Cognitive dysfunction in aging is a major biomedical challenge. Whether treatment with klotho, a longevity factor, could enhance cognition in human-relevant models such as in nonhuman primates is unknown and represents a major knowledge gap in the path to therapeutics.
We validated the rhesus form of the klotho protein in mice, showing it increased synaptic plasticity and cognition.
We then found that a single administration of low-dose, but not high-dose, klotho enhanced memory in aged nonhuman rhesus macaque primates.
Systemic low-dose klotho treatment may prove therapeutic in aging humans.
…To execute our primary goal, we treated aged rhesus macaques (mean age ~21.78 years; Supplementary Table 1, putative human age equivalent of mean ~65 years) with a single administration of vehicle or 10 μg kg−1 (s.c.) of rhesus KL and tested their cognition. We chose this dose for primary analysis because it produced similar increases of KL that are (1) effective in cognitive enhancement of mice and (2) present at birth in humans38.
Cognitive testing of aged rhesus macaques was performed using the spatial delayed response (SDR) task (Figure 2a), assessing frontal-temporal circuits and regions of the brain, including the hippocampus and PFC. This task assesses working and spatial memory for both a normal memory load (NML; easier task) and high memory load (HML; harder task)39 (Figure 2b). SDR is an ideal task to test KL because aging disrupts the specific cognitive domains that it probes31,40. In brief, monkeys were trained to achieve a stable baseline response for the NML task in remembering the spatial location of a food reward (Figure 2a). All macaques were then treated with vehicle (s.c.) to habituate to effects of the procedure and stress of an injection on cognitive performance. Finally, monkeys were treated with either vehicle or rhesus KL (s.c.); 4h later, monkeys underwent the HML task (with up to 9 wells) followed by a series of NML tasks (up to 7 wells) over 2 weeks (Figure 2a), ending with another HML task.
In the primary analysis, KL (10 μg kg−1, s.c.) enhanced cognition in aged rhesus macaques during both NML and HML testing. As expected, performance between baseline or vehicle treatment did not differ. KL (10 μg kg−1, s.c.) increased HML performance by 4h after treatment (Figure 2c), within the same rapid time frame it increased cognition in mice (Figure 1e). KL-mediated cognitive enhancement of HML, a harder memory task, persisted at 2 weeks (Figure 2d). KL (10 μg kg−1, s.c.) also enhanced the average NML performance (Figure 2e), an effect that persisted across multiple tests during the first and second weeks following treatment (Figure 2f). KL-mediated enhancement was observed independent of sex.
In exploratory analysis of higher KL doses (20 and 30 μg kg−1, s.c.), statistically-significant cognitive improvement was not observed in either the HML (Figure 2g) or the NML (Figure 2h) tasks. It is interesting to note that in contrast to monkeys, mice continued to show cognitive enhancement with much higher KL doses5. This species difference may be related to increased structural and network complexity of the monkey compared to the mouse brain.
Collectively, our data show that KL (10 μg kg−1) enhanced cognition in aging rhesus macaques, an effect that persisted for at least 2 weeks in both the NML and HML measures of memory.
(b) Diagram of the SDR cognitive task. Monkey is shown food reward (cue phase), screen is lowered (delay) and then screen is raised with all wells covered (test phase). Remembering location of hidden reward in NML and HML (more wells and longer delays) was measured.
(c) percentage correct choices by monkeys, representing spatial and working memory, in HML task at baseline (n = 19 sessions from 18 monkeys; 13 females and 5 males), at 4h following treatment with Veh (n = 26 sessions from 18 monkeys; 13 females and 5 males) or rhesus KL (10 μg kg−1) (n = 11 sessions from 9 monkeys; 6 females and 3 males). p = 0.0077 versus Veh (linear mixed-model ANOVA and Satterthwaite’s t-tests using sessions, two-tailed). Bars represent mean ± s.e.m. of test sessions; points are mean performance of each monkey.
(d) percentage correct choices by monkeys in the HML task 4h and 14–23 d after a single injection with Veh or rhesus KL (10 μg kg−1). n, same as Figure 1c. p = 0.0077 (4 h), p = 0.0035 (14–23d) versus Veh (linear mixed-model ANOVA and Satterthwaite’s t-tests using sessions, two-tailed). Points indicate sessions. Filled circles are mean ± s.e.m. of sessions. Dashed line shows mean Veh performance.
(e) percentage correct choices by monkeys in NML task 1–14 d after baseline or vehicle treatment (n = 71 sessions from 17 monkeys; 12 females and 5 males) or rhesus KL (10 μg kg−1) (n = 46 sessions from 9 monkeys; 6 females and 3 males), p = 0.0006 versus Veh + baseline (linear mixed-model ANOVA and Satterthwaite’s t-tests using session, two-tailed). Points show mean performance of each monkey. Bars represent mean ± s.e.m. of test sessions.
(f) percentage correct choices by monkeys in NML task averaged over the first and second weeks after baseline or Veh treatment (Ctrl) (n, same as Figure 1e) or rhesus KL (10 μg kg−1) (n, same as Figure 1e; 32 sessions between 1–7 d and 14 sessions between 8–14 d), p = 0.0080 (1–7 d) and p = 0.0021 (8–14 d) versus Veh + baseline (linear mixed-model ANOVA and Satterthwaite’s t-tests using sessions, two-tailed). Points indicate sessions. Filled circles indicate mean ± s.e.m. of sessions. Dashed line shows mean control performance.
(g) percentage increase in cognition in HML task at varying doses of rhesus KL treatment at 10 μg−1 averaged over the course of testing (n = 11 sessions from 9 monkeys; 6 female and 3 male), 20 μg kg−1 (n = 7 sessions from 7 monkeys; 5 female and 2 male) or 30 μg kg−1 (n = 13 sessions from 13 monkeys; 9 females and 4 males) compared to Veh treatment (n = 26 sessions from 18 monkeys; 13 females and 5 males). p = 0.0077 versus Veh (linear mixed-model ANOVA and Satterthwaite’s t-tests using sessions, two-tailed).
(h) percentage increase in cognition in NML task at varying doses of rhesus KL treatment at 10 μg kg−1 averaged over the course of testing (n = 46 sessions from 9 monkeys; 6 female and 3 male), 20 μg kg−1 (n = 31 sessions from 7 monkeys; 5 female and 2 male) or 30 μg kg−1 (n = 40 sessions from 13 monkeys; 9 females and 4 males) compared to Veh + baseline (n = 71 sessions from 17 monkeys; 12 females and 5 males) p = 0.0006 versus Veh (linear mixed-model ANOVA and Satterthwaite’s t-tests using sessions, two-tailed).
…As KL has pleiotropic actions, including on insulin7 and FGF signaling8, Wnt9 and NMDAR functions3,4,5, it is interesting to speculate that the specificity of its action at low doses represents a balanced, multimodal effect across signaling systems that inherently benefits biological substrates of cognition. Higher doses of KL, beyond what is experienced over the human lifespan, could differentially impact signaling systems to create imbalances that no longer enhance cognition. Whether even lower doses of KL than those tested could also enhance cognition remains to be determined. Further, because peripherally injected KL does not cross into the brain5,15, peripheral messengers that transduce its signals into the brain should be identified.