Deregulated expression of a longevity gene, Klotho, in the C9orf72 deletion mice with impaired synaptic plasticity and adult hippocampal neurogenesis

Hexanucleotide repeat expansion of C9ORF72 is the most common genetic cause of amyotrophic lateral sclerosis and frontotemporal dementia. Synergies between loss of C9ORF72 functions and gain of toxicities from the repeat expansions contribute to C9ORF72-mediated pathogenesis. However, how loss of C9orf72 impacts neuronal and synaptic functions remains undetermined. Here, we showed that long-term potentiation at the dentate granule cells and long-term depression at the Schaffer collateral/commissural synapses at the area CA1 were reduced in the hippocampus of C9orf72 knockout mice. Using unbiased transcriptomic analysis, we identified that Klotho, a longevity gene, was selectively dysregulated in an age-dependent manner. Specifically, Klotho protein expression in the hippocampus of C9orf72 knockout mice was incorrectly enriched in the dendritic regions of CA1 with concomitant reduction in granule cell layer of dentate gyrus at 3-month of age followed by an accelerating decline during aging. Furthermore, adult hippocampal neurogenesis was reduced in C9orf72 knockout mice. Taken together, our data suggest that C9ORF72 is required for synaptic plasticity and adult neurogenesis in the hippocampus and Klotho deregulations may be part of C9ORF72-mediated toxicity. Electronic supplementary material The online version of this article (10.1186/s40478-020-01030-4) contains supplementary material, which is available to authorized users.

2 positioned at an adequate distance within the stratum radiatum of the CA1 region for stimulating two independent synaptic inputs S1 and S2 to a single neuronal population, thus evoking field fEPSPs from Schaffer collateral/commissural-CA1 synapses. For recording the fEPSP (measured as its initial slope function), one electrode (5 MΩ; AM Systems) was placed in the CA1 apical dendritic layer. For dentate gyrus (DG), one monopolar lacquer-coated, stainless-steel electrode (5 MΩ; AM Systems, United States of America) was placed in the stratum moleculare of the DG to stimulate the medial perforant path input. About 200 μm apart, the recording electrode was lowered to the same level to record fEPSPs. The signals were amplified using a differential amplifier (Model 1700; AM Systems) and digitized by using a CED 1401 analog-to-digital converter (Cambridge Electronic Design).
After 3-hour of pre-incubation, an input-output curve (I-O curve), which uses afferent stimulation vs. fEPSP slope, was taken. Test stimulation intensity was adjusted to elicit fEPSP slope of 40% of the maximal EPSP response for synaptic inputs S1 and S2. Late-LTP was induced using three repeated high frequency stimulus trains of 100 pulses ("strong" tetanus (STET), 100 Hz; duration, 0.2 ms/polarity; intertrain intervals, 10 min). Late-LTP was induced in DG by a single theta burst stimulation (TBS) protocol paradigm consisting of 15 bursts of eight pulses, 200 Hz, interburst interval 200 ms. Late-LTD was induced in CA1 area using a strong low-frequency stimulation (SLFS) protocol of 900 bursts [one burst consisted of three stimuli at 20 Hz, and the interburst interval was 1 s (i.e., f = 1 Hz; stimulus duration, 0.2 ms/half wave; total number of stimuli, 2700)]. This stimulation pattern produced a stable late-LTD in vitro for 3 h. The slopes of the fEPSPs were monitored online. Four 0.2-Hz biphasic constantcurrent pulses (0.1 ms per polarity) were used for baseline recording at each time point. In all experiments, a stable baseline was recorded for at least 30 min.

Statistics
The average values of the slope function of the field EPSP (millivolts per milliseconds) per time point were analyzed using the Wilcoxon signed rank test when compared within one group, or the Mann-Whitney U-test when data were compared between groups; p < 0.05 was considered as statistically significantly different. The nonparametric test was used because the analyses of the prolonged recordings do not allow the use of parametric tests.

Immunofluorescence
Tissue preparation for immunohistochemistry was described previously [1,4]. In brief, mice were anesthetized with isoflurane and perfused transcardially with phosphate buffered saline (PBS), followed by 4% paraformaldehyde (PFA) in phosphate buffer for fixation. Brains were dissected and post-fixed in 4% PFA in PBS for 2 hours. Tissues were cryopreserved in 30% sucrose for over 24 hours and embedded in Tissue-Tek before sectioning. Brains were sectioned coronally into PBS at 30 μm using a cryostat or microtome.

EdU (5-ethynyl-2'deoxyuridine) injection and staining
EdU injection and staining was described previously with modifications [4]. EdU powder (Toronto Research Chemical, #T-E932175) was dissolved in sterile 1x PBS at the stock concentration of 2.5 mg/ml. It was further diluted to 1 mg/ml in sterile 1x PBS as working concentration. Mice at P90 were injected with EdU at a dosage of 50 mg/kg intraperitoneally for 2 consecutive days. Twelve day after the last injection, mice were perfused with 1x PBS, followed by 4% PFA in phosphate buffer, brain and spinal cord were dissected for further tissue sectioning. Brains were sectioned at 30 µm thickness for EdU staining. EdU staining was performed on spinal cord sections using EdU staining solution containing 100mM Tris pH 7.5, 4mM CuSO4, 1mg/ml Sulfo-Cyanide Azide and 100mM Sodium Ascorbate in milli-Q water.
Brain sections were incubated in reaction cocktail for 1 hour at room temperature in dark, followed by 3 times wash with 1x PBS. To combine with cell markers staining, sections were permeabilized with 0.3% PBST for 15 minutes, followed by blocking with blocking buffer (5% BSA, 0.5% Tween-20 in 1x PBS) for 1 hour at room temperature, before applying primary antibody overnight in dark. The following steps are the same as normal immunofluorescent staining. EdU signal was visualized using red fluorescence at 594 nm and cell markers were visualized using green fluorescence at 488 nm.

Image acquisition
Confocal images were acquired with a Zeiss LSM700 inverted confocal microscope with 4 laser lines (405/488/555/639 nm) with either a 20x/0.8 N.A. air or 63x/1.15 N.A. oil immersion objectives. Images were captured using a AxioCam MRm monochromatic CCD camera (Zeiss) run by Zeiss Zen software.

Statistics
For data with multiple measures from individual mice were observed (e.g. Fig 2H and 2I with n = 3 per genotype and 5-6 sections per mouse), the variance was fit using a linear mixedeffects models with genotype as a fixed effect and random effect of individual mice variation 5 using the lme4 R package [5]. The model with the lowest AICc score is selected [6] and further pairwise contrasts were performed using Tukey's HSD test from the emmeans R package with Kenward-Roger approximations of degrees of freedom [7].

RNA isolation and qRT-PCR
Total RNAs were extracted from hippocampi using Trizol reagent (Thermo Fisher Scientific) as described previously [1] according to manufacturer's instruction. After DNase treatment using RQ1 RNase-Free DNase (Promega), 1 μg RNA were reversely transcribed using Maxima First Strand cDNA Synthesis Kit for RT-qPCR (Thermo Fisher Scientific). All qRT-PCR reactions were performed with at least three biological replicates for each group and two technical replicates. mRNA levels were determined using Maxima SYBR Green qPCR master mix (Thermo Fisher Scientific). The forward and reverse primer sequences were based on pre-validated primers from PrimerBank (https://pga.mgh.harvard.edu/primerbank/) and further validated in-house. Primers for genes of interest were listed in Supplemental Table 3.
Expression values were normalized to two control genes: HRPT and GAPDH mRNA.
Expression values were expressed as a percentage of the average expression of control samples.

Microarray analysis
RNA quality was measured using the Agilent Bioanalyzer system. Samples with RIN (RNA integrity numbers) larger than 8.5 were used for microarray analysis according to the manufacturer's protocol. In brief, total RNAs (n=3 for each genotype) were reverse transcribed to produce cDNA/mRNA hybrid, which was subsequently used as a template to create double stranded cDNA. This double-stranded cDNA was then amplified via in vitro transcription to produce cRNA. In vitro transcription generated cRNA was then purified and subjected to 2 ndcylce single-stranded sense cDNA synthesis, which was later fragmented, labeled, and hybridized to the GeneChip Mouse Transcriptome 1.0 Array for 16 hours at 45 0 C. Arrays were then washed, stained and scanned using an Affymetrix 3000 7G scanner. Differentially 6 expressed genes were selected based on p < 0.05 and fold-change more than 2-fold increased or decreased. Microarray data have been deposited in NCBI's Gene Expression Omnibus.
genomic structure for endogenous mouse C9orf72 and null C9orf72 allele. Exon 2-6 was replaced with a gene-trap cassette. (b) Survival curve wild type and C9orf72 knockout mice.
(c) Open field test for wild type and C9orf72 knockout mice at 3 months of age (n=15 for wild type mice; n=27 for C9orf72 knockout mice).

Supplemental Table 1. Statistic analysis of DG-LTP, CA1-LTP, and CA1-LTD in wild type
(WT) and C9orf72 knockout (C9KO) mice. P values of statistical comparisons between pretetanization time points and post-tetanization time points (30 min, 60 min, 120 min, and 180 min; Wilcoxon test) and the statistical comparisons with its own control groups when applicable (at 30 min, 60 min, 120 min, and 180 min; U-test).