Leader: Antonio Frigeri (UNIBA); Other collaborator(s):
The aim of the study is to clarify the role of the glial component and water homeostasis in the pathogenesis of neurodegenerative diseases such as lateral amyotrophic sclerosis (LAS) and Alzheimer's. By the use of relevant models, we plan to: (i.) better understand the pathophysiological aspects of neurodegenerative diseases; (ii.) identify new molecular pathways useful for the development and validation of innovative therapies; (iii.) test their potential efficacy in restoring altered functions.
Brief description of the activities and of the intermediate results: The study is focused on the involvment of the different Aquaporin-4 (AQP4) isoforms in the aging mechanism. As known AQP4 is expressed as two main isoforms : M1 and M23. Recently a novel AQP4 isoform (AQP4ex) was discovered , longer 29 amino acids at C-terminus then the canonical one. Moreover it has been demonstrated that this isoform is highly phosphorylated at the C-terminus extension (p-AQP4ex). Phisiologically AQP4 isoforms are expressed mainly on the perivascular astrocytic end-feet, allowing the brain water homeostasis. and in the removing waste products from the CNS through the “glymphatic system”, and as previously reported AQP4 absence in aged mice reduces solutes clearance including misfolded proteins like β-amyloid, impairing glymphatic function.To assess this we firstly analyzed AQP4 isoforms expression levels in the brain cortex of young mice (3 months) vs old mice(19 months) . Interestingly we found a statistically significant reduction in AQP4 expression of aged mice compared with young ones. Moreover we observed a strong reduction in both AQP4ex and p-AQP4ex in aged mice compared to controls by both immunoblot and IF experiments. Preliminary experiments conducted on the glymphatic system, revealed an alteration of the glymphatic mechanism in old animals . Taken together, these data suggest that the reduction of AQP4 isoforms play a crucial role in the progressive decline of brain astrocytes functions, causing an impairment of the glymphatic system, as confirmed by the accumulation of b-amyloid fluorophore. Further studies will be carried out on different aged mice (6 and 12 months) to assess if during aging there will be changes in the distribution and expression of AQP4 isoforms and to investigate its contribution in the glymphatic functions.
During the current experimental period (March 30, 2024 – December 10, 2024), we performed similar experiments as reported previously, adding mice of additional age groups to assess age-related changes in the distribution and expression of AQP4 isoforms and their contribution to glymphatic function. Specifically, we analyzed mice aged 6, 10, 13, 24, and 32 months. Our data showed no significant changes in global AQP4 expression until 6 months, compared to 3-month-old mice, which were used as controls. However, beyond 6 months, AQP4 expression progressively decreased with increasing age, showing the most pronounced reduction in 32-month-old mice. Interestingly, we observed an increase in the expression of both AQP4ex and p-AQP4ex isoforms until 6 months, followed by a sharp decline in these extended isoforms during aging. Experiments assessing glymphatic function revealed a progressive reduction in β-amyloid accumulation in young mice until 6-10 months, indicating complete maturation and efficient clearance of waste solutes. Notably, we observed increased β-amyloid fluorescence in 19-month-old mice, suggesting impaired glymphatic function during aging. Taken together, these findings indicate that the maturation and maintenance of glymphatic function are highly correlated with AQP4 isoform expression during aging.
Further studies will be conducted to investigate the potential regulatory mechanisms of AQP4 during aging. Recently, a regulatory pathway involving long non-coding RNAs (lncRNAs) has been proposed to control AQP4 expression. These molecules influence gene expression by modulating processes such as mRNA splicing, transcription, translation, and genomic imprinting. Specifically, for AQP4, a lncRNA known as AQP4-AS1, transcribed from the antisense strand of AQP4, appears to play a key role in regulating its expression. Investigating the levels of such lncRNAs could provide valuable insights into the regulation of AQP4 and offer potential strategies for modulating AQP4 channel activity and improving glymphatic function.
During the current experimental period (March 30, 2024 – December 10, 2024), we performed similar experiments as reported previously, adding mice of additional age groups to assess age-related changes in the distribution and expression of AQP4 isoforms and their contribution to glymphatic function. Specifically, we analyzed mice aged 6, 10, 13, 24, and 32 months. Our data showed no significant changes in global AQP4 expression until 6 months, compared to 3-month-old mice, which were used as controls. However, beyond 6 months, AQP4 expression progressively decreased with increasing age, showing the most pronounced reduction in 32-month-old mice. Interestingly, we observed an increase in the expression of both AQP4ex and p-AQP4ex isoforms until 6 months, followed by a sharp decline in these extended isoforms during aging. Experiments assessing glymphatic function revealed a progressive reduction in β-amyloid accumulation in young mice until 6-10 months, indicating complete maturation and efficient clearance of waste solutes. Notably, we observed increased β-amyloid fluorescence in 19-month-old mice, suggesting impaired glymphatic function during aging. Taken together, these findings indicate that the maturation and maintenance of glymphatic function are highly correlated with AQP4 isoform expression during aging. Further studies will be conducted to investigate the potential regulatory mechanisms of AQP4 during aging. Recently, a regulatory pathway involving long non-coding RNAs (lncRNAs) has been proposed to control AQP4 expression. These molecules influence gene expression by modulating processes such as mRNA splicing, transcription, translation, and genomic imprinting. Specifically, for AQP4, a lncRNA known as AQP4-AS1, transcribed from the antisense strand of AQP4, appears to play a key role in regulating its expression. Investigating the levels of such lncRNAs could provide valuable insights into the regulation of AQP4 and offer potential strategies for modulating AQP4 channel activity and improving glymphatic function.
During this period, we expanded our analysis by including mice aged 6, 10, 13, 24, and 32 months to investigate age-related changes in AQP4 isoforms and glymphatic function. These results highlight the correlation between AQP4 isoforms and glymphatic efficiency, as well as a glymphatic decline coupled with the mis-localization e downregulation of AQP4 expression. Since AQP4 is inolved in water brain balance, and since mis-localization and expression of AQP4 and its isoform, seems to be crucial in the glympahtic functions, we moved on a possible mechanisms implicated in the regulation of AQP4 expression, particularly during the ageing phase. To this purpose, we examined the levels of long non-coding RNAs (lncRNAs) in aged mice, as these molecules are known to influence gene expression through various mechanisms, including mRNA splicing, transcriptional and translational regulation, and genomic imprinting. Among them, AQP4-AS1, a lncRNA transcribed from the antisense strand of AQP4, has been reported to play a key role in modulating AQP4 expression. Our analysis revealed a progressive decrease in AQP4 mRNA levels with aging, accompanied by a similar reduction in AQP4-AS1 expression. This suggests the involvement of additional regulatory mechanisms or alternative AQP4-AS1 transcripts in the aging process. Further studies will be needed to elucidate the early phases of this regulatory interplay between AQP4 and AQP4-AS1, particularly in the context of brain
development. Additionally, we plan to increase the sample size and extend our analysis to other brain regions, such as the hippocampus, hypothalamus, and cerebellum. These areas may be more susceptible to age-related alterations in AQP4 regulation, providing deeper insights into the mechanisms underlying glymphatic dysfunction during aging.
• Abbrescia, P., Signorile, G., Valente, O. et al. Crucial role of Aquaporin-4 extended isoform in brain water Homeostasis and Amyloid-β clearance: implications for Edema and neurodegenerative diseases. acta neuropathol commun 12, 159 (2024). https://doi.org/10.1186/s40478-024-01870-4
• Aquaporin-4 expression levels and mis-localization are inversely linked to peritumoral edema in gliomas of varying aggressiveness Onofrio Valente, Pasqua Abbrescia, Gianluca Signorile, Raffaella Messina, Luigi de Gennaro, Maria Teresa Bozzi, Giuseppe Ingravallo, Antonio D’amati, Domenico Sergio Zimatore, Claudia Palazzo, Grazia Paola Nicchia, Antonio Frigeri, Francesco Signorelli bioRxiv 2024.09.15.613106; doi: https://doi.org/10.1101/2024.09.15.613106