Doug W. Smith

Blood-CNS barriers protect the CNS from circulating immune cells and damaging molecules. It is thought barrier integrity becomes disrupted with aging, contributing to impaired CNS function. Using genome-wide and targeted molecular approaches, we found aging affected expression of predominantly immune invasion and pericyte-related genes in CNS regions investigated, especially after middle age, with spinal cord being most impacted. We did not find significant perturbation of endothelial cell junction genes or proteins, nor were vascular density or pericyte coverage affected by aging. We evaluated barrier paracellular permeability using small molecular weight tracers, serum protein extravasation, CNS water content, and iron labelling measures. We found no evidence for age-related increased barrier permeability in any of these tests. We conclude that blood–brain (BBB) and blood-spinal cord barrier (BSCB) paracellular permeability does not increase with normal aging in mouse. Whilst expression changes were not associated with increased permeability, they may represent an age-related primed state whereby additional insults cause increased leakiness.

3 Summary: Blood-CNS barriers protect the CNS from circulating immune cells and damaging molecules. It is thought barrier integrity becomes disrupted with aging, contributing to impaired CNS function. Using genome-wide and targeted molecular approaches, we found aging affected expression of predominantly immune invasion and pericyte-related genes in most CNS regions investigated, especially after middle age, with spinal cord being most impacted. We did not find significant perturbation of tight junction genes, nor were vascular density or pericyte coverage affected by aging. We evaluated barrier paracellular permeability using small molecular weight tracers, serum protein extravasation, CNS water content, and iron labelling measures. We found no evidence for age-related increased barrier permeability in any of these tests. We conclude that blood-brain (BBB) and blood-spinal cord barrier (BSCB) paracellular permeability does not increase with normal aging in mouse. Whilst expression changes were not associated with increased permeability, they may represent an age-related primed state whereby additional insults cause increased leakiness.

Cholinergic circuits in the central nervous system are vulnerable to age-related functional decline, but it is not known if aging impacts cholinergic signaling in the vestibular sensory organs, which are critically important to balance maintenance and visual gaze stability. We have previously shown cholinergic neurotransmission between vestibular efferent terminals and type II mechanosensory hair cells requires the alpha9 (Chrna9) nicotinic receptor subunit. Homozygous knockout of the alpha9 subunit causes vestibulo-ocular reflex adaptation deficits that mirror those observed in aged mice. This prompted examination of cholinergic signaling in the vestibular sensory organs of aged mice. We confirmed older (>24 months) mice had impaired performance in a balance beam task compared to young (3–4 months) adult mice. While there was no qualitative loss of cholinergic axon varicosities in the crista ampullaris of old mice, qPCR analysis revealed reduced expression of nicotinic receptor subunit genes Chrna1, Chrna9, and Chrna10 in the cristae of old relative to young mice. Functionally, single-cell patch clamp recordings taken from type II vestibular hair cells exposed to acetylcholine show reduced conductance through alpha9/10 subunit-containing nicotinic receptors in older mice, despite preserved passive membrane properties and voltage-activated conductances. These findings suggest that cholinergic signaling in the peripheral vestibular sensory organs is vulnerable to aging processes, manifesting in dynamic molecular and functional age-related changes. Given the importance of these organs to our everyday activities, and the dramatic increase in fall incidence in the older, further investigation into the mechanisms of altered peripheral vestibular function in older humans is warranted.

Background: Deterioration in vestibular function occurs with ageing and is linked to age-related falls. Sensory hair cells located in the inner ear vestibular labyrinth are critical to vestibular function. Vestibular hair cells rely predominantly on oxidative phosphorylation (OXPHOS) for energy production and contain numerous mitochondria. Mitochondrial DNA (mtDNA) mutations and perturbed energy production are associated with the ageing process. Objective: We investigated the effects of ageing on mtDNA in vestibular hair and support cells, and vestibular organ gene expression, to better understand mechanisms of age-related vestibular deficits. Methods: Vestibular hair and supporting cell layers were microdissected from young and old rats, and mtDNA was quantified by qPCR. Additionally, vestibular organ gene expression was analysed by microarray and gene set enrichment analyses. Results: In contrast to most other studies, we found no evidence of age-related mtDNA deletion mutations. However, we found an increase in abundance of major arc genes near the mtDNA control region. There was also a marked age-related reduction in mtDNA copy number in both cell types. Vestibular organ gene expression, gene set enrichment analysis showed the OXPHOS pathway was down regulated in old animals. Conclusion: Given the importance of mtDNA to mitochondrial OXPHOS and hair cell function, our findings suggest the vestibular organs are potentially on the brink of an energy crisis in old animals.

The causative link between UV exposure and melanoma development is well known, however the mechanistic relationship remains incompletely characterised. UVA and UVB components of sunlight are implicated in melanomagenesis; however the majority of studies have focused on the effects of UVB and UVC light. Interestingly, melanoma tumour sequencing has revealed an overrepresentation of mutations signature of unrepaired UV-induced DNA damage. Repair of UVA-induced DNA damage is thought to occur primarily through the Nucleotide Excision Repair (NER) pathway, which recognises and repairs damage either coupled to transcription (Transcription Coupled Repair; TCR), or through global genome scanning (Global Genome Repair; GGR). Current literature suggests NER is deficient in melanoma, however the cause of this remains unknown; and whether reduced NER activity in response to UVA may be involved in melanoma development remains uncharacterised. In this study we aimed to determine if melanoma cells exhibit reduced levels of NER activity in response to UVA. Melanocyte and melanoma cell lines were UVA-irradiated, and DNA damage levels assessed by immunodetection of Cyclobutane Pyrimidine Dimer (CPD) and (6-4) Photoproduct [(6-4)PP] lesions. Expression of NER pathway components and p53 following UVA treatment was quantified by qPCR and western blot. UVA did not induce detectable induction of (6-4)PP lesions, consistent with previous studies. Repair of CPDs induced by UVA was initiated at 4 h and complete within 48 h in normal melanocytes, whereas repair initiation was delayed to 24 h and >40 % of lesions remained in melanoma cell lines at 48 h. This was coupled with a delayed and reduced induction of GGR component XPC in melanoma cells, independent of p53. These findings support that NER activity is reduced in melanoma cells due to deficient GGR. Further investigation into the role of NER in UVA-induced melanomagenesis is warranted and may have implications for melanoma treatment.