Patients with AD from the Clinical Trials Program at the Geropsychiatric Clinic at Samsung Medical Center were included in the study. These patients were a completely separate sample from our previous report.⁸ Patients were diagnosed as "probable Alzheimer's disease" using the NINCDS-ADRDA (The National Institute of Neurological and Communicative Disorders and Stroke and the Alzheimer's Disease and Related Disorders Association) diagnostic criteria. All patients underwent a neuropsychological evaluation, routine laboratory tests, a neurological examination, and neuroimaging (brain magnetic resonance imaging) to rule out other causes of dementia. The Korean version of the Mini Mental State Examination (K-MMSE),¹³ Clinical Dementia Rating (CDR),¹⁴ Seoul-Instrumental Activity of Daily Living (S-IADL),¹⁵ and Seoul-Activity of Daily Living Scale (S-ADL)¹⁶ were administered. Patients with other significant psychiatric, neurological, or medical illnesses were excluded.

The normal control subjects consisted of elderly volunteers who responded to an advertisement. They were evaluated by a psychiatrist based on the Health Screening Exclusion Criteria¹⁷ to ensure the absence of medical, neurological, and psychiatric problems. All subjects met the following inclusion criteria: 1) free of memory and cognitive disorders; 2) lived independently without difficulty; 3) no active psychiatric illness, alcohol/drug history; and 4) no active medical or neurological disorder including human cytomegalovirus infection, hypoxia, and certain cancers that could potentially affect cognition and the G1/S transition.^18-21 In addition, the K-MMSE, the 7 Minute Screening Test,²² S-IADL, and S-ADL were administered by an independent clinical psychologist.

With the approval of the Institutional Review Board at the Samsung Medical Center, 26 patients with AD and 28 normal controls participated and completed this study. Signed informed consent was obtained from all participants.

Blood samples from the patients and normal controls were collected to separate peripheral lymphocytes. Blood samples were heparinized, stored at 4℃ immediately after venipuncture, and assayed within 24 hr. Peripheral blood mononuclear cells were isolated by centrifugation at 300×g for 15 min on a Ficoll-Hypaque (Pharmacia Biotech, Uppsala, Sweden) density gradient. The cells were washed twice with phosphate buffered saline (Sigma, St. Louis, MO, USA), and the lymphocytes were separated in Hank's balanced salt solution (Sigma). We defined this point as T₀ (baseline). After 20 µg/mL of PHA (Gibco, Grand Island, NY, USA) was added to reactivate the cell cycle, the lymphocytes were cultured in RPMI1640 (Gibco) containing 10% fetal bovine serum at 37℃ with 5% CO₂ for 24 hr. These lymphocytes were then treated with 100 ng/mL rapamycin (Sigma) and cultured for an additional 24 hr under the previous conditions. We defined this point as T₄₈. Rapamycin is an immunosuppressant drug used to prevent organ transplant rejection. It prevents lymphocytes from activating by inhibiting their response to interleukin-2. We assumed that rapamycin would inhibit the G1/S transition.²³ We measured cell numbers in the cell cycle stages, as indicated below, to assess the G1/S transition control at T₀ and T₄₈.

The cultured lymphocytes were fixed in 70% ethanol at 4℃ for 10 min to measure cell number in each stage. The samples (1×10⁶ cells for each) were washed twice with ice-cold phosphate buffered saline, and incubated with RNase, DNA intercalating dye, and 2.0 mL of DAPI (Cystain DNA 1step, Partec, Görlitz Germany). We used a PAS flow cytometer (Partec) and the ModFit LT 3.0 Partec software to measure the proportion of cells in each cell cycle phase (Fig. 1A). At least 1×10⁴ events were analyzed, and the assay was repeated twice. We evaluated the proportion of G1 and S phases at T₀ and T₄₈ from the patients with AD and the normal controls.

We used the independent two-sample t-test and the Mann-Whitney test with Bonferroni's correction to evaluate the difference in the proportion of G1 and S phase cells between the patients with AD and normal controls at each point. Pearson's partial correlation and Spearman's partial correlation analyses were applied to adjust for age. The paired t-test and Wilcoxon's signed-rank test with Bonferroni's correction were used to evaluate the differences in the cell cycle phase between T₀ and T₄₈ in each group. The relationships between the proportion of G1 phase cells at T₄₈ and the clinical scores reflecting disease severity (CDR, K-MMSE, S-IADL, S-IADL, and duration of illness) in patients with AD were tested using Pearson's correlation and Spearman's correlation analyses. We divided the patients with AD into three groups according to their CDR score (CDR score 0.5 or 1, CDR score 2, and CDR score 3). The differences in the proportion of G1 phase cells at T₄₈ among these groups were evaluated using the Kruskal-Wallis test.

No significant differences were observed in the female/male ratio (AD 16/10 vs. control 19/9, p=0.62) or educational status (AD 8.76±5.42 years vs. control 10.39±4.21 yr, p=0.22) between the patients with AD and normal controls. The patients with AD were older than that of the normal controls (AD 74.03±7.90 yr vs. control 68.28±6.21 yr, p=0.004), so all results were adjusted for age. The patients with AD had mild to severe dementia with a mean K-MMSE score of 17.34 (SD, 6.26). Sixteen patients had CDR scores of 0.5 or 1, eight had a CDR score of 2, and two had a CDR score of 3 (Table 1). A total of 24 patients received cognitive enhancers [donepezil (n=7), galantamine (n=13), or memantine (n=4)] and two patients received sertraline. We divided the patients into subgroups according to their medications. No significant differences were observed for gender, age, education, K-MMSE, or CDR scores among the subgroups taking cognitive enhancers, as well as among the four subgroups taking any medication.

We found (i) A 24 hr PHA incubation followed by a 24 hr rapamycin incubation resulted in no net change in the G1 and S phases in control subjects; (ii) there was evidence of activation of the cell cycle (fewer in G1 and more in S phases) in patients with AD. The cell cycle analysis at T₀ did not show any differences between the AD and control groups (G1 phase; p>0.99, S phase; p>0.99). After cell cycle reactivation at T₄₈ and following the rapamycin treatment, the proportion of G1 phase cells in the AD group was lower than that in the control group (70.29±6.32% vs. 76.03±9.05%, p=0.01) and the proportion of S phase cells in the AD group was higher than that in the control group (12.45±6.09% vs. 6.03±5.11%, p=0.001) after adjusting for age (Table 2 and Fig. 1B).

We compared the cell cycle between T₀ and T₄₈ in each group. The cell cycle in the control group at T₄₈ was not different from that at T₀ (G1 phase; p>0.99, S phase; p>0.99). The proportion of G1 phase cells at T₄₈ in the AD group was significantly lower than that at T₀ (70.29±6.32% vs. 77.05±11.69%, p=0.02), and the proportion of S phase cells at T₄₈ was higher than that at T₀ (12.45±6.09% vs. 5.80±7.40%, p=0.002) (Fig. 1B). We also compared the cell cycle at T₀ and T₄₈ in each subgroup according to the medications to exclude the influence of medication, and no significant differences in the cell cycle analysis were observed among the subgroups.

No correlation was found between the proportion of G1 phase cells at T₄₈ and the clinical scores reflecting dementia severity (CDR, K-MMSE, S-IADL, S-ADL, and duration of illness) in the AD group (CDR, ρ=0.14, p=0.49; K-MMSE, r=0.25, p=0.22; S-IADL, r=-0.19, p=0.34; S-ADL, ρ=-0.05, p=0.80; duration of illness, r=-0.13, p=0.53).