Deficiency of tPA Exacerbates White Matter Damage, Neuroinflammation, Glymphatic Dysfunction and Cognitive Dysfunction in Aging Mice
Peng Yu1,2,3, Poornima Venkat2, Michael Chopp2,4, Alex Zacharek2, Yi Shen2, Linlin Liang2,5, Julie Landschoot-Ward1, Zhongwu Liu2, Rongcai Jiang1,*, Jieli Chen2,*
1Department of Neurosurgery, Tianjin Medical University General Hospital, and Tianjin Neurological institute, Key Laboratory of Post-Neurotrauma Neurorepair and Regeneration in Central Nervous System, Ministry of Education and Tianjin City, Tianjin, China. 2Neurology, Henry Ford Hospital, Detroit, MI, USA. 3Department of Neurosurgery, Shanghai Tenth People’s Hospital, Tongji University, Shanghai, China. 4Department of Physics, Oakland University, Rochester, MI, USA. 5Reproductive Medical Center, Henan Provincial People’s Hospital, Zhengzhou, China
Tissue plasminogen activator (tPA) is a serine protease primarily involved in mediating thrombus breakdown and regulating catabolism of amyloid-beta (Aβ). The aim of this study is to investigate age-dependent decline of endogenous tPA and the effects of tPA decline on glymphatic function and cognitive outcome in mice. Male, young (3m), adult (6m) and middle-aged (12m) C57/BL6 (wild type) and tPA knockout (tPA-/-) mice were subject to a battery of cognitive tests and white matter (WM) integrity, neuroinflammation, and glymphatic function were evaluated. Adult WT mice exhibit significantly decreased brain tPA level compared to young WT mice and middle-aged WT mice have significantly lower brain tPA levels than young and adult WT mice. Middle-aged WT mice exhibit significant neuroinflammation, reduced WM integrity and increased thrombin deposition compared to young and adult mice, and increased blood brain barrier (BBB) permeability and reduced cognitive ability compared to young WT mice. In comparison to adult WT mice, adult tPA-/- mice exhibit significant BBB leakage, decreased dendritic spine density, increased thrombin deposition, neuroinflammation, and impaired functioning of the glymphatic system. Compared to age-matched WT mice, adult and middle-aged tPA-/- mice exhibit significantly increased D-Dimer expression and decreased perivascular Aquaporin-4 expression. Compared to age-matched WT mice, young, adult and middle-aged tPA-/- mice exhibit significant cognitive impairment, axonal damage, and increased deposition of amyloid precursor protein (APP), Aβ, and fibrin. Endogenous tPA may play an important role in contributing to aging induced cognitive decline, axonal/WM damage, BBB disruption and glymphatic dysfunction in the brain.
Peng Yu,Poornima Venkat,Michael Chopp, et al. Deficiency of tPA Exacerbates White Matter Damage, Neuroinflammation, Glymphatic Dysfunction and Cognitive Dysfunction in Aging Mice[J]. Aging and disease,
2019, 10(4): 770-783.
Figure 1. Aging and tPA deficiency induce progressive cognitive deficits
(A) Significant short-term memory loss evaluated by novel object recognition test is evident in middle-aged WT mice compared to young WT mice, and in young and middle-aged tPA-/- mice compared to age-matched WT mice. (B) Long-term memory deficits evaluated by odor test are evident in middle-aged WT mice compared to young WT mice as well as young, adult and middle-aged tPA-/- mice compared to corresponding age-matched WT mice. (C) Aging decreases tPA expression level in brain of WT mice. Adult mice (6m) have significantly lower tPA expression in brain compared to young mice (3m), and middle-aged (12m) mice have significantly lower tPA levels than young and adult mice. (D) tPA-/- mice and middle-aged WT mice exhibit significantly increased spatial learning and memory deficits measured by Morris Water maze test. *p<0.05, n=6/group.
Figure 2. Aging and deficiency of tPA induce WM damage and loss of synaptic plasticity
Middle-aged WT and tPA-/- mice exhibit significantly increased myelin rarefaction as indicated by Luxol fast blue immunostaining (A-F) compared to young WT and young tPA-/- mice, respectively. Luxol fast blue quantification data is presented in panel (G. H-M) Middle-aged WT and tPA-/- mice exhibit significantly increased axon damage indicated by decreased axon density in Bielschowsky silver immunostaining compared to young WT and young tPA-/- mice respectively. Young, adult and middle-aged tPA-/- mice exhibit significantly increased axonal damage compared with age-matched WT mice. Bielschowsky silver quantification data are presented in panel N. (O-Q) Analysis of Golgi staining under a 40× objective indicates that there were no changes in the primary neuronal branching in tPA-/- mice compared to WT mice. (R-T) Analysis of Golgi staining under a 100× objective indicates that the spine density in cortical neurons was significantly decreased in adult tPA-/- mice compared to adult WT mice. *p<0.05, n=6/group.
Figure 3. Deficiency of tPA increases age induced Aβ and APP aggregation in the brain
(A-G) Aβ and (H-N) APP deposition significantly increases in brain of young, adult and middle-aged tPA-/- mice compared to corresponding age matched WT mice. However, no significant age dependent changes were observed within WT or tPA-/- groups of varying age. Aβ and APP quantification data is presented in panels G and N, respectively. *p<0.05, n=6/group.
Figure 4. Aging and deficiency of tPA induce increased fibrin deposition in vessels and thrombin deposition in the brain
(A-G) Fibrin deposition in brain of adult and middle-aged tPA-/- mice significantly increases compared to young tPA-/- mice, and the fibrin accumulation is significantly greater in young, adult and middle-aged tPA-/- mice compared to corresponding age-matched WT mice. Blood vessels are stained using vWF (Von Willebrand factor) immunostaining and DAPI is used to stain nuclei. Quantification data for fibrin immunostaining (A-F) is presented in panel (G. H) ELISA data indicate that D-Dimer expression levels were significantly increased in adult and middle-aged tPA-/- mice compared to age-matched WT mice. (I-O) Thrombin deposition significantly increased in middle-aged WT and tPA-/- mice compared to young and adult WT or tPA-/- mice, respectively. In addition, thrombin deposition was significantly higher in adult tPA-/- mice compared to adult WT mice. Quantification data for thrombin immunostaining (I-N) is presented in panel O. *p<0.05, n=6/group.
Figure 5. Aging and deficiency of tPA increases number of microglia, astrocytes and macrophages
Immunostaining and quantification data indicate that middle-aged WT and tPA-/- mice have significantly increased (A-G) number of microglia (IBA1) and (H-N) macrophages (CD68) compared to young WT or tPA-/- mice, respectively. In addition, adult tPA-/- mice exhibit significantly increased numbers of microglia (IBA1, A-G), macrophages (CD68, H-N) and astrocytes (GFAP, O-U) compared to adult WT mice. Quantification data for microglia, macrophages and astrocytes are presented in panels G, N and U respectively. *p<0.05, n=6/group.
Figure 6. Aging and deficiency of tPA induces BBB disruption
BBB leakage (Albumin) is significantly increased in middle-aged WT mice compared to young WT mice and adult tPA-/- mice compared to adult WT mice. Quantification data for albumin immunostaining (A-F) is presented in panel G. *p<0.05, n=6/group.
Figure 7. Deficiency of tPA decreased AQP-4 expression and induced glymphatic system dysfunction
(A-H) Compared with adult WT mice, adult tPA-/- mice exhibit significantly delayed clearance of dyes injected into the cisterna magna indicating impaired functioning of the glymphatic system. (I-O) AQP-4 expression was significantly decreased in adult and middle-aged tPA-/- mice compared to age matched WT mice respectively. Quantification data for AQP-4 immunostaining is presented in panel O. *p<0.05, n=6/group.
Figure 8. Summary scheme indicating age-dependent changes in wild type and tPA-/- mice.
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