Researchers are beginning to uncover genetic factors that modify the ability of amyloid plaques to spur tau pathology. Now, scientists led by Michael Ewers at Ludwig-Maximilians University, Munich, add a longevity gene into the mix. In a preprint posted July 31 to medRxiv, they propose that for a given amount of amyloid, people who carry one copy of the protective VS klotho variant accumulate fewer neurofibrillary tangles than noncarriers. This lighter tangle load correlated with better episodic memory. The finding sheds light on how klotho protects the brain, and could point to therapeutic strategies, the authors suggest. “This study adds evidence that the association between amyloid and tau pathologies depends on genotype,” Bernard Hanseeuw at Massachusetts General Hospital, Boston, wrote to Alzforum.

  • People with one copy of the klotho VS variant have fewer tangles for a given amyloid load.
  • Their memories are better, too.
  • These protective effects are stronger in APOE4 carriers.

Others found the study a significant advance. “[The authors] are the first to publish a beneficial klotho-tau-cognition relationship,” Carmela Abraham at Boston University School of Medicine wrote to Alzforum. Abraham, who first implicated klotho in brain aging, called the work elegant and said it strengthened the rationale for boosting klotho to treat AD (full comment below). Michael Belloy at Stanford University noted that klotho initially was believed to be a resilience factor that helped the brain cope with plaques and tangles, but recent evidence suggests it directly protects against their accumulation. “It would be promising to figure out the molecular machinery behind this,” Belloy said.

High klotho levels associate with longer life and sharper cognition (Dec 2007 conference news; May 2013 conference newsMay 2014 news). The protective VS variant occurs in about 25 percent of the population. It comprises two SNPs, F352V and C370S, that are inherited together. One copy of VS boosts klotho expression, leading to bigger brains and better memory (Yokoyama et al., 2015; de Vries et al., 2017). Curiously, two copies of the VS variant lower klotho production and confer no benefit.

Tangle Suppression. The relationship between plaques and tangles seen in controls (gray), is weaker in people who carry the protective klotho-VS variant (blue). [Courtesy of Neitzel et al.]

Other groups previously found that the VS variant best protects APOE4 carriers, who are more at risk for amyloid accumulation. The variant reduces their plaque burden and cuts Alzheimer’s risk by a third (Erickson et al., 2019; Apr 2020 news). However, no one had examined klotho’s effect on tangles.

To investigate, first author Julia Neitzel analyzed data from 354 participants in ADNI3, all of whom had amyloid and tau PET scans. Of these, 213 were cognitively healthy, 111 had mild cognitive impairment, and 30 had AD. Their average age was 71. About a quarter of the cohort, 92 people, were heterozygous VS carriers.

At a given amyloid burden, the VS carriers bound less flortaucipir tau tracer in their brains than did noncarriers. This held true in both the inferior temporal cortex, where tau pathology usually starts, and in the whole brain. The attenuation remained significant after adjusting for age, sex, education, disease status, and APOE genotype.

Within this overall signal, the researchers found hints of stronger protection in APOE4 carriers. When they stratified the cohort by APOE genotype, having the VS variant correlated with less brain-wide flortaucipir uptake, indicating fewer tangles, in the 130 APOE4 carriers, but not in the 224 people with other APOE genotypes. The authors noted that the protective effect of the VS variant on tangles seems to be limited to people who either have a high amyloid burden or who carry APOE4. Both these groups are at higher risk of tangles.

A regional analysis of the brain hinted that klotho may act locally. The gene is most strongly expressed in mesotemporal, inferior temporal, and posterior cingulum cortices, according to the Allen Brain Atlas. In the ADNI3 cohort, the VS variant conferred a stronger protective effect against tangles in those brain regions than in others.

Does this matter for cognition? To address this question, the researchers limited their analysis to the 258 participants who were amyloid-positive, since they are most likely to have cognitive decline. In this subgroup, the 67 VS carriers outperformed the 191 noncarriers on an episodic memory measure in the ADNI-MEM composite. This remained significant even after adjusting for APOE4 genotype and other confounders. Statistical mediation analysis, which can help tease out cause and effect, showed the effect was mediated by the lower tau tracer uptake.

Previous studies had reported that VS correlated with less amyloid accumulation. Neitzel and colleagues did not see this in the 354 ADNI3 participants who had a tau PET scan; however, in a larger ADNI3 cohort of all 959 participants who had had amyloid scans and genotyping, heterozygous VS carriers did have less amyloid in the brain than did VS noncarriers, at least among those younger than 80. In agreement with prior studies, the amyloid burden difference was driven by APOE4 carriers. Because the authors saw a reduction in tangles in a small subset of the cohort, they concluded that the klotho variant more strongly affects tau than amyloid. They calculated a Cohen’s effect size of 0.069 for amyloid, and 0.118 for tangles.

In ongoing research, Neitzel and colleagues are testing serum and cerebrospinal fluid samples from the same study participants to see if the protective effects in VS carriers are mediated by klotho protein levels. They will also try to decipher the mechanism of how klotho prevents tangles. The protein takes part in numerous cellular processes, including redox reactions and autophagy, and it regulates calcium, insulin, and growth factor signaling. In a mouse model of amyloidosis, klotho overexpression reduced plaque buildup by activating autophagy (Zeng et al., 2019). Ewers and colleagues will analyze proteomes from volunteers to evaluate whether autophagic proteins are upregulated in VS carriers.

The findings imply that somehow raising klotho levels might stave off AD. This is supported by work in mouse models of amyloidosis, where injecting different forms of klotho protein into blood or brain sharpens memory (Aug 2017 news; Nov 2017 news). Ligustilide, a small molecule found in essential oils extracted from plants, promotes klotho processing, increasing the secreted form of the protein, and could have therapeutic potential, Ewers said. In mouse models of amyloidosis, ligustilide treatment lowered amyloid load and improved memory (Kuang et al., 2017). As the primary active component in the Chinese herbal extract Rhizoma Chuanxiong, the compound is used to treat cardiovascular ailments, but the liver rapidly breaks it down, reducing its bioavailability (Yan et al., 2008). 

Beyond therapeutic considerations, the finding elucidates the relationship between plaques and tangles. Other genetic variants have been found to weaken that association. In a woman with the presenilin 1 Paisa mutation that causes familial AD, the Christchurch mutation in APOE3 was reported to protect from cognitive decline. Now in her 70s, her brain is full of plaques but contains few tangles (Nov 2019 news). The Christchurch mutation is rare, however, while klotho-VS is common. “Our data show the potential for tangles to be modulated by common genetic variants,” Ewers told Alzforum.

Recently, researchers led by Alfredo Ramirez at the University of Cologne, Germany, and Agustín Ruiz at the International University of Catalonia in Barcelona, Spain, identified another gene that intervenes in the pathogenic cascade at the step between plaques and tangles. They reported that a rare coding variant in the microglial phospholipase-C-γ2 (PLCG2) gene protected against AD. In 1,261 MCI patients, those with the variant had less CSF p-tau181 and slower cognitive decline than noncarriers. This marker rises early in AD and thus far appears specific for amyloid-driven tau pathology (Mar 2020 news). The drop in p-tau181 predominantly occurred in MCI patients who did have amyloid buildup, as determined by low CSF Aβ42. The effect size of this protective association was similar to that of APOE4, although in the opposite direction.

This suggests that, like klotho, PLCG2 may intervene between amyloid and tau, the authors noted. In protein network analysis, PLCG2 interacts with other known AD risk factors such as TREM2, APOE, and complement (Kleineidam et al., 2020). Ewers noted that PLCG2 acts downstream of fibroblast growth factor. FGF interacts with klotho, hinting that klotho and PLCG2 could be part of the same mechanistic pathway (Mar 2019 news). 

“Future studies should investigate how the combination of distinct genetic protective and risk factors impacts the interaction between amyloid and tau, and whether these effects are additive or multiplicative,” Hanseeuw wrote.—Madolyn Bowman Rogers

Comments

  1. This paper by Neitzel and colleagues explores the effects of the klotho genetic polymorphism on AD pathophysiology as measured using Florbetapir (amyloid) and Flortaucipir (tau) PET imaging in the ADNI3 cohort. They found the klotho variant to be associated with lower tau burden for a given level of amyloid, consistent with the previous observation that this variant decreases the risk of developing AD. The effect of klotho on tau was mostly observed in the brain regions where klotho mRNA is highly expressed, such as the temporal lobe, suggesting a local effect of klotho on tau pathology and/or on the interaction between amyloid and tau.

    This study adds evidence that the association between amyloid and tau pathologies is dependent on genotype. Previous studies (Altmann et al., 2014; Buckley et al., 2019; Hohman et al., 2018) have observed that the E4 genotype increases tau burden for a given level of amyloid, particularly in women. Future studies should investigate how the combination of distinct genetic protective and risk factors impacts the interaction between amyloid and tau, and whether these effects are additive or multiplicative. This will—more than ever—require large samples of well-characterized older adults. Most importantly, we should study the biological pathways underlying this genetic variability and develop new ways of intervening in the risk of developing AD.

    References:

    . Sex modifies the APOE-related risk of developing Alzheimer disease. Ann Neurol. 2014 Apr;75(4):563-73. Epub 2014 Apr 14 PubMed.

    . Associations between baseline amyloid, sex, and APOE on subsequent tau accumulation in cerebrospinal fluid. Neurobiol Aging. 2019 Jun;78:178-185. Epub 2019 Mar 7 PubMed.

  2. I read with great satisfaction and interest Neitzel and colleagues’ elegant report on reduced tau accumulation and lower memory impairment, associated with KL-VS heterozygosity in Alzheimer’s disease patients. This work, and the plethora of other recent high-impact published papers (Belloy et al., 2020; Erickson et al., 2019), all highlighting the potential of Klotho as a worthwhile target in the search for Alzheimer’s disease therapeutics, is especially gratifying for me to see, since our lab at Boston University School of Medicine was the first to publish (starting in 2007) that Klotho is neuroprotective (Zeldich et al., 2014), that Klotho protein level in the brain decreases with age (Duce et al., 2008), and that increasing Klotho levels would be beneficial in neurodegenerative diseases (Abraham et al., 2013; King et al., 2012). We have known since 2002 that KL-VS (a Klotho polymorphism that changes two amino acids in the protein) is protective (Arking et al., 2002). Since then, many articles were published on the positive effects of KL-VS heterozygosity on lifespan and health span extension, for a number of age-related diseases, and on human cognition (Dubal et al., 2014). This new work raises a number of important questions for Klothologists and other researchers interested in aging and Alzheimer’s disease:

    1. Which KL-VS isoform is involved in the protective effect seen by Neitzel and colleagues?

    Klotho is mainly expressed in kidney and brain. It is a type I transmembrane protein which is shed from its membrane by ADAM10 and 17 (Chen et al., 2007). As such, it circulates in blood and CSF and is found in urine as well. A third isoform produced by differential splicing of the Klotho gene generates a shorter, secreted form, found mostly in the brain (Massó et al., 2015). All three isoforms contain the VS sequence in KL-VS carriers, who represent 25 percent of the human population. The functions of the transmembrane and circulating Klotho are different. The circulating isoform may act as a ligand to a receptor to initiate signaling that could affect tau phosphorylation. In the kidney, circulating Klotho inhibits IGF-1, Wnt, and TGF-β signaling. In the kidney, transmembrane Klotho serves as a co-receptor with the FGF receptor for FGF-23 signaling. In the brain, the signaling of all isoforms is still unknown.

    2. Is KL-VS enzymatic activity part of the mechanism?

    Klotho may also act as a glucuronidase/sialidase to modify receptors or ion channels that would indirectly affect tau phosphorylation positively, for example, less phosphorylation at positions 217 and 181 that were just recently found to be specific for AD tau (Barthélemy et al., 2020; Palmqvist et al., 2020). Interestingly, p-tau217 appears to be the most robust plasma biomarker for Alzheimer’s disease yet. As an enzyme, Klotho activates the calcium channels TRPV5 and 6 and the potassium channel, ROMK1, which could also participate in intracellular signaling. We reported that Klotho activates AKT in primary neurons and oligodendrocyte progenitor cells (Chen et al., 2013; Zeldich et al., 2014). A recent article reports that sulfhydration of AKT triggers tau phosphorylation by activating glycogen synthase kinase 3β in Alzheimer's disease (Sen et al., 2020). 

    3. Does Klotho act via β-amyloid or directly on tau?

    Since in patients with lots of amyloid KL-VS is protective via tau, we may conclude that KL-VS is bypassing amyloid to influence tau post-translational modifications such as phosphorylation.

    4. Is KL-VS more protective because there is more of it, or because KL-VS possesses a protective gain of function due to the two amino acid substitutions?

    KL-VS levels in conditioned medium of transfected cells and in human blood are slightly higher than those of wild-type Klotho, likely due to more shedding. We have reported that, in vitro, KL-VS exhibits stronger binding to FGFR and FGF23, which leads to an increased ERK phosphorylation (Tucker Zhou et al., 2013). Moreover, when comparing the glucuronidase activity of recombinant KL-VS with that of recombinant wild-type Klotho, we found that they differ (unpublished data). The aberrant enzymatic activity of KL-VS could also affect tau phosphorylation.

    5. Is KL-VS more anti-inflammatory than wild-type Klotho?

    We and others showed that Klotho has anti-inflammatory properties (Zeldich et al., 2019). If Klotho inhibits microglia activation, it may affect the propagation of tau from neuron to neuron (Asai et al., 2015). However, we don’t have evidence that KL-VS is more anti-inflammatory than wild-type Klotho.

    Neitzel et al. are the first to publish a beneficial Klotho-tau-cognition relationship. While there is so much more we need to learn before we fully understand the mysterious mechanisms of this neuroprotective pleiotropic protein, we do know that increasing its levels will be beneficial in neurodegenerative and other diseases of aging.

    References:

    . The anti-aging protein Klotho as a therapeutic target for neurodegenerative diseases. Paper presented at: Journal of neurochemistry, 2013.

    . 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.

    . Depletion of microglia and inhibition of exosome synthesis halt tau propagation. Nat Neurosci. 2015 Nov;18(11):1584-93. Epub 2015 Oct 5 PubMed.

    . Blood plasma phosphorylated-tau isoforms track CNS change in Alzheimer's disease. J Exp Med. 2020 Nov 2;217(11) PubMed.

    . 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.

    . Insulin stimulates the cleavage and release of the extracellular domain of Klotho by ADAM10 and ADAM17. Proc Natl Acad Sci U S A. 2007 Dec 11;104(50):19796-801. PubMed.

    . The antiaging protein Klotho enhances oligodendrocyte maturation and myelination of the CNS. J Neurosci. 2013 Jan 30;33(5):1927-39. PubMed.

    . Life extension factor klotho enhances cognition. Cell Rep. 2014 May 22;7(4):1065-76. Epub 2014 May 10 PubMed.

    . Gene profile analysis implicates Klotho as an important contributor to aging changes in brain white matter of the rhesus monkey. Glia. 2008 Jan 1;56(1):106-17. PubMed.

    . KLOTHO heterozygosity attenuates APOE4-related amyloid burden in preclinical AD. Neurology. 2019 Apr 16;92(16):e1878-e1889. Epub 2019 Mar 13 PubMed.

    . Identification of novel small molecules that elevate Klotho expression. Biochem J. 2012 Jan 1;441(1):453-61. PubMed.

    . Secreted and Transmembrane αKlotho Isoforms Have Different Spatio-Temporal Profiles in the Brain during Aging and Alzheimer's Disease Progression. PLoS One. 2015;10(11):e0143623. Epub 2015 Nov 24 PubMed.

    . Discriminative Accuracy of Plasma Phospho-tau217 for Alzheimer Disease vs Other Neurodegenerative Disorders. JAMA. 2020 Aug 25;324(8):772-781. PubMed.

    . Sulfhydration of AKT triggers Tau-phosphorylation by activating glycogen synthase kinase 3β in Alzheimer's disease. Proc Natl Acad Sci U S A. 2020 Feb 25;117(8):4418-4427. Epub 2020 Feb 12 PubMed.

    . 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.

    . Klotho Is Neuroprotective in the Superoxide Dismutase (SOD1G93A) Mouse Model of ALS. J Mol Neurosci. 2019 Oct;69(2):264-285. Epub 2019 Jun 27 PubMed.

    . The neuroprotective effect of Klotho is mediated via regulation of members of the redox system. J Biol Chem. 2014 Aug 29;289(35):24700-15. Epub 2014 Jul 18 PubMed.

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References

News Citations

  1. San Diego: Klotho Spins Threads Linking Aging and AD Research
  2. Could Longevity Factor, Epilepsy Med, Treat AD One Day?
  3. Longevity Gene Boosts Brainpower, Even in the Young
  4. Klotho Variant Cuts ApoE4’s Alzheimer Risk by a Third
  5. Shot of Klotho Boosts Memory In Aging and Diseased Mice
  6. Brain-Specific Klotho Isoform Fortifies Memory
  7. Can an ApoE Mutation Halt Alzheimer’s Disease?
  8. Different CSF Phospho-Taus Match Distinct Changes in Brain Pathology
  9. Is Klotho’s Partner FGF23 a Cognition Protein?

Mutations Citations

  1. PSEN1 E280A (Paisa)

Paper Citations

  1. . Variation in longevity gene KLOTHO is associated with greater cortical volumes. Ann Clin Transl Neurol. 2015 Mar;2(3):215-30. Epub 2015 Jan 26 PubMed.
  2. . Klotho, APOEε4, cognitive ability, brain size, atrophy, and survival: a study in the Aberdeen Birth Cohort of 1936. Neurobiol Aging. 2017 Jul;55:91-98. Epub 2017 Mar 7 PubMed.
  3. . KLOTHO heterozygosity attenuates APOE4-related amyloid burden in preclinical AD. Neurology. 2019 Apr 16;92(16):e1878-e1889. Epub 2019 Mar 13 PubMed.
  4. . Lentiviral vector-mediated overexpression of Klotho in the brain improves Alzheimer's disease-like pathology and cognitive deficits in mice. Neurobiol Aging. 2019 Jun;78:18-28. Epub 2019 Feb 13 PubMed.
  5. . Neuroprotective Effect of Ligustilide through Induction of α-Secretase Processing of Both APP and Klotho in a Mouse Model of Alzheimer's Disease. Front Aging Neurosci. 2017;9:353. Epub 2017 Nov 2 PubMed.
  6. . Pharmacokinetics and metabolism of ligustilide, a major bioactive component in Rhizoma Chuanxiong, in the rat. Drug Metab Dispos. 2008 Feb;36(2):400-8. Epub 2007 Nov 26 PubMed.
  7. . PLCG2 protective variant p.P522R modulates tau pathology and disease progression in patients with mild cognitive impairment. Acta Neuropathol. 2020 Jun;139(6):1025-1044. Epub 2020 Mar 12 PubMed.

External Citations

  1. Allen Brain Atlas

Further Reading

Primary Papers

  1. . KL-VS heterozygosity is associated with reduced tau accumulation and lower memory impairment in Alzheimer’s disease. medRxiv July 31, 2020.