Nicotinamide mononucleotide (NMN) supplementation rescues cerebromicrovascular endothelial function and neurovascular coupling responses and improves cognitive function in aged mice

Abstract

Adjustment of cerebral blood flow (CBF) to neuronal activity via neurovascular coupling (NVC) has an essential role in maintenance of healthy cognitive function. In aging increased oxidative stress and cerebromicrovascular endothelial dysfunction impair NVC, contributing to cognitive decline. There is increasing evidence showing that a decrease in NAD⁺ availability with age plays a critical role in a range of age-related cellular impairments but its role in impaired NVC responses remains unexplored. The present study was designed to test the hypothesis that restoring NAD⁺ concentration may exert beneficial effects on NVC responses in aging. To test this hypothesis 24-month-old C57BL/6 mice were treated with nicotinamide mononucleotide (NMN), a key NAD⁺ intermediate, for 2 weeks. NVC was assessed by measuring CBF responses (laser Doppler flowmetry) evoked by contralateral whisker stimulation. We found that NVC responses were significantly impaired in aged mice. NMN supplementation rescued NVC responses by increasing endothelial NO-mediated vasodilation, which was associated with significantly improved spatial working memory and gait coordination. These findings are paralleled by the sirtuin-dependent protective effects of NMN on mitochondrial production of reactive oxygen species and mitochondrial bioenergetics in cultured cerebromicrovascular endothelial cells derived from aged animals. Thus, a decrease in NAD⁺ availability contributes to age-related cerebromicrovascular dysfunction, exacerbating cognitive decline. The cerebromicrovascular protective effects of NMN highlight the preventive and therapeutic potential of NAD⁺ intermediates as effective interventions in patients at risk for vascular cognitive impairment (VCI).

Keywords: Endothelial dysfunction; Functional hyperemia; Microcirculation; Oxidative stress; ROS.

Graphical abstract

Fig. 1

NMN supplementation improves microvascular endothelial function and rescues NO mediation of neurovascular coupling responses in aged mice. A) Representative traces of cerebral blood flow (CBF; measured with a laser Doppler probe above the whisker barrel cortex) during contralateral whisker stimulation (30 s, 5 Hz) in the absence and presence of the NO synthase inhibitor l-NAME in young (3 month old), aged (24 month old) and NMN treated aged mice. B) Summary data showing that in aged mice NMN supplementation restores NO mediated component of NVC responses. C) In aged mice NMN supplementation improves endothelium-mediated CBF responses elicited by topical perfusion of acetylcholine. D) NMN supplementation decreases protein 3-nitrotyrosine content in the aged cortex, indicating decreased peroxynitrite formation. E-G) In aged mouse aortas NMN supplementation rescues acetylcholine-induced endothelium-mediated relaxation (E), increases tissue NAD⁺ levels (F) and attenuates oxidative stress (G; see Methods). Data are mean ± S.E.M. (n = 5–8 for each data point).*P < 0.05 vs. Young; ^#P < 0.05 vs. Aged. (one-way ANOVA with post-hoc Tukey's test). n.s.: not significant.

Fig. 2

Treatment with NMN improves mitochondrial energetics and attenuates mitochondrial ROS production in aged cerebromicrovascular endothelial cells (CMVECs). A) Treatment with NMN (5 × 10⁻⁴ moL/L; for 5 days) restores NAD⁺ levels in primary CMVECs derived from aged rats. B) Treatment with NMN (5 × 10⁻⁴ moL/L; for 1–5 days) attenuates age-related increases in mtROS production in CMVECs (MitoSox fluorescence, assessed by flow cytometry). C) shRNA knockdown of SIRT1/SIRT2 prevents NMN-induced attenuation of mtROS in aged CMVECs. D-E) Treatment of aged CMVECs with NMN rescues cellular NO production (D; DAF fluorescence, assessed by flow cytometry) and increases mitochondrial membrane potential (E; JC-1 mitochondrial membrane potential probe) to levels observed in young cells. shRNA knockdown of SIRT1/SIRT2 prevents the NMN effect. F) Treatment of aged CMVECs with NMN restores cellular ATP levels. Data are mean ± S.E.M (n = 5–10 for each data point in A-F). *P < 0.05 vs. Young; ^#P < 0.05 vs. Aged. G) Attenuation of mtROS production and improved mitochondrial membrane potential in NMN treated aged CMVECs were associated with significant improvement of cellular oxygen consumption rate (OCR; a marker of oxidative phosphorylation; measured using the Seahorse XFe96 analyzer). Vertical dashed lines indicate assay drug injections. OCR in untreated young and aged CMVECs is shown for reference. Note the marked NMN-induced increase in both basal and maximal respiration in aged CMVECs. Right panel shows the effects of shRNA knockdown of SIRT1/SIRT2 on NMN-induced changes in OCR in aged CMVECs. OCR in aged CMVECs transfected with scrambled shRNA is shown for reference. H) Summary data showing the effects of aging and NMN on basal respiration, ATP-linked respiration and maximal respiration. Data are mean ± S.E.M., n = 9 for each data point.*P < 0.05 vs. Young; ^#P < 0.05 vs. Aged. $ P < 0.05 vs. Aged + NMN (one-way ANOVA with post-hoc Tukey's test). n.s.: not significant.

Fig. 3

Treatment with NMN rescues age-related downregulation of mitochondrially encoded subunits of the electron transport chain without promoting mitochondrial biogenesis. A-C) Representative electronmicrographs showing mitochondria in cerebromicrovascular endothelial cells in young (A), aged (B) and NMN treated aged mice (C); (arrowheads, mitochondria; nu, nucleus; bm, basal lamina; AC, astrocyte; scale bar: 500 nm). D) Summary data showing that NMN treatment does not affect mitochondrial volume density in aged cerebromicrovascular endothelial cells. E) NMN treatment of aged mice does affect mtDNA content in cerebral arteries. F) NMN treatment (5 × 10⁻⁴ moL/L; for 5 days) of aged CMVECs does not affect mtDNA content. G) NMN supplementation rescues age-related decreases in mRNA expression of mitochondrially encoded subunits of the electron transport chain in cerebral arteries. Data are mean ± SEM (n = 5–6 for each data point in D-G). *P < 0.05 vs. Young; ^#P < 0.05 vs. Aged. (one-way ANOVA with post-hoc Tukey's test). n.s.: not significant.

Fig. 4

In NMN treated aged mice rescue of neurovascular coupling responses associates with improved performance in the radial-arm water maze (RAWM). Young (3 month old), aged (24 month old) and NMN treated aged mice were tested in the RAWM. A) Heatmap representing the percentage of time spent in different locations in the maze for a randomly selected animal from each group during experimental day 3. Note that the untreated aged mouse required a greater amount of time and a longer path length in order to find the hidden escape platform. Older mice also re-enter a previously visited arm multiple time, accruing working memory errors. B) Older animals have higher combined error rates throughout day 2 and 3 of the learning phase. Combined error rate is calculated by adding 1 error for each incorrect arm entry as well as for every 15 s spent not exploring the arms. C) Older animals make significantly more working memory errors (repetitive incorrect arm entries) as compared to young mice. In contrast, aged mice treated with NMN perform this task significantly better than untreated aged mice. D) The ratio of successful escapes, averaged across trial blocks, is shown for each group. Note day-to-day improvement in the performance of young mice, which was significantly delayed in aged mice. Although aged mice treated with NMN tended to be more successful at finding the hidden escape platform in comparison to untreated age-matched controls, the difference did not reach statistical significance. Average path length (Panel E) and escape latencies (Panel F) required to reach the hidden platform in the RAWM for trial blocks 1–6. Young mice find the hidden platform sooner while swimming significantly less than aged animals. In aged mice treated with NMN the escape latencies and the average path length required to reach the hidden platform did not differ from that in aged mice. G) NMN had only marginal effect on the swimming speed. H) Aged control mice exhibited longer non-exploratory behavior compared to young mice. Treatment with NMN partially reduces the non-exploratory time to young levels. All data are shown as mean ± SEM. (n = 20 for each data point).

Fig. 5

In NMN treated aged mice rescue of neurovascular coupling responses associates with improved cognitive performance. A) NMN treatment improved learning ability in aged mice, as assessed using the elevated plus maze-based learning protocol (see Methods section). For young mice, transfer latency on day 2 was significantly decreased compared to day 1, indicating an intact learning effect. For aged mice the transfer latency on day 1 and day 2 were similar, indicating impaired learning capability. NMN supplementation in aged mice restored learning performance to youthful levels. B) NMN treatment restored recognition memory in aged mice as measured by the novel object recognition test (see Methods). Recognition memory is expressed as a recognition index which is defined as the ratio of time spent exploring the novel object over the total time spent exploring both familiar and novel objects. C) NMN supplementation in aged mice does not affect mean latencies to fall from the rotarod. All data are shown as mean ± SEM. (n = 20 for each data point). Statistical significance was calculated using one-way ANOVA with Tukey's post hoc test to determine differences among groups. *P < 0.05 vs. Young; ^#P < 0.05 vs. Aged control. D) NMN supplementation improves gait performance in aged mice. Shown is the 3D triplot of first three principal components (PC) identified by PCA on the correlation matrix of spatial and temporal indices of gait. Each point represents an individual mouse. Note, that mice in the same age groups clustered together. Differences between young and aged mouse gait were evident. NMN supplementation partially reverses age-related changes in mouse gait (MANOVA; P < 0.01 Aged vs. Aged treated; P < 0.01 Young vs. Aged). E) Scheme showing proposed role for increased NAD⁺ deficiency and mitochondrial oxidative stress in cerebromicrovascular endothelial impairment and neurovascular dysfunction in aging and their pathophysiological consequences.

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