Myosin 5a in the Urinary Bladder: Localization, Splice Variant Expression, and Functional Role in Neurotransmission

Abstract

Dysregulation of neurotransmission is a feature of several prevalent lower urinary tract conditions, but the mechanisms regulating neurotransmitter release in the bladder are not completely understood. The unconventional motor protein, Myosin 5a, transports neurotransmitter-containing synaptic vesicles along actin fibers towards the varicosity membrane, tethering them at the active zone prior to reception of a nerve impulse. Our previous studies indicated that Myosin 5a is expressed and functionally relevant in the peripheral nerves of visceral organs such as the stomach and the corpora cavernosa. However, its potential role in bladder neurotransmission has not previously been investigated. The expression of Myosin 5a was examined by quantitative PCR and restriction analyses in bladders from DBA (dilute-brown-nonagouti) mice which express a Myosin 5a splicing defect and in control mice expressing the wild-type Myosin 5a allele. Functional differences in contractile responses to intramural nerve stimulation were examined by ex vivo isometric tension analysis. Data demonstrated Myosin 5a localized in cholinergic nerve fibers in the bladder and identified several Myosin 5a splice variants in the detrusor. Full-length Myosin 5a transcripts were less abundant and the expression of splice variants was altered in DBA bladders compared to control bladders. Moreover, attenuation of neurally-mediated contractile responses in DBA bladders compared to control bladders indicates that Myosin 5a facilitates excitatory neurotransmission in the bladder. Therefore, the array of Myosin 5a splice variants expressed, and the abundance of each, may be critical parameters for efficient synaptic vesicle transport and neurotransmission in the urinary bladder.

Differential Myosin 5a splice variants in innervation of pelvic organs

“…Our analysis indicated that variant ACEF was not abundantly expressed in pelvic tissues, although its presence can be inferred in distal colon by a degree of resistance of the ∼600 bp fragment to Ban II digestion, and its presence was inferred as a minor species in bladder ( Carew et al, 2022 ). In contrast, the ∼600 bp fragment from bladder and CCP was extensively cleaved by Ban II, suggesting it was comprised mostly of ACDE in these tissues.…”

“…The ABCE variant of Myo5a is so abundantly expressed in neurons of the central nervous system that it is referred to as the brain form and might be expected to be the most prevalent form in the peripheral nervous system as well. However, our previous work implied that this was not the case: the ABCE variant is detectable in stomach sections but undetectable in bladder, while variants containing the alternative exons D and F were identified in both tissues ( Carew et al, 2021 ; Carew et al, 2022 ). Based on immunohistochemistry data, Myo5a protein is found predominantly in nerve trunks and extensions where it colocalizes with axonal markers such as synaptophysin and β-III-tubulin.…”

“…Since in situ hybridization had indicated that Myo5a mRNA is primarily localized to nerve soma/dendrites within the myenteric plexus ( Carew et al, 2021 ), and immunohistochemistry indicated extensive colocalization with neural markers (such as β-III-tubulin, synaptophysin or NeuN) ( Carew et al, 2021 ; Carew et al, 2022 ), Myo5a expression in CCP, distal colon and MPG, as well as bladder, was expected to be largely restricted to nerves. But because variant-specific anti-Myo5a antibodies are unavailable, it was not possible to assign expression of a particular mRNA variant detected to a particular location by immunohistochemistry.…”

“…However, Myo5a is thought to play important roles in transport of synaptic vesicles within varicosities and their retention near active zones until a depolarization event, both critical functions for optimal regulation of neurotransmitter release. Our previous work characterized impairments of both inhibitory and excitatory neurotransmission in the fundus, CCP and bladder of the dilute-brown-nonagouti, or DBA, mouse, a strain carrying a defect that reduces the splicing of exon D and exons D+F in Myo5a transcripts ( Chaudhury et al, 2014 ; Carew et al, 2021 ; Carew et al, 2022 ). It is tempting to speculate that these impairments arose from the inability of Myo5a lacking exons D and F to carry out essential neurotransmission functions in peripheral nerve axons.…”

“…The Myo5a splice variants expressed in peripheral tissues other than skin melanocytes, have not yet been extensively characterized. We have recently identified splice variants expressed by stomach and bladder using PCR methodologies ( Carew et al, 2021 ; Carew et al, 2022 ). In all stomach sections (antrum, fundus, body and pylorus), the exon arrangement identified in brain Myo5a, ABCE, was detected along with other arrangements such as ACDE, ACEF, and ACDEF.…”