Shibata K, Chen C, Tai XY, Manohar SG, Husain M.
Impact of APOE, Klotho, and sex on cognitive decline with aging.
Proc Natl Acad Sci U S A. 2025 Feb 11;122(6):e2416042122. Epub 2025 Feb 4
PubMed.
This study examined the sex-dependent effects of APOE ε4 genotype and a KLOTHO haplotype on aging-associated differences in cognitive performance. It stands out for testing the genetic associations in a large sample including more than 350 000 subjects between 40 – 70 years of age from the UK Biobank, and is thus well suited to settle some of the questions previous, less powered studies have left to address.
A surprising finding was that the KL-VS haplotype was not associated with a cognitive benefit at any age. Over 25 years ago, the KLOTHO gene was serendipitously discovered when transgenic mutation of it elicited accelerated physical aging in mice (Kuro-o et al., 1977), but overexpressing Klotho led to increased live span (Kurosu et al., 2005). A first study in humans suggested that the KL-VS haplotype is associated with increased longevity (Arking et al., 2002), although other studies yielded mixed results. This current study provides compelling evidence against beneficial effect of the Klotho haplotype on cognition in normal aging.
It should be mentioned, though, that Klotho protein levels are also subject to the influence of factors other than the KL-VS haplotype. This raises the question of whether alterations in Klotho levels attributable to the KL-VS haplotype are sufficiently large to cause beneficial effects in normal aging, or, alternatively, a larger increased in Klotho levels, such as induced by pharmaceutical intervention, is necessary to affect cognition, such as shown recently in non-human primates treated with Klotho protein (Grøntvedt et al., 2022).
Another remaining question is whether the KL-VS haplotype is beneficial in disease conditions. For Alzheimer’s disease, we and several others groups showed that the KL-VS heterozygosity is associated with lower levels of Aβ and tau pathology in patients with AD (Belloy et al., 2020; Neitzel et al., 2021). Furthermore, biofluid levels of the Klotho protein assessed in the CSF were decreased in AD, and higher CSF levels of Klotho were associated with less abnormal CSF levels of p-tau and Aβ4. Therefore, Klotho may exert beneficial effects in disease conditions including AD.
Klotho is involved in various biological processes such as growth factor signaling phosphate/calcium metabolism. However, little is known about what factors drive Klotho protein levels and which biological processes downstream of Klotho are crucial in conditions such as AD. Future studies are needed to address the biological pathways of Klotho that are potentially disease-specifically effective to enhance cognitive resilience.
References:
Kuro-o M, Matsumura Y, Aizawa H, Kawaguchi H, Suga T, Utsugi T, Ohyama Y, Kurabayashi M, Kaname T, Kume E, Iwasaki H, Iida A, Shiraki-Iida T, Nishikawa S, Nagai R, Nabeshima YI.
Mutation of the mouse klotho gene leads to a syndrome resembling ageing.
Nature. 1997 Nov 6;390(6655):45-51.
PubMed.
Kurosu H, Yamamoto M, Clark JD, Pastor JV, Nandi A, Gurnani P, McGuinness OP, Chikuda H, Yamaguchi M, Kawaguchi H, Shimomura I, Takayama Y, Herz J, Kahn CR, Rosenblatt KP, Kuro-O M.
Suppression of aging in mice by the hormone Klotho.
Science. 2005 Sep 16;309(5742):1829-33.
PubMed.
Arking DE, Krebsova A, Macek M Sr, Macek M Jr, Arking A, Mian IS, Fried L, Hamosh A, Dey S, McIntosh I, Dietz HC.
Association of human aging with a functional variant of klotho.
Proc Natl Acad Sci U S A. 2002 Jan 22;99(2):856-61. Epub 2002 Jan 15
PubMed.
Grøntvedt GR, Sando SB, Lauridsen C, Bråthen G, White LR, Salvesen Ø, Aarsland D, Hessen E, Fladby T, Waterloo K, Scheffler K.
Association of Klotho Protein Levels and KL-VS Heterozygosity With Alzheimer Disease and Amyloid and Tau Burden.
JAMA Netw Open. 2022 Nov 1;5(11):e2243232.
PubMed.
Belloy ME, Napolioni V, Han SS, Le Guen Y, Greicius MD, Alzheimer’s Disease Neuroimaging Initiative.
Association of Klotho-VS Heterozygosity With Risk of Alzheimer Disease in Individuals Who Carry APOE4.
JAMA Neurol. 2020 Jul 1;77(7):849-862.
PubMed.
Neitzel J, Franzmeier N, Rubinski A, Dichgans M, Brendel M, Alzheimer’s Disease Neuroimaging Initiative (ADNI), Malik R, Ewers M.
KL-VS heterozygosity is associated with lower amyloid-dependent tau accumulation and memory impairment in Alzheimer's disease.
Nat Commun. 2021 Jun 22;12(1):3825.
PubMed.
The longevity gene Klotho enhances cognition prolongs lifespan in mice and humans (Kuro-o et al., 2009). Leveraging data from the UK Biobank, Shibata et al. challenge this notion, demonstrating that carriers of the “protective” Klotho-VS haplotype, which is associated with enhanced longevity (Arking et al., 2002) and improved cognition (Dubal et al., 2014), exhibit no cognitive advantage over non-carriers. The authors reasonably suggest that this discrepancy may reflect a weak correlation between the Klotho genotype and the actual levels of the Klotho brain protein. Notably, while the Klotho-VS genotype associates with increased levels of the serum Klotho protein (Yokoyama et al., 2017), its effect on the Klotho protein levels in the brain is yet to be established.
Adding to this complexity, Klotho is a pleiotropic gene, affecting multiple biological pathways, and its mutations correspondingly trigger widespread downstream effects. In a recent study, we demonstrated that Klotho knockout induces a profound shift in the murine brain transcriptome, altering the expression of 10 percent of all transcripts, including various non-coding RNAs, such as long non-coding RNAs, microRNAs, and short tRNA fragments, that regulate transcription and splicing (Dubnov et al., 2024). Given this extensive regulatory landscape, which appears to be activated to perturb Klotho downstream processes, it stands to reason that the impact of the Klotho-VS genotype may be blocked by multiple layers of molecular modulation. A follow-up comparison of the brain transcriptomic profiles from Klotho-VS carriers and age-matched controls may uncover the regulatory mechanism(s) that mediate the cognitive impact of the Klotho-VS genotype.
References:
Kuro-o M.
Klotho and aging.
Biochim Biophys Acta. 2009 Oct;1790(10):1049-58. Epub 2009 Feb 20
PubMed.
Arking DE, Krebsova A, Macek M Sr, Macek M Jr, Arking A, Mian IS, Fried L, Hamosh A, Dey S, McIntosh I, Dietz HC.
Association of human aging with a functional variant of klotho.
Proc Natl Acad Sci U S A. 2002 Jan 22;99(2):856-61. Epub 2002 Jan 15
PubMed.
Dubal DB, Yokoyama JS, Zhu L, Broestl L, Worden K, Wang D, Sturm VE, Kim D, Klein E, Yu GQ, Ho K, Eilertson KE, Yu L, Kuro-o M, De Jager PL, Coppola G, Small GW, Bennett DA, Kramer JH, Abraham CR, Miller BL, Mucke L.
Life extension factor klotho enhances cognition.
Cell Rep. 2014 May 22;7(4):1065-76. Epub 2014 May 10
PubMed.
Yokoyama JS, Marx G, Brown JA, Bonham LW, Wang D, Coppola G, Seeley WW, Rosen HJ, Miller BL, Kramer JH, Dubal DB.
Systemic klotho is associated with KLOTHO variation and predicts intrinsic cortical connectivity in healthy human aging.
Brain Imaging Behav. 2016 Oct 6;
PubMed.
Dubnov S, Bennett ER, Yayon N, Yakov O, Bennett DA, Seshadri S, Mufson E, Tzur Y, Greenberg D, Kuro-O M, Paldor I, Abraham CR, Soreq H.
Knockout of the longevity gene Klotho perturbs aging and Alzheimer's disease-linked brain microRNAs and tRNA fragments.
Commun Biol. 2024 Jun 11;7(1):720.
PubMed.
This did not find any significant effects of Klotho-VS heterozygosity on cognitive function across aging, nor on brain gray matter volume in healthy individuals.
These data do not confirm previous findings that reported improved memory and executive function and a larger prefrontal cortex in Klotho-VS carriers. Furthermore, according to the Shibata data, Klotho-VS heterozygosity did not protect individuals with ApoE4, contrary to earlier findings (Belloy et al., 2020; Porter et al., 2019)
What may explain the conflicting results?
We commented in the past on Alzforum, when previous papers on this topic were published, that it is unclear if the effects seen with the Klotho-VS heterozygosity are due to higher levels of Klotho in blood, or the result of the two amino acid substitutions (FC to VS) leading to a modified structure and bioactivity of the Klotho protein. For example, we have reported that KL-VS interacts with the FGF receptor differently than the wildtype Klotho (Tucker-Zhou et al., 2013).
Shibata et al. correctly mentioned as a drawback that the UKB database used in the study did not have Klotho levels in blood (or CSF, which may be even more relevant), thus we cannot conclude with certainty that it is the KL-VS genotype versus the Klotho blood levels that affect cognition and cortex volume.
Additional potential explanations for the conflicting results (previous studies versus the recent Shibata study) include:
Size of the cohort (almost three orders of magnitude larger in the new study) which has an enhanced statistical power
Different number and types of cognition and behavioral studies
Differences in age range (for example 52-85 in Dubal et al., 2014 versus 40-70 in Shibata et al.), which may indicate brain changes despite the description of the individuals as cognitively “healthy”
There is a time window for the effects of KL-VS which was possibly missed in some case
The precise brain regions found to be affected in KL-VS carriers by MRI
Differences in statistical analyses among studies
Klotho is an enigmatic protein with pleiotropic functions affecting all organs in a beneficial way. Its protective roles, which decrease with age, lead to enhanced health span and lifespan (Abraham and Li., 2022). Despite more than 4,000 articles published on Klotho, there is still much to be discovered.
References:
Belloy ME, Napolioni V, Han SS, Le Guen Y, Greicius MD, Alzheimer’s Disease Neuroimaging Initiative.
Association of Klotho-VS Heterozygosity With Risk of Alzheimer Disease in Individuals Who Carry APOE4.
JAMA Neurol. 2020 Jul 1;77(7):849-862.
PubMed.
Porter T, Burnham SC, Milicic L, Savage G, Maruff P, Lim YY, Ames D, Masters CL, Martins RN, Rainey-Smith S, Rowe CC, Salvado O, Groth D, Verdile G, Villemagne VL, Laws SM.
Klotho allele status is not associated with Aβ and APOE ε4-related cognitive decline in preclinical Alzheimer's disease.
Neurobiol Aging. 2019 Apr;76:162-165. Epub 2019 Jan 6
PubMed.
Tucker Zhou TB, King GD, Chen C, Abraham CR.
Biochemical and functional characterization of the klotho-VS polymorphism implicated in aging and disease risk.
J Biol Chem. 2013 Dec 20;288(51):36302-11. Epub 2013 Nov 11
PubMed.
Dubal DB, Yokoyama JS, Zhu L, Broestl L, Worden K, Wang D, Sturm VE, Kim D, Klein E, Yu GQ, Ho K, Eilertson KE, Yu L, Kuro-o M, De Jager PL, Coppola G, Small GW, Bennett DA, Kramer JH, Abraham CR, Miller BL, Mucke L.
Life extension factor klotho enhances cognition.
Cell Rep. 2014 May 22;7(4):1065-76. Epub 2014 May 10
PubMed.
Abraham CR, Li A.
Aging-suppressor Klotho: Prospects in diagnostics and therapeutics.
Ageing Res Rev. 2022 Dec;82:101766. Epub 2022 Oct 22
PubMed.
This study represents an important addition to the ongoing research into the interaction of age, sex, Klotho-VS, and APOE4, on cognition and brain resilience. The authors leverage a large sample of cognitively normal subjects, ages 40-70, in the UK Biobank, where they had access to cross-sectional cognitive test scores from automated touch-screen tasks (N=320k) and brain imaging metrics (N=29k). This large sample size in a single cohort study provides a unique platform to investigate these questions in healthy subjects during mid and early late life, prior to the typical age range during which cognitive symptoms and dementia become more prevalent.
The authors evaluated different APOE strata, APOE2/2+2/3, APOE3/3+3/4, and APOE4/4, as well Klotho-VS heterozygotes versus non-heterozygotes. One primary result was the age-specific sex differences for the effect of APOE4/4 on cognition and volumes of the hippocampus and amygdala. The protective effect of APOE2/2+2/3 also appeared to be age-specific (55-65 years) but was quite modest, especially considering that the APOE2/2+2/3 group was contrasted to the APOE3/3+3/4 group (rather than APOE3/3 carriers only). The anticipated protective effect of Klotho-VS heterozygosity was not observed at any age or APOE group.
There have indeed been some variable reports regarding the protective effect of Klotho-VS heterozygosity with regard to cognition and brain resilience. Some of the prominent factors that may explain this heterogeneity across studies are the considered age range, cross-sectional versus longitudinal designs, sample sizes, and stratification according to APOE4 status. Most notably, the effect of Klotho-VS heterozygosity may be age and APOE4 specific.
In the context of Alzheimer's disease pathology and risk, we and others have shown that Klotho-VS heterozygosity is associated with reduced amyloid and tau burden during the age range of 60-80 years in cognitively normal APOE4 positive (APOE2/4+3/4+4/4) subjects (Erickson et al., 2019; Belloy et al., 2020; Belloy et al., 2021; Neitzel et al., 2021; Ali et al., 2022). We previously also showed, with longitudinal data, that Klotho-VS heterozygosity was associated with reduced conversion rates to mild cognitive impairment or Alzheimer's disease in APOE4 carriers (APOE2/4+3/4+4/4). The latter effect became apparent around 75 years of age, which is five years past the oldest age considered by Shibata et al.
Further, Shibata et al. observed a quite modest protective effect of APOE2/2+2/3 on cognition, but the protective effect on cognition and Alzheimer's disease risk appears to be stronger at later ages (Walters et al. 2023; Belloy et al. 2023). Given the expected prominent protective effect of APOE2, this may partially explain the lack of a protective effect of Klotho-VS heterozygosity in the study by Shibata et al.
Lastly, from the preceding literature, it is not clear whether the effect of Klotho-VS heterozygosity differs in APOE4/4 compared to APOE3/4 carriers, as those studies only evaluated the aggregate group of APOE4 subjects (APOE2/4+3/4+4/4). It would thus be interesting to know what effect Shibata et al. would observe using similar APOE4 stratification, or when considering just APOE3/4 subjects.
In summary, Shibata et al. contribute importantly to our understanding of the potential protective effect of Klotho-VS heterozygosity on cognition and brain resilience. Their cross-sectional findings suggest Klotho-VS heterozygosity confers no benefit during the age range of 40 to 70 years, regardless of APOE genotype status. When combined with knowledge of other studies conducted at later ages that did observe positive effects, this emphasizes an important need for future research to longitudinally track the association of Klotho-VS heterozygosity with cognition at the transition of 60 to 80 years of age (and beyond). Large-scale cross-sectional studies or meta-analyses with subjects ages 60 to 100 years of age may also help resolve some of the previous heterogeneity observed by smaller studies in this age range. Lastly, with increasing cohort sample sizes, it becomes possible and relevant to parse out the potential modifying effect of APOE into it's individual genotypes (APOE2/2, 2/3, 3/3, 2/4, 3/4, 4/4).
References:
Erickson CM, Schultz SA, Oh JM, Darst BF, Ma Y, Norton D, Betthauser T, Gallagher CL, Carlsson CM, Bendlin BB, Asthana S, Hermann BP, Sager MA, Blennow K, Zetterberg H, Engelman CD, Christian BT, Johnson SC, Dubal DB, Okonkwo OC.
KLOTHO heterozygosity attenuates APOE4-related amyloid burden in preclinical AD.
Neurology. 2019 Apr 16;92(16):e1878-e1889. Epub 2019 Mar 13
PubMed.
Belloy ME, Napolioni V, Han SS, Le Guen Y, Greicius MD, Alzheimer’s Disease Neuroimaging Initiative.
Association of Klotho-VS Heterozygosity With Risk of Alzheimer Disease in Individuals Who Carry APOE4.
JAMA Neurol. 2020 Jul 1;77(7):849-862.
PubMed.
Belloy ME, Eger SJ, Le Guen Y, Napolioni V, Deters KD, Yang HS, Scelsi MA, Porter T, James SN, Wong A, Schott JM, Sperling RA, Laws SM, Mormino EC, He Z, Han SS, Altmann A, Greicius MD, A4 Study Team, Insight 46 Study Team, Australian Imaging Biomarkers and Lifestyle (AIBL) Study, Alzheimer's Disease Neuroimaging Initiative.
KL∗VS heterozygosity reduces brain amyloid in asymptomatic at-risk APOE∗4 carriers.
Neurobiol Aging. 2021 May;101:123-129. Epub 2021 Jan 23
PubMed.
Neitzel J, Franzmeier N, Rubinski A, Dichgans M, Brendel M, Alzheimer’s Disease Neuroimaging Initiative (ADNI), Malik R, Ewers M.
KL-VS heterozygosity is associated with lower amyloid-dependent tau accumulation and memory impairment in Alzheimer's disease.
Nat Commun. 2021 Jun 22;12(1):3825.
PubMed.
Ali M, Sung YJ, Wang F, Fernández MV, Morris JC, Fagan AM, Blennow K, Zetterberg H, Heslegrave A, Johansson PM, Svensson J, Nellgård B, Lleó A, Alcolea D, Clarimon J, Rami L, Molinuevo JL, Suárez-Calvet M, Morenas-Rodríguez E, Kleinberger G, Haass C, Ewers M, Levin J, Farlow MR, Perrin RJ, Alzheimer’s Disease Neuroimaging Initiative (ADNI), Dominantly Inherited Alzheimer Network (DIAN), Cruchaga C.
Leveraging large multi-center cohorts of Alzheimer disease endophenotypes to understand the role of Klotho heterozygosity on disease risk.
PLoS One. 2022;17(5):e0267298. Epub 2022 May 26
PubMed.
Walters S, Contreras AG, Eissman JM, Mukherjee S, Lee ML, Choi SE, Scollard P, Trittschuh EH, Mez JB, Bush WS, Kunkle BW, Naj AC, Peterson A, Gifford KA, Cuccaro ML, Cruchaga C, Pericak-Vance MA, Farrer LA, Wang LS, Haines JL, Jefferson AL, Kukull WA, Keene CD, Saykin AJ, Thompson PM, Martin ER, Bennett DA, Barnes LL, Schneider JA, Crane PK, Hohman TJ, Dumitrescu L, Alzheimer’s Disease Neuroimaging Initiative, Alzheimer’s Disease Genetics Consortium, and Alzheimer’s Disease Sequencing Project.
Associations of Sex, Race, and Apolipoprotein E Alleles With Multiple Domains of Cognition Among Older Adults.
JAMA Neurol. 2023 Sep 1;80(9):929-939.
PubMed.
Belloy ME, Andrews SJ, Le Guen Y, Cuccaro M, Farrer LA, Napolioni V, Greicius MD.
APOE Genotype and Alzheimer Disease Risk Across Age, Sex, and Population Ancestry.
JAMA Neurol. 2023 Dec 1;80(12):1284-1294.
PubMed.
Comments
Ludwig Maximilian University
This study examined the sex-dependent effects of APOE ε4 genotype and a KLOTHO haplotype on aging-associated differences in cognitive performance. It stands out for testing the genetic associations in a large sample including more than 350 000 subjects between 40 – 70 years of age from the UK Biobank, and is thus well suited to settle some of the questions previous, less powered studies have left to address.
A surprising finding was that the KL-VS haplotype was not associated with a cognitive benefit at any age. Over 25 years ago, the KLOTHO gene was serendipitously discovered when transgenic mutation of it elicited accelerated physical aging in mice (Kuro-o et al., 1977), but overexpressing Klotho led to increased live span (Kurosu et al., 2005). A first study in humans suggested that the KL-VS haplotype is associated with increased longevity (Arking et al., 2002), although other studies yielded mixed results. This current study provides compelling evidence against beneficial effect of the Klotho haplotype on cognition in normal aging.
It should be mentioned, though, that Klotho protein levels are also subject to the influence of factors other than the KL-VS haplotype. This raises the question of whether alterations in Klotho levels attributable to the KL-VS haplotype are sufficiently large to cause beneficial effects in normal aging, or, alternatively, a larger increased in Klotho levels, such as induced by pharmaceutical intervention, is necessary to affect cognition, such as shown recently in non-human primates treated with Klotho protein (Grøntvedt et al., 2022).
Another remaining question is whether the KL-VS haplotype is beneficial in disease conditions. For Alzheimer’s disease, we and several others groups showed that the KL-VS heterozygosity is associated with lower levels of Aβ and tau pathology in patients with AD (Belloy et al., 2020; Neitzel et al., 2021). Furthermore, biofluid levels of the Klotho protein assessed in the CSF were decreased in AD, and higher CSF levels of Klotho were associated with less abnormal CSF levels of p-tau and Aβ4. Therefore, Klotho may exert beneficial effects in disease conditions including AD.
Klotho is involved in various biological processes such as growth factor signaling phosphate/calcium metabolism. However, little is known about what factors drive Klotho protein levels and which biological processes downstream of Klotho are crucial in conditions such as AD. Future studies are needed to address the biological pathways of Klotho that are potentially disease-specifically effective to enhance cognitive resilience.
References:
Kuro-o M, Matsumura Y, Aizawa H, Kawaguchi H, Suga T, Utsugi T, Ohyama Y, Kurabayashi M, Kaname T, Kume E, Iwasaki H, Iida A, Shiraki-Iida T, Nishikawa S, Nagai R, Nabeshima YI. Mutation of the mouse klotho gene leads to a syndrome resembling ageing. Nature. 1997 Nov 6;390(6655):45-51. PubMed.
Kurosu H, Yamamoto M, Clark JD, Pastor JV, Nandi A, Gurnani P, McGuinness OP, Chikuda H, Yamaguchi M, Kawaguchi H, Shimomura I, Takayama Y, Herz J, Kahn CR, Rosenblatt KP, Kuro-O M. Suppression of aging in mice by the hormone Klotho. Science. 2005 Sep 16;309(5742):1829-33. PubMed.
Arking DE, Krebsova A, Macek M Sr, Macek M Jr, Arking A, Mian IS, Fried L, Hamosh A, Dey S, McIntosh I, Dietz HC. Association of human aging with a functional variant of klotho. Proc Natl Acad Sci U S A. 2002 Jan 22;99(2):856-61. Epub 2002 Jan 15 PubMed.
Grøntvedt GR, Sando SB, Lauridsen C, Bråthen G, White LR, Salvesen Ø, Aarsland D, Hessen E, Fladby T, Waterloo K, Scheffler K. Association of Klotho Protein Levels and KL-VS Heterozygosity With Alzheimer Disease and Amyloid and Tau Burden. JAMA Netw Open. 2022 Nov 1;5(11):e2243232. PubMed.
Belloy ME, Napolioni V, Han SS, Le Guen Y, Greicius MD, Alzheimer’s Disease Neuroimaging Initiative. Association of Klotho-VS Heterozygosity With Risk of Alzheimer Disease in Individuals Who Carry APOE4. JAMA Neurol. 2020 Jul 1;77(7):849-862. PubMed.
Neitzel J, Franzmeier N, Rubinski A, Dichgans M, Brendel M, Alzheimer’s Disease Neuroimaging Initiative (ADNI), Malik R, Ewers M. KL-VS heterozygosity is associated with lower amyloid-dependent tau accumulation and memory impairment in Alzheimer's disease. Nat Commun. 2021 Jun 22;12(1):3825. PubMed.
The Hebrew University of Jerusalem
The longevity gene Klotho enhances cognition prolongs lifespan in mice and humans (Kuro-o et al., 2009). Leveraging data from the UK Biobank, Shibata et al. challenge this notion, demonstrating that carriers of the “protective” Klotho-VS haplotype, which is associated with enhanced longevity (Arking et al., 2002) and improved cognition (Dubal et al., 2014), exhibit no cognitive advantage over non-carriers. The authors reasonably suggest that this discrepancy may reflect a weak correlation between the Klotho genotype and the actual levels of the Klotho brain protein. Notably, while the Klotho-VS genotype associates with increased levels of the serum Klotho protein (Yokoyama et al., 2017), its effect on the Klotho protein levels in the brain is yet to be established.
Adding to this complexity, Klotho is a pleiotropic gene, affecting multiple biological pathways, and its mutations correspondingly trigger widespread downstream effects. In a recent study, we demonstrated that Klotho knockout induces a profound shift in the murine brain transcriptome, altering the expression of 10 percent of all transcripts, including various non-coding RNAs, such as long non-coding RNAs, microRNAs, and short tRNA fragments, that regulate transcription and splicing (Dubnov et al., 2024). Given this extensive regulatory landscape, which appears to be activated to perturb Klotho downstream processes, it stands to reason that the impact of the Klotho-VS genotype may be blocked by multiple layers of molecular modulation. A follow-up comparison of the brain transcriptomic profiles from Klotho-VS carriers and age-matched controls may uncover the regulatory mechanism(s) that mediate the cognitive impact of the Klotho-VS genotype.
References:
Kuro-o M. Klotho and aging. Biochim Biophys Acta. 2009 Oct;1790(10):1049-58. Epub 2009 Feb 20 PubMed.
Arking DE, Krebsova A, Macek M Sr, Macek M Jr, Arking A, Mian IS, Fried L, Hamosh A, Dey S, McIntosh I, Dietz HC. Association of human aging with a functional variant of klotho. Proc Natl Acad Sci U S A. 2002 Jan 22;99(2):856-61. Epub 2002 Jan 15 PubMed.
Dubal DB, Yokoyama JS, Zhu L, Broestl L, Worden K, Wang D, Sturm VE, Kim D, Klein E, Yu GQ, Ho K, Eilertson KE, Yu L, Kuro-o M, De Jager PL, Coppola G, Small GW, Bennett DA, Kramer JH, Abraham CR, Miller BL, Mucke L. Life extension factor klotho enhances cognition. Cell Rep. 2014 May 22;7(4):1065-76. Epub 2014 May 10 PubMed.
Yokoyama JS, Marx G, Brown JA, Bonham LW, Wang D, Coppola G, Seeley WW, Rosen HJ, Miller BL, Kramer JH, Dubal DB. Systemic klotho is associated with KLOTHO variation and predicts intrinsic cortical connectivity in healthy human aging. Brain Imaging Behav. 2016 Oct 6; PubMed.
Dubnov S, Bennett ER, Yayon N, Yakov O, Bennett DA, Seshadri S, Mufson E, Tzur Y, Greenberg D, Kuro-O M, Paldor I, Abraham CR, Soreq H. Knockout of the longevity gene Klotho perturbs aging and Alzheimer's disease-linked brain microRNAs and tRNA fragments. Commun Biol. 2024 Jun 11;7(1):720. PubMed.
ADvantage
This did not find any significant effects of Klotho-VS heterozygosity on cognitive function across aging, nor on brain gray matter volume in healthy individuals.
These data do not confirm previous findings that reported improved memory and executive function and a larger prefrontal cortex in Klotho-VS carriers. Furthermore, according to the Shibata data, Klotho-VS heterozygosity did not protect individuals with ApoE4, contrary to earlier findings (Belloy et al., 2020; Porter et al., 2019)
What may explain the conflicting results?
We commented in the past on Alzforum, when previous papers on this topic were published, that it is unclear if the effects seen with the Klotho-VS heterozygosity are due to higher levels of Klotho in blood, or the result of the two amino acid substitutions (FC to VS) leading to a modified structure and bioactivity of the Klotho protein. For example, we have reported that KL-VS interacts with the FGF receptor differently than the wildtype Klotho (Tucker-Zhou et al., 2013).
Shibata et al. correctly mentioned as a drawback that the UKB database used in the study did not have Klotho levels in blood (or CSF, which may be even more relevant), thus we cannot conclude with certainty that it is the KL-VS genotype versus the Klotho blood levels that affect cognition and cortex volume.
Additional potential explanations for the conflicting results (previous studies versus the recent Shibata study) include:
Klotho is an enigmatic protein with pleiotropic functions affecting all organs in a beneficial way. Its protective roles, which decrease with age, lead to enhanced health span and lifespan (Abraham and Li., 2022). Despite more than 4,000 articles published on Klotho, there is still much to be discovered.
References:
Belloy ME, Napolioni V, Han SS, Le Guen Y, Greicius MD, Alzheimer’s Disease Neuroimaging Initiative. Association of Klotho-VS Heterozygosity With Risk of Alzheimer Disease in Individuals Who Carry APOE4. JAMA Neurol. 2020 Jul 1;77(7):849-862. PubMed.
Porter T, Burnham SC, Milicic L, Savage G, Maruff P, Lim YY, Ames D, Masters CL, Martins RN, Rainey-Smith S, Rowe CC, Salvado O, Groth D, Verdile G, Villemagne VL, Laws SM. Klotho allele status is not associated with Aβ and APOE ε4-related cognitive decline in preclinical Alzheimer's disease. Neurobiol Aging. 2019 Apr;76:162-165. Epub 2019 Jan 6 PubMed.
Tucker Zhou TB, King GD, Chen C, Abraham CR. Biochemical and functional characterization of the klotho-VS polymorphism implicated in aging and disease risk. J Biol Chem. 2013 Dec 20;288(51):36302-11. Epub 2013 Nov 11 PubMed.
Dubal DB, Yokoyama JS, Zhu L, Broestl L, Worden K, Wang D, Sturm VE, Kim D, Klein E, Yu GQ, Ho K, Eilertson KE, Yu L, Kuro-o M, De Jager PL, Coppola G, Small GW, Bennett DA, Kramer JH, Abraham CR, Miller BL, Mucke L. Life extension factor klotho enhances cognition. Cell Rep. 2014 May 22;7(4):1065-76. Epub 2014 May 10 PubMed.
Abraham CR, Li A. Aging-suppressor Klotho: Prospects in diagnostics and therapeutics. Ageing Res Rev. 2022 Dec;82:101766. Epub 2022 Oct 22 PubMed.
Washington University in St. Louis
This study represents an important addition to the ongoing research into the interaction of age, sex, Klotho-VS, and APOE4, on cognition and brain resilience. The authors leverage a large sample of cognitively normal subjects, ages 40-70, in the UK Biobank, where they had access to cross-sectional cognitive test scores from automated touch-screen tasks (N=320k) and brain imaging metrics (N=29k). This large sample size in a single cohort study provides a unique platform to investigate these questions in healthy subjects during mid and early late life, prior to the typical age range during which cognitive symptoms and dementia become more prevalent.
The authors evaluated different APOE strata, APOE2/2+2/3, APOE3/3+3/4, and APOE4/4, as well Klotho-VS heterozygotes versus non-heterozygotes. One primary result was the age-specific sex differences for the effect of APOE4/4 on cognition and volumes of the hippocampus and amygdala. The protective effect of APOE2/2+2/3 also appeared to be age-specific (55-65 years) but was quite modest, especially considering that the APOE2/2+2/3 group was contrasted to the APOE3/3+3/4 group (rather than APOE3/3 carriers only). The anticipated protective effect of Klotho-VS heterozygosity was not observed at any age or APOE group.
There have indeed been some variable reports regarding the protective effect of Klotho-VS heterozygosity with regard to cognition and brain resilience. Some of the prominent factors that may explain this heterogeneity across studies are the considered age range, cross-sectional versus longitudinal designs, sample sizes, and stratification according to APOE4 status. Most notably, the effect of Klotho-VS heterozygosity may be age and APOE4 specific.
In the context of Alzheimer's disease pathology and risk, we and others have shown that Klotho-VS heterozygosity is associated with reduced amyloid and tau burden during the age range of 60-80 years in cognitively normal APOE4 positive (APOE2/4+3/4+4/4) subjects (Erickson et al., 2019; Belloy et al., 2020; Belloy et al., 2021; Neitzel et al., 2021; Ali et al., 2022). We previously also showed, with longitudinal data, that Klotho-VS heterozygosity was associated with reduced conversion rates to mild cognitive impairment or Alzheimer's disease in APOE4 carriers (APOE2/4+3/4+4/4). The latter effect became apparent around 75 years of age, which is five years past the oldest age considered by Shibata et al.
Further, Shibata et al. observed a quite modest protective effect of APOE2/2+2/3 on cognition, but the protective effect on cognition and Alzheimer's disease risk appears to be stronger at later ages (Walters et al. 2023; Belloy et al. 2023). Given the expected prominent protective effect of APOE2, this may partially explain the lack of a protective effect of Klotho-VS heterozygosity in the study by Shibata et al.
Lastly, from the preceding literature, it is not clear whether the effect of Klotho-VS heterozygosity differs in APOE4/4 compared to APOE3/4 carriers, as those studies only evaluated the aggregate group of APOE4 subjects (APOE2/4+3/4+4/4). It would thus be interesting to know what effect Shibata et al. would observe using similar APOE4 stratification, or when considering just APOE3/4 subjects.
In summary, Shibata et al. contribute importantly to our understanding of the potential protective effect of Klotho-VS heterozygosity on cognition and brain resilience. Their cross-sectional findings suggest Klotho-VS heterozygosity confers no benefit during the age range of 40 to 70 years, regardless of APOE genotype status. When combined with knowledge of other studies conducted at later ages that did observe positive effects, this emphasizes an important need for future research to longitudinally track the association of Klotho-VS heterozygosity with cognition at the transition of 60 to 80 years of age (and beyond). Large-scale cross-sectional studies or meta-analyses with subjects ages 60 to 100 years of age may also help resolve some of the previous heterogeneity observed by smaller studies in this age range. Lastly, with increasing cohort sample sizes, it becomes possible and relevant to parse out the potential modifying effect of APOE into it's individual genotypes (APOE2/2, 2/3, 3/3, 2/4, 3/4, 4/4).
References:
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Belloy ME, Andrews SJ, Le Guen Y, Cuccaro M, Farrer LA, Napolioni V, Greicius MD. APOE Genotype and Alzheimer Disease Risk Across Age, Sex, and Population Ancestry. JAMA Neurol. 2023 Dec 1;80(12):1284-1294. PubMed.
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