Cell culture assays

MTT

MTT assays were used to measure SH-SY5Y cell proliferation. The neuroblastoma cells were seeded at 3 × 104 per well in 96-well plates in DMEM/F12 (Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (Omega Scientific) until 85% confluent. Cells were treated with 100 uM of freshly reconstituted humanin in Milli-Q water (GenScript) with and without Aβ 1-42 (CPC Scientific), prepared as previously described1, for 24 hours. To prevent humanin-serum interference and to support cell viability for the long 24-hour treatment duration, treatments were in DMEM/12 media supplemented with N2 (Thermo Fisher Scientific). MTT (Sigma-Aldrich) reagent (5 mg/ml) was added to each well after treatments for four hours and lysed before absorbance values were read using the SpectrMax M3 microplate reader.

Cell Cytotoxicity Assay

A two-color fluorescence cell viability assay (LIVE/DEAD Viability/Cytotoxicity Kit; Invitrogen (cat. L3224) was used to distinguish live cells from dead cells after humanin and Aβ 1-42 treatment. The ratio of live to dead cells can be quantified since live cells retain the Calcein AM dye and dead cells with damaged membranes permit entry of the ethidium homodimer dye. SH-SY5Y cells were seeded at 3 × 104 per well in 96-well plates in DMEM/F12 (Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (Omega Scientific) until 85% confluent. Cells were treated with 100 uM of freshly reconstituted humanin in Milli-Q water (GenScript) with and without Aβ 1-42 (CPC Scientific), prepared as previously described1, for 24 hours. To prevent humanin-serum interference and to support cell viability for the long 24-hour treatment duration, treatments were in DMEM/12 media supplemented with N2 (Thermo Fisher Scientific). Cytotoxicity assays were performed immediately after the 24-hour incubations according to the provided protocol.

Live Mitochondria Isolation

Cortical brain mitochondria were isolated from male 16-week old C57BL/6 mice. Brains were removed and rinsed in ice cold buffer containing 210 mM mannitol, 70 mM sucrose, 20 mM HEPES, 2 mM EGTA, and 0.2% (w/v) fatty acid free bovine serum albumin, pH 7.4 (ME Buffer). Cortical tissue was extracted, minced, and immediately homogenized in MEB with 5-10 Teflon pestle strokes. Homogenates were centrifuged at 1000 × g for 5 minutes (4 °C) to remove tissue debris. Supernatants were transferred to a clean tube and centrifuged at 3500 × g for 10 minutes (4 °C). Centrifugations were repeated twice. The mitochondrial pellet was re-suspended in a buffer containing 220 mM mannitol, 8 mM sucrose, 10 mM KH 2 PO 4 , 5 mM MgCl 2 , 2 mM HEPES, 1 mM EGTA, and 0.2% (w/v) fatty acid free bovine serum albumin, 10 mM succinate, and 2 uM rotenone, pH 7.2 (MAS Buffer) to a final concentration of 400 ug/ml per well49. To confirm the ability of our protocol to isolate mitochondria, we repeated the procedure using Western blotting and probed for mtCOX2 (SC-23984 Santa Cruz Biotechnology) and GAPDH (#7074 Cell Signaling).

Incubation of Live Mitochondria with HNG and Aβ 1-42

Cortical mitochondria suspensions in MAS Buffer (250 mg/ml) were plated in a 96-well plate. To prime the mitochondria, 10 uM of HNG was administered to wells and incubated at 37 °C 15 minutes. HNG is a potent analogue of humanin that contains a glycine substitution for serine at the 14th amino acid. HNG was dissolved in Milli-Q water immediately prior to mitochondria administration. Following HNG exposure, 3uM or 30 uM of Aβ 1-42 (CPC Scientific), prepared as previously described1, was dispensed into wells and incubated at 37 °C for 45 minutes.

Live Mitochondrial-derived H 2 0 2 Detection by Amplex Red

Mitochondria readily produce superoxide in the mitochondrial matrix, which is dismutated to H 2 0 2 by superoxide dismutase 2. H 2 0 2 quickly diffuses across the mitochondrial membrane to the medium and can be detected using Amplex Red (10-acetyl-3,7-dihydroxyphenoxazine, Thermo Fisher Scientific). In the presence of horseradish peroxidase (HRP), Amplex Red reacts with H 2 0 2 and is converted to resorufin, which emits light at 590 nm when excited at 530 nm. Both Amplex Red (50 uM) and HRP (0.1 U/ml) were added to MAS Buffer during HNG and Aβ 1-42 incubations. Fluorescence emission was detected using a SpectrMax M3 microplate reader.

Longitudinal Study of Humanin Administration in Healthy Mice

We conducted a 14-month study of 100 female, C57BL/6N mice obtained from the NIA aged mouse colony starting at 18 months of age. The mice were given twice-weekly injections of HNG at 4-mg/kg/BW or water/vehicle IP. This dose was chosen after protective effects were recently demonstrated by a Swedish group that administered humanin in twice-weekly treatments. Survival was monitored daily and body weight and daily food intake measured weekly50. All experiments with mice were performed in accordance with the appropriate guidelines and regulations and approved by the University of Southern California Institutional Animal Care and Use Committee (IACUC) under protocol #20787.

Mouse cognitive tests

Accelerating Rotarod

This procedure was the same as described previously43. At 24 months of age, the mice were placed on the rod rotating at an initial speed of 4 rpm. The rotation of the rotarod was gradually increased to 40 rpm over a 5 minute session. The time at which they fall was recorded and analyzed.

Y-maze: A Y-maze test was performed when the mice were 28 months of age. The procedure was the same as described previously43. In short, mice were placed in one arm of the maze and allowed to freely explore for 8 minutes. An entry into an arm was recorded only if both forepaws and hindpaws fully enter the arm. Quantification of spontaneous alternation behavior was calculated as the proportion of alternations, defined as an arm choice different from her previous two choices, to the total number of alternations.

Barnes Maze: Barnes maze tests were performed as previously described43. 28 month old Mice were placed in a start chamber in the middle of the maze and allowed to habituate for 30 seconds. The start chamber was then removed and the mouse was allowed to explore the maze and the time until the mouse finds the escape box was recorded. A maximum of 2 minutes was allowed for each trial.

Strategy Assessment: A random strategy was characterized by localized searches to holes separated by crosses through the maze center. A serial search strategy was characterized by a systematic search of consecutive holes (every hole or every other hole) with no maze center crosses. A spatial search strategy consisted of navigating directly to the escape hole with both error and deviation scores less or equal to 3.

BrdU and Doublecortin Staining

At approximately 32 months of age, mice were injected with BrdU for 8 days before sacrifice (50 mg/kg). Fixed hemibrains were sectioned coronally on a vibratome at 40 μm and then the brains were processed for BrdU and doublecortin (DCX) staining. Briefly, for BrdU staining sections were pretreated with 2N HCl at 37 °C, washed, and incubated overnight in primary antibody against BrdU (1:500; Abd Serotec). For DCX staining, sections were washed and incubated overnight in primary antibody against DCX (1:2500; Santa Cruz). Sections were then washed and incubated with biotinylated anti-rat secondary antibody for BrdU or anti-goat secondary antibody for DCX (Vector) for 1 hour and processed for diaminobenzidene staining using Vectastain ABC Elite kit (Vector). Stained sections were air-dried overnight, dehydrated, then coverslipped with Krystalon (EMD Millipore). Counting was done in the dentate gyrus (all layers) and quantified per unit area (mm2) for BrdU and per section for DCX.

Iba-1 staining and microglial quantification

Sections for microglia staining were processed as above without the HCl and were incubated overnight in primary antibody against Iba-1 (1:2000; Wako) and biotinylated anti-rabbit secondary antibody (Vector). For microglia morphological analyses, brain sections containing the hippocampus (bregma −1.95 to −2.18) (wt n = 6; n = 7), defined regions of interest (ROI) were identified for the CA1 and CA3 regions and the morphology of Iba-1+ microglia evaluated. Brain sections were scanned under 40x magnification (Aperio ScanScope T2 scanner, Aperio Technologies, Inc., Vista, CA) and viewed using Aperio ImageScope v.6.25.0.1117. Images were assigned random numbers and blinded for evaluation. Activation state of microglia was based on morphological analysis of Iba-1 staining in a manner consistent with prior reports51,52. The number of Iba-1 immunoreactive cells in the hippocampus was estimated by two-dimensional cell counts using random-sampling based on the optical dissector technique. Briefly, an Olympus BX50 microscope equipped with a motorized stage and computer-guided CASTGrid software (Olympus) was used for unbiased sampling. In sections containing well-defined CA1-CA3 subregions of hippocampus, the hippocampus (excluding the dentate gyrus) was outlined, and high magnification microscopic fields were randomly sampled. Within each field, cells within a counting frame (3000 μm2) was used for analysis. Microglia were classified as either type 1, (many thin, ramified processes), type 2 (short, thick processes and a rod-shaped cell body), or type 3 (no or few short non-ramified processes or many filapodial processes) cells. Type 2 and 3 microglia were considered activated.

Proinflammatory plasma biomarkers measurement

Proinflammantory biomarkers including IL-6, IL-8, IL-10, TNF-α and INF-γ were measured by a custom metabokine 8-plex kit provided by Meso Scale Discovery (Catalog#: N051A-1, Rockville, Maryland). Briefly, 25ul of standards and samples were added and incubated for 2 hours on the MSD plate. After 3 washes, 25ul of detection antibody was added into each well and incubated for 1.5 hours. After the final wash, read buffer was added and the plate was analyzed on the Quick plex SQ120.

Human studies

Using samples from the B-Vitamin Atherosclerosis Intervention Trial (BVAIT) approved by the University of Southern California Institutional Review Board27, we selected 75 AA and 75 CA participants that were matched by randomized treatment assignment (B-vitamins or placebo), gender and age. Written informed consent was obtained from all subjects before their inclusion in the study and all methods were performed in accordance with the relevant guidelines and regulations. We then measured plasma humanin levels by ELISA in these patients as previously described53.

DNA extraction and next generation sequencing

DNA was extracted from buffy coats collected from blood samples of the 150 BVAIT participants using the DNeasy blood and tissue kit (Qiagen) according to the manufacturer’s directions. In short, the buffy coats were lysed using proteinase K and buffer added to optimize the DNA binding conditions. These lysates were pipetted into a column that binds the DNA using silica-membrane technology, washed, and then the DNA was eluted in water. The mitochondrial DNA was minimally amplified by PCR using two different, overlapping sets of primers using TaKaRa LA TaqDNA Polymerase. The PCR products were run on a gel to check for correct amplification and then excised and purified using the QIAquick gel extraction kit (Qiagen). Illumina sequencing libraries were prepared in a 96-well plate format according to Dunham & Friesen (2013) with the exception that DNA was sheared with dsDNA Shearase Plus (Zymo: Irvine, CA, USA) and cleaned using Agencourt AMPure XP beads (Beckman- Coulter: Indianapolis, IN, USA). Fragment size selection was also carried out using beads instead of gel electrophoresis. Libraries were quantified using the Qubit 2·0 Fluorometer (Thermo Fisher Scientific Inc.), and the fragment size distribution was determined with an Agilent Bioanalyzer 2100. The libraries were then pooled equimolarly, and the final pool was quantified via qPCR using the Kapa Biosystems Library Quantification Kit, according to manufacturer’s instructions. The pool was then sequenced in a Rapid ver. 1 Flowcell, in an Illumina HiSeq 2500 platform. Final file formatting, demultiplexing and fastq generation was carried out using CASAVA v.1·8. Sequencing reads were further sorted by 4 bp barcode using the Novobarcode application within Novocraft (www.novocraft.com). The libraries preparation, pooling, quality control, sequencing and initial file processing were all performed at the UPC Genome Core (University of Southern California, Los Angeles, CA, USA).

SNP analysis

For each sample, we first trimmed the raw sequencing reads by quality using the SolexaQA package (ver. 1·12)54. All bases with a quality score of less than Q = 13 were trimmed and all reads that were less than 25 bp after trimming were removed from further analysis. We then mapped the quality-trimmed reads to the revised Cambridge reference sequence of the human mitochondrial genome (NC_012920·1) using the BWA aligner (ver. 0·7·5a)55. Following mapping, local realignment around insertion/deletions was performed using the IndelRealigner tool as part of the GATK package (ver. 2·6–5)56. After mapping and realignment, we applied the GATK UnifiedGenotyper (ver. 2·6–5) to all samples simultaneously to identify single nucleotide polymorphisms (SNPs) in our sample set (we identified 791 SNPs in the mitochondria). Having identified various SNPs in our sequencing data, the next step of analysis was to correlate the SNPs to humanin levels. In particular, we focused on SNPs contained within the small open reading frame of the humanin peptide within the 16S gene of the mitochondrial genome.

The SNP was then identified in the HRS and analyzed to determine whether the SNP was correlated with humanin-related phenotypes.

Cognitive scores

The humanin genotype was extracted from bed, bim, and fam files and merged with cognitive scores of all individuals between 1998 and 2012. Humanin genotype was identified for 15,620 individuals and was regressed against the cognitive state score for self-respondents in 2012. Scores under 7, between 7–12, and over 12 delineate demented, cognitively impaired, and cognitively healthy status, respectively. Age, sex, race, and the previous year’s cognitive score was used in the lag-dependent variable regression model on 10,158 respondents. The beta coefficient for the humanin SNP on cognitive score was 0.195 (p = 0.006, 95% CI: 0.056–0.333).

Cognitive aging

Cognitive age measures were based on data from wave three (1996) through wave ten (2010) of the Health and Retirement Study, using four measures of cognitive functioning—delayed recall, immediate recall, serial 7 s, and backwards counting. A cognitive age was calculated for each participant at each wave based on an algorithm proposed by Klemera & Doubal57 that in prior work has been used for calculating biological ages from biomarker data58,59. This method has been validated using both real and simulated data. Cognitive age estimates combine information from equations of chronological age regressed on each of the four cognitive functioning markers. The equation for calculating cognitive age is:

$${\rm{CogAge}}=\frac{{\sum }_{j=1}^{m}({x}_{ji}-{q}_{j})\frac{{k}_{j}}{{s}_{j}^{2}}+\frac{C{A}_{i}}{{s}_{BA}^{2}}}{{\sum }_{j=1}^{m}{(\frac{{k}_{j}}{{s}_{j}})}^{2}+\frac{1}{{s}_{BA}^{2}}}$$