-CD14-coated beads. Expression of human DYSF in CD14+ cells is determined by Western, and the results used to aid in dysferlinopathy diagnosis where DYSF is typically reduced by more than 90%. In order to assess the efficacy of 4-PBA and other putative therapeutics in preclinical mouse models (and potentially in patients) where blood draw amounts are smaller, we plan to isolate fixed PBMCs from mouse blood samples from MMex38 and wild type control mice either untreated or treated with 4-PBA using a series of PBMC isolation/fixation/permeabilization buffers (BD Bioscience). Following permeabilization, DYSF expression among monocytes will be determined by flow cytometry using antibody which binds the mouse monocyte surface marker Ly6C and the a-dysferlin Hamlet antibody to quantify dysferlin expression. Ultimately, the status of DYSF expression in monocytes from blood samples taken from mice in our longitudinal 4-PBA drug study will be performed and correlated with the immunohistochemical analysis of DYSF expression in muscle explants.
Modelling 4-PBA responsive patient missense mutations (PMMs) in myoblasts/myotubes: Apart from L1341P and R555W, we have identified a number of other DYSFPMMs that increase plasma membrane (PM) localization in response to 4-PBA treatment in HEK cells (Tominaga et. al 2021, iScience). We plan to express these DYSFPMMs in DYSF deficient GREG myoblasts, and interrogate if 4-PBA increases their PM expression using a flow cytometry-based assay protocol similar to that used for HEK cells. DYSFPMMs showing highest PM expression with 4-PBA treatment will be prioritized for differentiation into myotubes, and determination of functional restoration of membrane repair following laser wounding in the presence or absence of 4-PBA. This information will help determine the patient population that may benefit from 4-PBA treatment, and also aid our understanding the functionality of DYSFPMMs at the PM.
Project Results
Previous Grant Period
09/22 – 08/23
MMex38 mice harbor the murine dysferlin mutation DYSFL1360P synonymous to the dysferlinopathy patient missense mutation DYSFL1341P. We previously found that 4-PBA could increase plasma membrane localization and restore membrane repair function to of DYSFL1341P in vitro and could also do the same after a 2-day treatment of MMex38 mice in vivo (Tominaga et. al. 2021, iScience). During this grant period we began a study to determine if the clinically approved drug, sodium phenylbutyrate (4-PBA), is efficacious in preventing or reducing the severity of dystrophic phenotypes in the MMex38 mouse model of dysferlinopathy.
We first undertook an aging study of MMex38 mice in order to establish a baseline for histological and physiological changes that occur over time in this model in order to be able to establish endpoints for a longitudinal study of 4-PBA. To these ends, we established an aging colony of MMex38 (MUT) and wildtype (WT) control animals ranging from 3 months of age to up to 2 years of age, and isolated hind limb muscles from at least three male mice from each age group; including Quadriceps (Q), Gluteus (GL), Gastrocnemius (GC), Tibialis Anterior (TA), Psoas (P), and Extensor Digitorum Longus (EDL) muscles. We found statistically significant reductions in the weights of P, GL, and Q muscles from MUT animals at or over 12 months, and in GC muscles in MUT mice at or over 16 months. TA muscle show weight reduction at or over 20 months of age, while no differences were noted in EDL. We prepared fixed and fresh frozen tissue samples from all muscle groups for histological analysis, including H&E staining, Mason’s Trichrome staining, Congo Red staining, and Dysferlin immunohistochemistry in order to quantify dystrophic histological markers common to dysferlinopathy such as reduced myofiber size, increased numbers of central nuclei caused by regenerating myofibers, increased fat and collagen content/distribution indicative of fat infiltration and fiber replacement, and fibrosis. Most of these samples have been processed and analysis underway to determine differences between the various WT and MUT muscle groups.
To access the physiological effects on hind limb muscles, we developed an in-house Inclined Balance Beam (IBB) assay that scores the amount of time it takes a mouse to traverse a specified distance on inclined beams of various widths (24mm, 12mm, 10mm, 8mm). We found statistically significant differences in cross times between WT and MUT animals at 13 and 14 months of age on 12mm, 10mm, and 8mm width beams. We therefore plan to use the balance beam assay as a measure of muscle function in our longitudinal 4-PBA drug study.
During this granting period we established a longitudinal 4-PBA drug treatment trial. Thirty-two 2-month-old individually caged MMEx38 animals (16 male and female), were placed on a drug treatment protocol where half receive water containing 4-PBA (2mg/mL) and the remaining are given untreated water ad libitum, with water changed weekly. Animals were placed on protocol as they aged in; full enrollment occurred over three months. All animals were found to consume equivalent amounts of water, and showed equivalent increases in body weight over time; no adverse health events were found. Animals have been/are trained for IBB at 10 months of age, and assayed on the IBB each month thereafter. During our next grant period, we plan to end the longitudinal 4-PBA drug study after IBB assay are performed at 14 months of age, whereupon the various muscle groups listed above will be isolated, weighed, and processed for muscle histology.
