Scientists Seek, Find, and Exploit AD Risk Genes

Series - Society for Neuroscience Annual Meeting 2014:

The genes involved in Alzheimer’s disease harbor both pathogenic and protective factors, and at the annual meeting of the Society for Neuroscience, November 15 to 19 in Washington, D.C., researchers proposed two new variants that might have a hand in causing disease. Genotyping members of a family with a history of late-onset AD turned up the netrin receptor UNC5C. Targeted sequencing revealed the Tmp21 gene, a proposed modulator of γ-secretase. Other researchers reported that whole-exome sequencing of people homozygous for the ApoE4 allele uncovered variants that may ward off late-onset disease. And data gleaned from genetic studies have led yet others to design a therapeutic that promotes clearance of Aβ in the brain.

All in the Family: UNC5C
Late-onset AD runs rampant in some families, yet in many cases the responsible genetic risk factors remain hidden. Monica Wetzel–Smith of San Francisco-based Genentech explained how she and her colleagues looked for variants that associate with AD in one extended family with a pattern of late-onset disease that resembled autosomal dominance. The researchers sequenced the entire genome of one affected family member and the exome of an aunt, who was the most distantly affected relative. Both turned out to share the T835M missense variant in UNC5C. Six relatives with the disease, but also two unaffected ones, had one copy of this variant.

Four other families from the LOAD family cohort (see Wijsman et al., 2012) carried T835M UNC5C as well. In one, all four people with the disease did. One person in each of two other families carried the allele, but neither had AD. Because one of these carriers was younger than 70, the scientists are uncertain whether he or she will get the disease. In the fourth family, three people carried the variant. All three were cognitively healthy but younger than 70, so Wetzel-Smith said she could not infer whether the allele was involved in the family’s disease. To see if the variant is enriched in other people with late-onset Alzheimer’s, the researchers compared the genotypes of more than 8,000 AD patients with almost 100,000 controls. These came from the Genentech cohort, Washington University in St. Louis, the Alzheimer’s Disease Genetics Consortium at the University of Pennsylvania, Philadelphia, and the Iceland-based deCODE Genetics study. Seventeen patients (0.2 percent) and 68 controls (0.07 percent) carried the mutation. The researchers concluded that the T835M variant doubled the odds of getting Alzheimer’s.

What does this variant do? UNC5C is an axon guidance receptor for netrins, which are proteins secreted into the cellular environment during brain development. The receptor is thought to influence apoptotic signaling, much like its cousin UNC5B (see Wang et al., 2009). Both receptors contain a cytosolic “death domain.” It remains buried inside the protein structure when a netrin is bound. In the absence of ligand, the protein undergoes a conformational change exposing the death domain; this recruits signaling molecules that relay apoptotic messages to the nucleus. T835M seems to alter the “hinge” region of the protein's intracellular domain, to keep the death domains constantly exposed. In culture, fewer HEK293T cells survived transfection with T835M UNC5C than with wild-type protein. Aβ and tau production were unaffected. Rat hippocampal neurons expressing the risk variant succumbed more quickly to Aβ-, glutamate-, or staurosporine-induced toxicity.

The researchers published the findings in the November 24 Nature Medicine, where they suggested that T835M UNC5C predisposes carriers to neurodegenerative diseases (see Wetzel et al., 2014). Robert Graham co-led the work with Ryan Watts at Genentech in San Francisco and Carlos Cruchaga and Alison Goate at Washington University in St. Louis. Graham told Alzforum that carrying the UNC5C variant may make neurons more susceptible to stress, exacerbating any underlying neurodegenerative processes. The authors found this protein highly expressed in neurons of people’s temporal lobe and hippocampus.

“While it is a rare variant, it clearly looks like the UNC5C mutation increases AD risk,” David Holtzman, WashU, wrote to Alzforum. He was particularly interested that the mechanism seemed unrelated to Aβ. “The data looked good,” agreed Steve Estus, University of Kentucky, Lexington. “This is another indication that Alzheimer’s is a multiple etiology disease," he said. "I wouldn’t have thought that a cell death pathway would be implicated in Alzheimer’s, but that’s how it looks.” Other researchers are skeptical. John Hardy, University College London, said he would reserve judgment until the findings are repeated. “Rare variant work is extremely difficult to prove, and just as difficult to refute.”

Zeroing In on Suspects
Other researchers are taking a targeted approach to finding risk alleles, looking in suspect genes. For example, the type I transmembrane protein TMP21 has been reported to modulate γ-secretase cleavage of APP (see Chen et al., 2006, and Vetrivel et al., 2007), but no one had found a genetic link between this receptor and Alzheimer’s.

Xiaojie Zhang, a Ph.D. student in the lab of Weihong Song, University of British Columbia, Vancouver, Canada, pored over intron and exon sequencing data from 261 AD patients and 236 controls. She found a single nucleotide polymorphism (SNP) in intron 4 of TMP21 that was associated with a 1.5 odds ratio for AD. The scientists have not looked for this association in any other cohorts yet, and Song would not say how many of the patients and controls carried the risk allele.

In HEK293 cells, the variant accelerated maturation of Tmp21 pre-mRNA to mRNA, meaning the SNP made splicing more efficient. Cells harboring the SNP made more Tmp21 protein. To see whether this SNP affected Aβ production, the researchers transfected HEK293 cells expressing APPSwe with plasmids encoding either wild-type Tmp21 or the variant. Production of both Aβ40 and 42 crept higher in the latter. Zhang suggested that perturbing TMP21 could affect the risk for Alzheimer’s disease. Holtzman wondered if Aβ40 or 42 levels differed among people with the variant and those without. Zhang said she had not looked at that yet.

“Reproducing this genetic association in larger cohorts is critical,” said Estus. “In the past, many candidate gene studies identified genetic risk factors in cohorts of this size that were not reproduced in other populations.”

Help Wanted: Seeking the Protectors
What about genetic variants that ward off disease? Researchers are studying those who are most at risk for developing Alzheimer’s, but still cognitively normal, to find variants that might delay AD. Aurelie N’Songo, of the Mayo Clinic in Jacksonville, Florida, who works in the labs of Nilüfer Ertekin-Taner and Guojun Bu, has examined the genetic makeup of people who are homozygous for the ApoE4 allele, older than 75, and cognitively intact. People with two copies of this allele have up to a 15-fold greater chance of developing late-onset Alzheimer’s disease by age 75. Only about 2 percent of the population is ApoE4 homozygote and those who are also older than 75 and have no cognitive impairment are even harder to find. Trolling the collection of the Mayo Clinics in Rochester, Minnesota, and Phoenix, N'Songo found 24 European-Americans who were ApoE4/4 with an average age of 81.

Sequencing the entire exome of these people, N'Songo found more than 200 variants enriched in this group compared with the general population (see NHLBI GO Exome Sequencing Project). Exome data from the Mayo Clinic Biobank of 88 people with mixed ApoE genotypes indicated 40 of those variants were specifically enriched in non-demented ApoE4 homozygotes. The preliminary data has not been validated yet, Ertekin-Taner emphasized. That said, it places some of these variants in genes previously associated with AD risk, such as HLA, ADAM9, ADAM29, and GSTM1. Other variants fit into pathways that may be associated with Alzheimer’s, such as cell signaling and oxidative stress.

Has the group checked ApoE4 homozygous people with dementia to see if they lack any of the 40 variants? N’Songo said they are trying to answer that question with ADNI data. They also plan to analyze data from 11,000 people in the AD Sequencing Project to see if those variants are enriched in non-demented ApoE4/4 carriers. Giulio Taglialatela, University of Texas Medical Branch at Galveston, asked if any of these 24 homozygous individuals had AD pathology in the brain. N’Songo said her group lacks histopathological information on these people, because they are still alive and only gave blood. “I am impressed by the innovative strategy they are using,” Taglialatela told Alzforum.

Capitalizing on the Data
Typically, genetic data do not lead directly to therapy development, but consider this project: Manasi Malik, from Estus' lab, is working on inhibiting the transmembrane receptor CD33, a known AD risk factor (see Aug 2013 news story). CD33 is present on immune cells in the periphery and the brain. This receptor prevents microglia from ingesting Aβ (see Gricuic et al., 2013). A protective, non-functional variant removes that brake and allows the cells to gobble up more Aβ. Malik tests whether antibodies to CD33 might do the same. One such antibody, Lintuzumab, has been used, to no avail, in people with leukemia, where CD33 is overexpressed.

Malik reported that 10 ng/ml of Lintuzumab reduced CD33 on the microglia cell surface by 80 percent. Estus said they are now testing whether this treatment improves microglial phagocytosis of amyloid and dead neurons, and affects cytokines released by microglia. He is unsure how much Lintuzumab crosses the blood-brain barrier, but thinks it may still be a viable therapeutic option even if it only acts in the periphery because peripheral monocytes cross into the brain to clear up Aβ deposits (see Dec 2014 news story).—Gwyneth Dickey Zakaib

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References

News Citations

  1. Protective Microglial Gene Variant Promotes Phagocytosis
  2. TREM2 Data Surprise at SfN Annual Meeting

Paper Citations

  1. Wijsman EM, Pankratz ND, Choi Y, Rothstein JH, Faber KM, Cheng R, Lee JH, Bird TD, Bennett DA, Diaz-Arrastia R, Goate AM, Farlow M, Ghetti B, Sweet RA, Foroud TM, Mayeux R, . Genome-wide association of familial late-onset Alzheimer's disease replicates BIN1 and CLU and nominates CUGBP2 in interaction with APOE. PLoS Genet. 2011 Feb;7(2):e1001308. PubMed.
  2. Wang R, Wei Z, Jin H, Wu H, Yu C, Wen W, Chan LN, Wen Z, Zhang M. Autoinhibition of UNC5b revealed by the cytoplasmic domain structure of the receptor. Mol Cell. 2009 Mar 27;33(6):692-703. PubMed.
  3. Wetzel-Smith MK, Hunkapiller J, Bhangale TR, Srinivasan K, Maloney JA, Atwal JK, Sa SM, Yaylaoglu MB, Foreman O, Ortmann W, Rathore N, Hansen DV, Tessier-Lavigne M, Alzheimer's Disease Genetics Consortium, Mayeux R, Pericak-Vance M, Haines J, Farrer LA, Schellenberg GD, Goate A, Behrens TW, Cruchaga C, Watts RJ, Graham RR. A rare mutation in UNC5C predisposes to late-onset Alzheimer's disease and increases neuronal cell death. Nat Med. 2014 Dec;20(12):1452-7. Epub 2014 Nov 24 PubMed.
  4. Chen F, Hasegawa H, Schmitt-Ulms G, Kawarai T, Bohm C, Katayama T, Gu Y, Sanjo N, Glista M, Rogaeva E, Wakutani Y, Pardossi-Piquard R, Ruan X, Tandon A, Checler F, Marambaud P, Hansen K, Westaway D, St George-Hyslop P, Fraser P. TMP21 is a presenilin complex component that modulates gamma-secretase but not epsilon-secretase activity. Nature. 2006 Apr 27;440(7088):1208-12. PubMed.
  5. Vetrivel KS, Gong P, Bowen JW, Cheng H, Chen Y, Carter M, Nguyen PD, Placanica L, Wieland FT, Li YM, Kounnas MZ, Thinakaran G. Dual roles of the transmembrane protein p23/TMP21 in the modulation of amyloid precursor protein metabolism. Mol Neurodegener. 2007;2:4. PubMed.
  6. Griciuc A, Serrano-Pozo A, Parrado AR, Lesinski AN, Asselin CN, Mullin K, Hooli B, Choi SH, Hyman BT, Tanzi RE. Alzheimer's disease risk gene CD33 inhibits microglial uptake of amyloid beta. Neuron. 2013 May 22;78(4):631-43. PubMed.

External Citations

  1. NHLBI GO Exome Sequencing Project
  2. HLA
  3. ADAM9
  4. GSTM1
  5. AD Sequencing Project
  6. CD33

Further Reading

Primary Papers

  1. Wetzel-Smith MK, Hunkapiller J, Bhangale TR, Srinivasan K, Maloney JA, Atwal JK, Sa SM, Yaylaoglu MB, Foreman O, Ortmann W, Rathore N, Hansen DV, Tessier-Lavigne M, Alzheimer's Disease Genetics Consortium, Mayeux R, Pericak-Vance M, Haines J, Farrer LA, Schellenberg GD, Goate A, Behrens TW, Cruchaga C, Watts RJ, Graham RR. A rare mutation in UNC5C predisposes to late-onset Alzheimer's disease and increases neuronal cell death. Nat Med. 2014 Dec;20(12):1452-7. Epub 2014 Nov 24 PubMed.

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