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mTOR drives cerebrovascular, synaptic, and cognitive dysfunction in normative aging

aging brain vasculature cerebral blood flow cognitive decline functional mri mtor

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#1 Engadin

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Posted 11 November 2019 - 10:54 AM






S O U R C E :   AgingCell


C O M P A C T E D    &   M O R E   A C C E S I B L E   S O U R C E :   MedicalXpress  (Study: Rapamycin prevents age-related brain vascular deterioration)







Cerebrovascular dysfunction and cognitive decline are highly prevalent in aging, but the mechanisms underlying these impairments are unclear. Cerebral blood flow decreases with aging and is one of the earliest events in the pathogenesis of Alzheimer's disease (AD). We have previously shown that the mechanistic/mammalian target of rapamycin (mTOR) drives disease progression in mouse models of AD and in models of cognitive impairment associated with atherosclerosis, closely recapitulating vascular cognitive impairment. In the present studies, we sought to determine whether mTOR plays a role in cerebrovascular dysfunction and cognitive decline during normative aging in rats. Using behavioral tools and MRI‐based functional imaging, together with biochemical and immunohistochemical approaches, we demonstrate that chronic mTOR attenuation with rapamycin ameliorates deficits in learning and memory, prevents neurovascular uncoupling, and restores cerebral perfusion in aged rats. Additionally, morphometric and biochemical analyses of hippocampus and cortex revealed that mTOR drives age‐related declines in synaptic and vascular density during aging. These data indicate that in addition to mediating AD‐like cognitive and cerebrovascular deficits in models of AD and atherosclerosis, mTOR drives cerebrovascular, neuronal, and cognitive deficits associated with normative aging. Thus, inhibitors of mTOR may have potential to treat age‐related cerebrovascular dysfunction and cognitive decline. Since treatment of age‐related cerebrovascular dysfunction in older adults is expected to prevent further deterioration of cerebral perfusion, recently identified as a biomarker for the very early (preclinical) stages of AD, mTOR attenuation may potentially block the initiation and progression of AD.
Normal brain aging predisposes vulnerable neurons to degeneration and is associated with cognitive decline and an increased likelihood of developing a neurodegenerative disorder (Mattson & Magnus, 2006). The prevalence of Alzheimer's disease (AD), the most common cause of dementia in the elderly, is expected to double approximately every 20 years, with 131.5 million cases expected worldwide by 2050 (Prince et al., 2015). The prevalence of AD is increasing rapidly, yet there is no disease‐modifying treatment currently available. Although age is a primary risk factor for AD, very little is known about the molecular mechanisms that link the regulation of brain aging to neurodegenerative diseases of advanced age.
Cerebrovascular dysfunction is a universal feature of aging (Zlokovic, 2011) that includes impaired endothelium‐dependent vasodilation and global and regional decreases in cerebral blood flow (CBF) (Martin, Friston, Colebatch, & Frackowiak, 1991). While decreased CBF does not indicate a particular disease state, reduced CBF is associated with impaired cognitive function (Wang et al., 2016) and decreased neuronal plasticity (Hamadate et al., 2011). Using functional magnetic resonance imaging (fMRI), which measures changes in CBF to infer neuronal activation, it is apparent that the human brain can reorganize and redistribute functional networks to compensate for nonpathologic age‐related impairments. In general, task‐related neural activation becomes more diffuse with advancing age as other brain regions are recruited to maintain task proficiency. However, when cognitive demand exceeds these compensatory mechanisms, performance becomes impaired (Cabeza et al., 1997). Because cerebrovascular dysfunction plays a critical role in the pathogenesis of age‐related neurodegenerative disorders, including AD (Csiszar et al., 2017; Lin et al., 2017, 2013; Van Skike et al., 2018), and manifests early in disease progression (Iturria‐Medina, Sotero, Toussaint, Mateos‐Perez, & Evans, 2016), it is important to investigate the mechanisms underlying cognitive decline and brain vascular deterioration driven by nonpathologic aging.
The mammalian/mechanistic target of rapamycin (mTOR) pathway regulates aging in mammals (Wilkinson et al., 2012). mTOR is also expressed throughout the brain, where it is linked to synaptic plasticity and learning and memory (Tang et al., 2002) through the regulation of protein synthesis (Tang et al., 2002) and autophagy (Mizushima, Levine, Cuervo, & Klionsky, 2008). In the aging brain, autophagy is reduced, contributing to the accumulation of aggregated proteins and neurodegeneration (Komatsu et al., 2006). Thus, dysregulation of the mTOR pathway during nonpathologic aging may contribute to cognitive decline, cerebrovascular dysfunction, and a predisposition toward developing neurodegenerative diseases associated with advanced age.
We have previously shown that mTOR inhibition attenuates cognitive dysfunction in aged mice (Halloran et al., 2012). We and others have also shown that chronic mTOR attenuation with rapamycin can prevent and reverse cognitive and cerebrovascular deficits in several independent mouse models of AD (Lin et al., 2017, 2013;Van Skike et al., 2018) and vascular cognitive impairment (Jahrling et al., 2018), leading to improved cerebrovascular function and preserved cognitive outcomes in these models of age‐related disease. The contribution of mTOR to cerebrovascular deficits is associated with normative aging, though its impact on cognitive outcomes remains unknown. The goal of this study was to test the hypothesis that mTOR drives cerebrovascular and synaptic dysfunction during aging and that chronic mTOR inhibition with rapamycin mitigates nonpathologic age‐related deterioration of cognition and cerebrovascular function in aged rats without underlying disease.
2.1 Age‐related decline in hippocampal‐dependent learning and memory is driven by mTOR
To determine the contribution of mTOR to cognitive deficits during normative aging, we used the Morris water maze to measure hippocampal‐dependent spatial learning and memory in adult (16 months old) and aged (34 months old) rats that were either fed either a diet with empty microcapsules or a diet containing microencapsulated rapamycin at 14 parts per million (ppm) for 15 months starting at 19 months of age. Consistent with prior studies (Novier, Van Skike, Diaz‐Granados, Mittleman, & Matthews, 2013), we found that adult rats swam significantly faster than aged rats, regardless of treatment condition (Figure 1a). Thus, we used path length as measure of performance during training in the MWM since this measure is not impacted by swim speed. Total distance swam declined progressively throughout the 4 days of training (Figure 1b), indicating effective spatial learning among the groups. However, 34‐month‐old aged rats had significantly longer path lengths than the 16‐month‐old adult rats on training days 3 and 4. No significant differences in path length were observed in aged rats treated with rapamycin as compared to adult rats on any training day. Additionally, there were no differences among the groups in the amount of slow swimming below 0.10 m/s during acquisition (Figure 1c).
Figure 1
Age‐associated cognitive decline in rats is driven by mTOR. (a) Thirty‐four‐month old aged rats, regardless of treatment condition, displayed slower swim speed compared with 16‐month‐old adults (for each training day, q(12)<4.60, *p < .018; ** indicates p < .01). Tukey's post hoc tests were applied to a significant main effect of group, F(2,36)=8.96, p = .0007 in two‐way repeated measures ANOVA analyses. (b) Aged rats exhibit spatial learning and memory impairments compared with adults (F(2, 36)=5.40, p = .009), especially on days 3 (**q(144)=4.23, p = .009) and 4 (*q(144)=3.35, p = .049) of training in the Morris water maze (MWM). Performance of aged rats in which mTOR was chronically attenuated with rapamycin (aged + rapa) did not significantly differ from that of adult rats for each training day (q(144)<2.56, p < .17, n.s.). Tukey's post hoc tests were applied to significant main effects of day (F(3,108) = 48.59, p < .0001) and group (F(2,36)=5.40, p = .009) in two‐way ANOVA (day x group) with repeated measures analyses. © The proportion of swimming slower than 0.10 m/s decreases with training (F(3,108)=12.87, p < .0001), but is not different among groups (F(2, 36)=1.93, p = .16). (d) Aged rats exhibit significant spatial memory impairment in the probe trial compared with adult rats (*q(144)=3.81, p = .021). Inhibition of mTOR restores spatial memory in aged rats (aged + rapa group) to a level indistinguishable from that of adult animals (q(144)=1.17, p = .69). Tukey's post hoc tests were applied to a significant main effect of group (F(3, 108)=24.86 p < .0001) via two‐way ANOVA. Data are presented as mean ± SEM (n = 10‐15/group)




During a 24‐hr probe trial, aged rats made significantly fewer passes over the learned location of the hidden platform as compared to adult rats during a 24‐hr recall probe trial (Figure 1d). Recall of the hidden platform location in aged rats treated with rapamycin, however, was indistinguishable from that of adult rats. Together, these data indicate that deficits in spatial learning and memory in aged rats can be negated by mTOR attenuation, suggesting that spatial learning and memory impairments in aged rats are at least partially driven by mTOR. Of note, chronic mTOR inhibition did not rescue age‐related decreases in swim speed, ruling out an impact of mTOR attenuation on neuromotor pathways or muscle function and activity required for swimming.
2.2 mTOR drives sensory‐evoked functional hyperemia impairments in rats of advanced age
Optimal brain function depends on regulation of CBF in response to neuronal activity, through a complex mechanism known as neurovascular coupling (NVC). Decreased NVC occurs during aging in both humans (Fabiani et al., 2014) and rodents (Toth et al., 2014). Functional MRI (fMRI) measures the hemodynamic response of NVC in response to a defined stimulus as an indicator of neuronal activation. Since decreased neuronal activation and impaired cognitive performance during aging have been linked in humans (Cabeza et al., 1997), we used fMRI to determine overall neuronal activity and neurovascular coupling in our rat model of advanced age measured as the hemodynamic response associated with neuronal activation in response to somatosensory (forepaw) stimulation. We found that evoked fMRI response to somatosensory stimulation was blunted in aged rats as compared to adult animals. Chronic mTOR attenuation by rapamycin, however, restored the fMRI response in aged rats to levels indistinguishable from those of adult animals (Figure 2). These results indicate that mTOR attenuation can restore profound deficits in neurovascular coupling responses in aged rats, suggesting that deficient neuronal network activation and/or impaired functional hyperemia during aging are mediated by mTOR.
Figure 2
mTOR drives impaired neuronal network activation during aging in rats. (a) Representative fMRI activation in the somatosensory cortex and (b) quantitative analysis demonstrates the response to forepaw stimulation is decreased with age (*** indicates p < .001 via t test). fMRI activation, however, is preserved in aged rats treated with rapamycin (***, p < .001 compared with age‐matched controls). The restoration of fMRI activation in response to forepaw stimulation by mTOR attenuation was complete since the magnitude of the evoked response in the rapamycin‐treated aged group was indistinguishable from that of adult rats. Evoked responses are shown as mean percent increase over baseline cerebral blood flow ± SEM
2.3 mTOR contributes to decreased presynaptic density with age
Since the decrease in functional hyperemia during somatosensory stimulation (i.e., neurovascular coupling) is dependent on the integrity of both neuronal and vascular responses, we quantified presynaptic density to provide a measure of neuronal integrity. Decreased presynaptic density is associated with mild cognitive impairment and AD (Scheff et al., 2015) and with cognitive impairment in rodents (Wang et al., 2014). To define whether changes in synaptic integrity occur during normative aging in rats and understand the role of mTOR, we measured presynaptic density in rats after completion of training and testing in the Morris water maze. Density (Figure 3a and b) and quantity (Figure 3a and c) of synaptophysin‐positive synaptic boutons in hippocampal CA1 were decreased with advanced age in rats. Both density and quantity of synaptophysin‐positive synaptic elements in aged rats treated with rapamycin to attenuate mTOR, however, were indistinguishable from those of adult animals (Figure 3a–c). Together, these findings indicate that chronic mTOR attenuation curtails an age‐related loss of synaptic boutons in the hippocampus, suggesting that preserved presynaptic integrity by mTOR attenuation may underlie the restoration of hippocampal‐dependent learning and memory and the maintenance of fMRI responses to somatosensory stimulation in aged rats. These data suggest that mTOR dysregulation drives age‐related structural remodeling of the hippocampus during aging in the rat and that mTOR attenuation may block age‐related impairments in hippocampal‐dependent memory through the preservation of presynaptic density.
Figure 3
mTOR‐dependent deterioration of presynaptic density during aging. (a) Representative images of synaptophysin immunofluorescent reactivity in hippocampal CA1. (b) Decreased synaptophysin density in aged as compared to adult rats (**, q(9)=7.25, p = .002) was significantly ameliorated by chronic mTOR attenuation using rapamycin in the aged + rapa treatment group (*q(9)= 4.31, p = .03 vs. aged + vehicle). Synaptophysin density was restored to levels not significantly different from those of adult rats (q(9)=2.93, p = .15) in the aged + rapa treatment group. Tukey's post hoc tests were applied to a significant omnibus one‐way ANOVA, F(2,9)=13.29, p = .002). Data are mean ± SEM of n = 4. © Aged rats have fewer synapses as shown by decreased synaptophysin reactive area (****q(9)=13.41, p < .0001), a difference that was abolished by mTOR attenuation in aged rats treated with rapamycin (q(9)=12.57, p < .0001). Tukey's post hoc tests were applied to a significant one‐way ANOVA (F(2,9)=56.44, p < .0001). Data are presented as mean ± SEM of n = 4
The beneficial effects of mTOR inhibition we observed in this study may also be related to factors that were not measured in the present study. For instance, mTOR regulates microglial activation states as well as the secretion of proinflammatory cytokines and chemokines (Li et al., 2016). Therefore, some effects may be due to the downregulation of mTOR‐driven neuroinflammatory responses, which increase during aging and negatively impact both neuronal and brain vascular function. Additionally, the central role of oxidative stress in age‐related cerebrovascular dysfunction (Sure et al., 2018) was recently demonstrated in studies showing that the age‐related deterioration of cerebrovascular endothelial cell function and neurovascular coupling responses were reversed by reducing oxidative stress in endothelial cells (Kiss et al., 2019; Tarantini et al., 2019, 2018). Inhibition of mTOR is protective against oxidative stress in vitro in vascular endothelial cells (Zheng et al., 2017), indicating that mTOR may also regulate oxidative stress. While we have not addressed the role of mTOR‐driven neuroinflammation or oxidative stress in our studies, these may represent important mechanisms through which mTOR inhibition ameliorates cerebrovascular dysfunction and cognitive impairment in the aging brain. Figure 6 illustrates the interaction and potential convergence of mTOR‐driven pathways of brain aging.
Figure 6
Proposed mTOR‐dependent mechanisms of brain aging. mTOR inhibitors, including rapamycin, rapalogs, and kinase inhibitors, can restore neuronal and cerebrovascular function by blocking specific mTOR‐dependent pathways of brain aging

Also tagged with one or more of these keywords: aging, brain vasculature, cerebral blood flow, cognitive decline, functional mri, mtor

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