Glymphatic inhibition exacerbates tau propagation in an Alzheimer's disease model

Abstract

Background: The aggregation and spread of misfolded amyloid structured proteins, such as tau and α-synuclein, are key pathological features associated with neurodegenerative disorders, including Alzheimer's and Parkinson's disease. These proteins possess a prion-like property, enabling their transmission from cell to cell leading to propagation throughout the central and peripheral nervous systems. While the mechanisms underlying their intracellular spread are still being elucidated, targeting the extracellular space has emerged as a potential therapeutic approach. The glymphatic system, a brain-wide pathway responsible for clearing extracellular metabolic waste from the central nervous system, has gained attention as a promising target for removing these toxic proteins.

Methods: In this study, we investigated the impact of long-term modulation of glymphatic function on tau aggregation and spread by chronically treating a mouse model of tau propagation with a pharmacological inhibitor of AQP4, TGN-020. Thy1-hTau.P301S mice were intracerebrally inoculated with tau into the hippocampus and overlying cortex, and subsequently treated with TGN-020 (3 doses/week, 50 mg/kg TGN-020, i.p.) for 10-weeks. During this time, animal memory was studied using cognitive behavioural tasks, and structural MR images were acquired of the brain in vivo prior to brain extraction for immunohistochemical characterisation.

Results: Our findings demonstrate increased tau aggregation in the brain and transhemispheric propagation in the hippocampus following the inhibition of glymphatic clearance. Moreover, disruption of the glymphatic system aggravated recognition memory in tau inoculated mice and exacerbated regional changes in brain volume detected in the model. When initiation of drug treatment was delayed for several weeks post-inoculation, the alterations were attenuated.

Conclusions: These results indicate that by modulating AQP4 function and, consequently, glymphatic clearance, it is possible to modify the propagation and pathological impact of tau in the brain, particularly during the initial stages of the disease. These findings highlight the critical role of the glymphatic system in preserving healthy brain homeostasis and offer valuable insights into the therapeutic implications of targeting this system for managing neurodegenerative diseases characterized by protein aggregation and spread.

Keywords: Glymphatic; MRI; Neurodegeneration; Propagation; Tau.

Fig. 1

Chronic pharmacological blockade of AQP4 results in sustained glymphatic inhibition. A Representative example of whole brain images with psuedocoloured fluorescence overlaid, illustrating the dramatic reduction of brain surface coverage of fluorescence tracer (TxR-d3, 3 kDa) following chronic treatment with TGN-020 (50 mg/kg) compared to vehicle. Quantification of this signal B shows that in all three orientations, TGN-020 treated mice exhibit a reduction in tracer coverage. Sectioning of brains and imaging by fluorescence microscopy demonstrated a similar difference; C representative examples of a brain section (~ 1 mm lateral to the midline) of a vehicle treated and a TGN-020 treated mouse brain, showing clear surface penetration of TxR-d3 in the vehicle treated but not TGN-020 treated animal. D Quantification of the % area of each section covered by the tracer demonstrates this difference more clearly (shaded area indicates SEM of animals examined). E Area under curve of data shown in (D). *=p < 0.05, **=p < 0.01, n = 3–4 per group

Fig. 2

Chronic glymphatic inhibition exacerbates tau propagation in a mouse model. A Representative examples of AT8 immunoreactivity in the hippocampus of treated mice groups. B Quantification of AT8 immunopositive staining (% area) in the hippocampus indicates a trend towards increased tau pathology in ^P301SBE injected transgenic mice, but no apparent difference between TGN-020 treated and vehicle treated animals. When subdivided into hemispheres however (C), TGN-020 treated ^P301SBE infused mice exhibited a significant increase in immunoreactive intensity compared to vehicle treated animals. Contralateral tau signal intensity was also significantly greater in TGN-020 treated mice compared to vehicle treatment, which was made more clear when calculating the interhemispheric intensity ratio in tau injected mice (D). A similar trend of increased tau load was observed in the cortex (E), i.e. the other region of the brain to receive tau inoculation. However no differences in tau signal intensity were seen here between treatment groups (F) or between hemispheres of tau injected mice (G). ▲=females, ■=males, *=p < 0.05, **=p < 0.01,, ***=p < 0.001, n = 5–8 per group

Fig. 3

Chronic glymphatic inhibition exacerbates long-term memory impairment in a mouse model of tau propagation. Following 4 weeks of TGN-020 treatment, TGN-020 treated ^P301SBE injected mice do not exhibit any strong preference towards the novel unfamiliar object in the 24 h delayed trial of the Novel Object Recognition test (A). This decline in object preference is significantly greater than that observed in vehicle treated ^P301SBE injected mice. B TGN-020 treated animals on average appear to spend more time immobile in the open field task, with almost half of the drug treated animals spending extended periods of the time immobile. Limb clasping scores in a subset of the animals (C) indicate that TGN-020 treated ^P301SBE injected mice exhibit significantly greater hind-limb clasping compared to ^WTBE injected mice. D Representative scored example images of hind-limb clasping behaviour exhibited by animals. ▲=females, ■=males, *=p < 0.05, **=p < 0.01, n = 7–8 per group. Asterisks over bars indicate differences from baseline, asterisks over brackets indicate differences between denoted groups

Fig. 4

Chronic glymphatic inhibition appears to exacerbate volumetric changes caused by tau propagation, in extra-hippocampal/cortical regions. Volcano plot of comparisons between groupwise regional interhemispheric volume ratios and hemispheric equality, indicating both an upper-left (accentuated ispi < contralateral difference) and upper-right (accentuated ispi > contralateral difference) shift away from the origin (bottom centre) of the plot in P301S ^P301SBE TGN-020 treated group datapoints compared to vehicle treated controls. For anatomical reference, horizontal slice images are shown (left, -4 from Bregma; right, 2 mm from Bregma) with regions colour scaled based on their significant interhemispheric differences (ipsi < contralateral in blue (left), ipsi > contralateral in red (right)) in each of the treatment groups. n = 8 per group (4♂:4♀)

Fig. 5

Delayed TGN-020 treatment impacts its effect on propagating tau pathology. A Study design of delayed treatment experiments. Novel Object Recognition is performed prior to tau infusion, which is then followed either immediately, or 2 or 4 weeks later by chronic TGN-020 treatment. At the end of the study (week 10), the NOR task is repeated, limb clasping is assessed, and the structural MRI of the mouse brain is acquired, prior to culling for immunohistochemistry. Immunohistochemical analysis of tau (AT8) immunoreactivity in the hippocampus, reveals that immunoreactive intensity (B) is reduced with TGN-020 treatment delayed. This results in rescue of novel objective exploration at week 10 (C) and sparing of limb clasping behaviour (D) as a function of the delay in treatment. Structural MRI also reveals alleviation of volumetric differences in regions of significant ipsi > contralateral (e.g. hippocampus (E)) and ipsi < contralateral (e.g. amygdala (F)) difference. ▲=females, ■=males, *=p < 0.05, **=p < 0.01, n = 7–8 per group