The aims of the present study were to determine whether nerves that contain nitric oxide synthase (NOS), calcitonin gene-related peptide (CGRP) or substance P (SP) are present in the human vagina and, if so, to determine the pattern of innervation relative to that of other neurotransmitters, particularly vasoactive intestinal polypeptide (VIP) and neuropeptide Y (NPY). Surgical specimens of vaginal tissue (n = 10) from pre- and postmenopausal women were fixed and processed for immunohistochemistry of peptides and NOS and for histochemistry of NADPH-diaphorase. SP-immunoreactive nerves were very sparse, being absent from 9 of the 10 tissue samples. For other peptides and NOS, the innervation of the deep arteries and veins was greater than that of blood vessels in the propria. Capillaries in the epithelial papillae also appeared to be innervated by nerves containing NOS, CGRP, NPY and VIP. Beneath the epithelium nerve fibres formed a subepithelial plexus; no nerve cell bodies were seen. The relative density of innervation by immunoreactive fibres was PGP-9.5 > NPY > VIP >> NOS > CGRP > SP. These results imply that nerves that utilise nitric oxide or NPY, VIP or CGRP as a neurotransmitter may play a role in controlling blood flow and capillary permeability in the human vagina. The origin and function of all these nerves is discussed.
Summary The DBA/2J (D2) mdx mouse has emerged as a more severe model of Duchenne muscular dystrophy when compared to the traditional C57BL/10 (C57) mdx mouse. Here, we questioned whether sarco(endo)plasmic reticulum Ca 2+ -ATPase (SERCA) function would differ in muscles from young D2 and C57 mdx mice. In gastrocnemius muscles, both D2- and C57 mdx mice exhibited signs of impaired Ca 2+ uptake, however, this was more severe in D2 mdx mice. Maximal SERCA activity was lowered only in D2 mdx gastrocnemius muscles and not C57 mdx muscles. Furthermore, in the left ventricle and diaphragm, Ca 2+ uptake was impaired in C57 mdx muscles with lowered rates of Ca 2+ uptake compared with C57 WT mice, whereas in muscles from D2 mdx mice, rates of Ca 2+ uptake were unattainable due to the severe impairments in their ability to transport Ca 2+ . Overall, our study demonstrates that SERCA function is drastically impaired in young D2 mdx mice.
Introduction The kynurenine (KYN) pathway has been implicated in depression and neurotoxicity. Derived from tryptophan, KYN can be further degraded along one of two distinct branches. The KYN-KYNA branch is regulated by the enzyme kynurenine aminotransferase (KAT) and is considered neuroprotective, as it degrades KYN into the non-blood brain barrier (BBB) transportable metabolite kynurenic acid (KYNA). The KYN-NAD branch is regulated by the enzyme kynurenine monooxygenase (KMO) and is considered neurotoxic as it degrades KYN into the BBB transportable metabolite 3-hydroxykynurenine (3-HK) and, further in the cascade, quinolinic acid (QUIN). Recent studies have shown the importance of muscle health on directing kynurenine metabolism towards the neuroprotective branch, highlighting a novel muscle-to-brain axis. Specifically, exercise induced increases in the transcription factor PCG-1⍺ amplifies the content of KAT enzymes that convert KYNA from KYN. Duchenne muscular dystrophy (DMD) is an X-linked severe muscle disorder caused by a loss of dystrophin leading to muscle fragility, wasting, and weakness. In light of recent evidence revealing the cognitive and depressive behaviours in DMD patients and in the preclinical mdx mouse, we sought to determine whether KYN metabolism as well as PGC-1α and KAT content would be altered in the mdx model. Methods 8-10 week old male mdx and wild-type (DBA/2J) mice were purchased from Jackson laboratories. Behavioural changes (ie., grooming activity, food and water intake) were measured using a Promethion metabolic cage system along with fear and anxiety-like behaviour during a novel object recognition test (NORT). Mice were euthanized and serum KYN and KYNA were measured using commercially available ELISA kits. Extensor digitorum longus muscle was collected, homogenized, and Western blotting was performed for PGC-1⍺, KAT1, and KAT3. Results Metabolic cage results showed that fine activity (-5%), water intake (-25%), and food intake (-60%) were lower across light and dark stages in mdx mice compared to WT mice (main effect of genotype, p<0.0001 for all measures). The mdx mice also spent more time in the corners of the NORT arenas compared to their WT counterparts (+270s, p<0.0001). Though the change in serum KYN was insignificant across mdx and WT mice, the concentration of serum KYNA was lower in the mdx mice (-57%, p = 0.01), therefore causing a lower KYN:KYNA ratio in mdx mice compared with WT (-56%, p = 0.01). Western blotting demonstrated a reduction in PGC-1⍺ (-65%, p = 0.002) and KAT1 (-35%, p = 0.02) content in mdx mice compared to WT mice, whereas the KAT3 content was elevated in mdx mice (1.5-fold, p = 0.05). Conclusion Our results of lowered serum KYN:KYNA concentrations from mdx mice (compared to WT) correspond well with changes in affective and anxiety-related behaviours. The observed reduction in muscle PGC-1⍺ and KAT1 content likely contributes to these changes in KYN:KYNA ratio. Though KAT3 was upregulated in mdx muscle compared to WT, this could represent a failed compensatory response.
Background Duchenne Muscular Dystrophy (DMD) is an X-linked muscle wasting disease caused by an absence of dystrophin. Secondary mechanisms including elevated myoplasmic Ca2+ as a result of dysfunctional sarcoendoplasmic reticulum Ca2+-ATPase (SERCA) pumps and leaky ryanodine receptors (RyR) can perpetuate disease pathology. Although the widely used preclinical C57BL/10 (C57) mdx mouse model displays moderate muscle damage and weakness, this model falls short in its ability to recapitulate the more severe human phenotype. Conversely, the DBA/2J (D2) mdx mouse model displays severe and early-onset muscle weakness, damage and histopathology; and therefore, more closely mimics human DMD. To date, there has been no characterization of muscle Ca2+ handling in the D2-mdx mouse. Thus, we examined sarcoplasmic reticulum (SR) Ca2+ handling in both D2-mdx and C57-mdx mice. Methods Eight-week-old male C57-WT, C57-mdx, D2-WT and D2-mdx mice were purchased from Jackson Laboratories. All mice underwent a hangwire test and were temporarily housed (48 hr) in a Promethion Metabolic Cage System to measure energy expenditure and cage activity. At 10 weeks of age, mice were euthanized, and gastrocnemius muscles were collected to measure Ca2+ uptake and leak via an Indo-1 fluorimetric assay. Serum creatine kinase (CK) activity was measured using a commercially available kit. Results Consistent with previous literature, serum CK was elevated in both C57-mdx and D2-mdx mice compared with WT, but to a greater extent in the former (8.9-fold vs 3.3 fold, p = 0.04). While both C57-mdx and D2-mdx mice had lowered hangwire time, these impairments were more severe in the D2-mdx mouse (-30% in C57-mdx vs -50% in D2-mdx, p = 0.09). Similarly, while both C57-mdx (-15%) and D2-mdx (-33%) mice had lowered total cage activity, the D2-mdx mice were most affected (p = 0.03). Interestingly, metabolic cage analyses further revealed that D2-mdx (1.3-fold increase) but not C57-mdx mice, had significantly elevated daily energy expenditure compared to their respective WT groups. When examining SR Ca2+ uptake, we found that the D2-mdx mice had significantly elevated starting Ca2+ levels compared with D2-WT mice (5500 vs 2300 nM). When activated by ATP, there was significantly less removal of Ca2+ in the D2-mdx muscles compared with WT (2.6-fold greater area-under-the-curve). However, none of these effects were observed in the C57-mdx mice. Furthermore, when SERCA was inhibited with cyclopiazonic acid, the rate of SR Ca2+ leak (-50%) and the amount of SR Ca2+ released back into the cytosol (-48%) were significantly lowered in the D2-mdx but not in the C57-mdx mice. This is likely reflective of impaired SR Ca2+ uptake and filling. Conclusions Unlike the C57-mdx, the D2-mdx mice displayed early onset muscle weakness with lowered cage ambulation and hangwire time when compared with their respective WT groups. These findings were associated with impaired SR Ca2+ handling in the D2-mdx mice but not the C57-mdx mice. Together, the extensive muscle damage and impaired SR Ca2+ handling in the D2-mdx mice likely enhances their daily energy expenditure.
Duchenne Muscular Dystrophy (DMD) is a progressive muscle wasting disorder which affects approximately 1 in 3500 male births and is caused by a mutation in the dystrophin gene. Although most research focuses on the role of dystrophin in skeletal and cardiac muscle, dystrophin is also expressed in the brain, specifically in memory-associated brain regions like the hippocampus and cortex. Furthermore, patients with DMD and mdx mice (rodent model of DMD) exhibit significant memory and learning impairments, implicating dystrophin loss in neurodegeneration. A recent study found patients with DMD had higher serum amyloid-β42, with this marker being heavily associated with cognitive impairments and Alzheimer's disease pathology. The purpose of this study was to characterize the neuropathology associated with DMD, focusing on markers of amyloidogenesis and synaptic function, in a preclinical mdx mouse model. Male mdx and age matched (8-9 weeks old) wild-type (DBA-2J) mice were purchased from Jackson laboratories (n = 12 per group). Novel object recognition testing (NORT) was performed to determine cognitive function. Prefrontal cortex and hippocampus brain regions were extracted for analysis. NORT testing demonstrated that mdx mice had reduced exploration time of the novel object (WT 35.2 sec vs mdx 18.7 sec; p = 0.02) and a lower exploration index (-25%, p < 0.05) compared to WT mice. Western blot analysis revealed higher soluble APP β (+40%) and total APP (+20%) in the prefrontal cortex of mdx mice compared to WT, and lower soluble APP α (-30%) fragment in the hippocampus (p <0.05). Furthermore, PSD95 content was nearly 2-fold higher in the prefrontal cortex of mdx mice compared to WT (p <0.05). This study provides novel information about the cellular pathways associated with memory impairment with DMD. Specifically, we show a shift towards amyloidogenesis in the prefrontal cortex and hippocampus brain regions of mdx mice, suggesting the possibility of similar pathogenesis to Alzheimer's disease. These changes are accompanied by reduced performance in the NORT test. Importantly, our results demonstrate that these changes occur at a relatively young age in D2 mdx mice, similar to what is observed in patients with DMD.
Duchenne muscular dystrophy (DMD) is a progressive muscle wasting disorder caused by a mutation in the dystrophin gene. In addition to muscle pathology, some patients with DMD will exhibit cognitive impairments with severity being linked to age and type of genetic mutation. Likewise, some studies have shown that mdx mice display impairments in spatial memory compared with wild-type (WT) controls, while others have not observed any such effect. Most studies have utilized the traditional C57BL/10 (C57) mdx mouse, which exhibits a mild disease phenotype. Recently, the DBA/2J (D2) mdx mouse has emerged as a more severe and perhaps clinically relevant DMD model; however, studies examining cognitive function in these mice are limited. Thus, in this study we examined cognitive function in age-matched C57 and D2 mdx mice along with their respective WT controls. Our findings show that 8- to 12-week-old C57 mdx mice did not display any differences in exploration time when challenged with a novel object recognition test. Conversely, age-matched D2 mdx mice spent less time exploring objects in total as a well as less time exploring the novel object, suggestive of impaired recognition memory. Biochemical analyses of the D2 mdx brain revealed higher soluble amyloid precursor protein β (APPβ) and APP in the prefrontal cortex of mdx mice compared with WT, and lower soluble APPα in the hippocampus, suggestive of a shift towards amyloidogenesis and a similar pathogenesis to Alzheimer's disease. Furthermore, our study demonstrates the utility of the D2 mdx model in studying cognitive impairment.
Introduction Duchenne muscular dystrophy (DMD) is an X-linked disorder caused by an absence of dystrophin that compromises membrane integrity, ultimately resulting in muscle weakness, wasting, and premature death. The fast glycolytic muscle fibres are known to be most susceptible to dystrophic pathology, while slow oxidative fibres are less affected. Thus, promoting the slow oxidative phenotype has become a viable therapeutic strategy. Recent work from our lab has shown that inhibiting the enzyme glycogen synthase kinase 3 (GSK3) can promote the slow oxidative phenotype leading to enhancements in fatigue resistance. Furthermore, we have shown that inhibiting GSK3 augments muscle specific force production and myoblast fusion. Therefore, inhibiting GSK3 may aid in alleviating dystrophic pathology. Tideglusib is a potent GSK3 inhibitor currently undergoing clinical trials for myotonic dystrophy, another form of muscular dystrophy. Here, we tested whether treating the DMD preclinical mdx mouse with tideglusib would alter muscle oxidative phenotype, improve muscle function, and reduce serum creatine kinase (CK) levels, a marker of cellular damage. Methods Male DBA/2J wild type (WT) and mdx mice were ordered from Jackson Laboratories at 5-6 weeks of age. Three groups were included in this study; 1) WT healthy control, 2) mdx tideglusib (10 mg/kg/day, via oral gavage), and 3) mdx vehicle (26% peg400, 15% Chremaphor EL and water). The mdx-tideglusib and vehicle mice underwent their respective treatments for 2 weeks with ad libitum access to food and water. Subsequently, all mice were subjected to a hangwire test to assess muscle function. Mice were then euthanized and their serum extracted for CK activity analyses using a commercially available kit that was fitted onto a 96-well plate. Extensor digitorum longus (EDL) muscles were collected and homogenized for Western blotting to investigate phosphorylated (serine 9) and total GSK3b content along with myosin heavy chain (MHC) I and IIa levels. Results The hangwire test results showed that mdx mice treated with tideglusib were able to sustain hanging on the wire for a significantly longer period of time compared with the mdx vehicle group (p = 0.03). Serum CK analyses revealed that while the mdx vehicle group had significantly higher levels of activity compared with WT (p = 0.009), there was a 30% reduction in the mdx tideglusib mice that was no longer significantly different from WT. Western blotting revealed that though phosphorylated GSK3b levels were unaltered, tideglusib treatment led to a significant reduction in GSK3b content compared with vehicle (p = 0.04). Additionally, there was a significant increase in the oxidative MHC isoforms (I and IIa, p = 0.02) in the mdx tideglusib group compared with vehicle. Conclusions Our results demonstrate the potential use of tideglusib for DMD. We show that tideglusib treatment inhibited GSK3 in mdx mice through a reduction in total GSK3 content. In turn, we also found a significant increase in oxidative MHC isoforms (I and IIa), which was associated with enhanced muscle performance and reduced muscle damage.
Kennedy's disease (KD) is a rare, late onset X-linked lower motor neuron disease, affecting 1 in 40 000 males (Breza & Koutsis, 2019). KD is caused by a polymorphic expansion mutation of a CAG trinucleotide repeat, encoding for glutamine in the first exon of the androgen receptor gene (Breza & Koutsis, 2019). Both neurons and muscles are affected by the toxicity of the mutant protein, due to an androgen-dependent gain of function in the receptor. Androgen binding to the mutant receptor leads to aggregation and formation of nuclear inclusions, resulting in impaired neuromuscular function in affected muscles (Breza & Koutsis, 2019). The most common motor-related symptom is lower limb weakness, with onset beginning around 40 years of age and muscular strength declining at 2% annually. Symptoms appearing later in disease progression include tremors, cramps, gynecomastia and neuromuscular degradation (Breza & Koutsis, 2019). Brain-derived neurotrophic factor (BDNF) is part of the neurotrophic factor family of growth factors primarily expressed in neuronal tissues. BDNF plays key roles in regulating cellular growth, neuronal maintenance, synaptic plasticity and metabolism (Kowianski et al. 2018). Following its synthesis within the Golgi apparatus, translocation and cleavage results in mature, biologically active isoforms pro-BDNF and m-BDNF. Upon release BDNF binds to its receptor, tropomyosin-related kinase B (TrkB), resulting in auto-phosphorylation (Tyr515 and Tyr816) and homodimerization, with subsequent downstream activation of the MAPK and PI3K/Akt pathways, mediating dendritic growth/branching and synaptic plasticity, respectively (Kowianski et al. 2018). Both structural and functional aspects of synaptic transmission are influenced by BDNF through increased activity-induced changes, enhancing the efficiency of pre- and post-signal transfer at the synapse (Kowianski et al. 2018). BDNF is also expressed and released by skeletal muscle in response to contraction. Muscle-derived BDNF is thought to regulate local muscular development through increased protein synthesis and neuromuscular synaptic transmission (Kowianski et al. 2018; Halievski et al. 2020). BDNF expression is higher in more oxidative (type I) slow-twitch muscle such as the soleus, in comparison to fast-twitch glycolytic (type II) fibres (Just-Borras et al. 2019). At the neuromuscular synapse, BDNF/TrkB signalling has an important role in bidirectional communication. Given the expression, location and synaptic role of BDNF in muscle, it has potential for rescuing dysfunctional neuromuscular transmission observed in KD. Herein lies the novelty of the paper by Halievski et al. 2020 published in a recent issue of The Journal of Physiology, which provides insight into the role of muscle-derived BDNF in KD and its therapeutic potential. We highlight their findings here while discussing a potential effect of muscle BDNF on fibre type shifts during neuromuscular disease states and ways in which this could be used as a therapeutic strategy. The 97Q mouse used primarily throughout this study exhibits progressive muscle weakness, atrophy and neuropathological features similar to human KD disease – achieved by the overexpression of 97 CAG repeats for a full human androgen receptor (Katsuno et al. 2002). Novel to this study, the authors developed a 97Q mouse with Cre-dependent overexpression of BDNF (97Q/BDNF) specifically in muscle. This resulted in significant increases in muscle-specific BDNF expression by 150-fold in the fast-twitch tibialis anterior (TA) and 45-fold in the slow-twitch soleus. The hang test, used to determine disease progression, demonstrates neuromuscular impairment and motor coordination in mouse models by measuring how long a mouse can hold their bodyweight using an overhanging bar. In the current study, disease onset was defined as a hang time <120 s for two consecutive days and a hang-time <30 s represented disease end-stage. Overexpression of BDNF was found to significantly increase time to disease onset, end-stage and further, doubled survival time. The effects of muscle BDNF on neurotransmission and neuromuscular transmission in 97Q and 97Q/BDNF mice were also studied. Muscle-specific overexpression of BDNF rescued quantal content, miniature end-plate potential (mEPP) amplitude, end-plate potential (EPP) delay and EPP failure rates in the slow-twitch soleus muscles. However, evidence that BDNF improved EDL synapses was limited. EDL benefits were only detectable at the population level, unlike soleus synapse, suggesting that muscle-specific BDNF overexpression benefits synapses in slow- but not fast-twitch muscles. There were also small, but significant effects of disease on neuromuscular transmission in the soleus, causing increased failure rates that were reversed by BDNF expression. In contrast, healthy EDL muscle synapses reached near 100% failure by the first 45 s of stimulus, whereas diseased synapses had slower and lower rates of neuromuscular transmission failure. BDNF overexpression improved EDL neuromuscular transmission in the fast EDL muscle. Taken together, these findings suggest that BDNF overexpression in the 97Q/BDNF mice displays fibre type specific improvements in nerve conduction, synaptic transmission reliability, and neuromuscular transmission, leading to later onset and slowed progression of neuro-muscular deficits in KD, as evident from the hang-time tests. This study also investigated the effect of BDNF both directly and indirectly (via nerve stimulation) on muscle contractility in fast and slow muscle types. As expected, muscle from 97Q disease control mice showed reduced force production both directly and indirectly compared to wild-type mice in the soleus and EDL. In the soleus, BDNF overexpression had no therapeutic effect on contractility. However, BDNF overexpression was able to significantly increase muscle contractility in the EDL compared to 97Q disease control mice when stimulated directly. Conversely, indirect stimulation revealed no differences in EDL contractility between 97Q/BDNF and 97Q disease control mice. Collectively, these data indicated that the benefit of BDNF overexpression in the EDL was due to enhancement of postsynaptic mechanisms rather than benefit to the muscles indirectly via nerve-evoked force. In human skeletal muscle samples, it was discovered that BDNF was present in significantly higher amounts compared to disease-control subjects. The authors hypothesized that this discrepant result was due to variation in disease severity between human samples and the 97Q mice, with the 97Q mice experiencing a more severe phenotype. Therefore, BDNF mRNA content was investigated in an early-stage KD knock-in (KI) mouse model, where KD was less advanced than in the 97Q BDNF mice, and BDNF VI and IX isoforms were indeed found in significantly increased amounts. This suggested that increasing BDNF expression may be an early compensatory mechanism in KD that later depletes, explaining the reduction in BDNF in the 97Q disease control mice. Alternatively, BDNF levels may be different between the models due to fibre type switching throughout disease progression. It is possible that a shift from fast to slow fibres occurred in human samples and KI mice, as BDNF expression is higher in slow fibres, while a change from slow to fast fibre type may have occurred in the 97Q mice, causing lowered BDNF expression (Just-Borras et al. 2019). This study demonstrated that by increasing BDNF in a murine model of KD, disease progression could be significantly slowed, extending time to disease onset and end-stage while improving contractility via pre- and postsynaptic mechanisms. These results are novel and reveal potential therapeutic promise of BDNF for KD. Though upregulated BDNF expression was observed in samples from human KD patients and the KI model, it is possible that this may be a physiological adaptive response that could be harnessed to slow disease and temporal investigations of BDNF mRNA in the 97Q mice are required in the future. In our view, the upregulation of BDNF mRNA in the human KD and murine KI samples may represent an adaptive response that, based on the findings from the 97Q/BDNF mice, could aid in combatting disease pathology. Future studies could investigate the effects of muscle-specific BDNF deletion in the KI mouse model to determine whether disease progression would be worsened. This would be consistent with muscle-specific BDNF overexpression and could further highlight its therapeutic use. Additionally, future studies could investigate BDNF expression in KD patients starting from disease onset through endpoint, compared to healthy, age-matched controls to better elucidate the role of BDNF throughout disease progression. The role of BDNF in different fibre types could also be investigated. Many neuromuscular measures throughout the Halievski et al. study highlighted that BDNF overexpression differentially affects fast- and slow-twitch fibre types. While this study discovered that BDNF can enhance multiple measures of synaptic reliability in soleus muscles, synapses in the EDL were much less affected by BDNF. Data reported demonstrated that BDNF had no effect on force production in the soleus, while increasing force in the EDL. As a next step, quantifying fibre type from the 97Q disease model and 97Q/BDNF mice to determine if any fibre type shifts are provoked by BDNF overexpression would be of merit, as previous research found that a loss of BDNF promoted a shift from fast to slow fibre phenotype (Just-Borras et al. 2019). Insight into BDNF's mechanism in enhancing neuromuscular function in the soleus will be beneficial to further determine the role of this neurotrophin in KD. In conclusion, the Halievski et al. 2020 paper reports important findings suggesting for the first time that muscle BDNF may delay disease onset and endpoint in the 97Q mouse model of KD. While these findings are promising, further preclinical work in the 97Q mouse and other mouse models of KD should be pursued to determine if muscle BDNF can play a further role in treating KD. None declared. All authors have approved the final version of the manuscript and agree to be accountable for all aspects of the work. All persons designated as authors qualify for authorship, and all those who qualify for authorship are listed. None.
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