*2.1. Animals*

Homozygous triple-transgenic 3xTg-AD mice harboring human PS1/M146V, APPSwe, and tauP301L transgenes were genetically engineered at the University of California Irvine, as previously described [9]. Briefly, two independent transgenes (encoding human APPSwe and human tauP301L, both under control of the mouse Thy1.2 regulatory element) were co-injected into single-cell embryos harvested from homozygous mutant PS1M146V knockin (PS1KI) mice. The PS1 knock-in mice were originally generated after embryonic transfer into pure C57BL/6.

A total of thirty-six 14-month-old homozygous male and female mice from the Spanish colonies of 3xTg-AD (*n* = 19, 10 males, 9 females) and C57BL/6 (*n* = 17, 10 males and 7 females) wild-type mice (from now, referred as non-transgenic mice, NTg) from litters of a breeding program established after embryonic transfer to C57BL/6 strain background were used in this study. All the animals were housed three to four per cage and maintained (Makrolon, 35 × <sup>35</sup> × 25 cm 3) under standard laboratory conditions (12 h light/dark, cycle starting at 8:00 h, food and water available ad libitum, 22 ± 2 ◦C, 50–60% humidity) at the Universitat Autònoma de Barcelona. Behavioral tests were performed from 9:00 h to 13:00 h. Assessments were performed blind to the experiment in a counterbalanced manner.

All procedures were in accordance with Spanish legislation on "Protection of Animals Used for Experimental and Other Scientific Purposes" and the EU Council directive (2010/63/EU) on this subject. The protocol CEEAH 3588/DMAH 9452 was approved the 8th of March 2019 by Departament de Medi Ambient i Habitatge, Generalitat de Catalunya. The study complies with the ARRIVE guidelines developed by the NC3Rs and aims to reduce the number of animals used [25].

#### *2.2. Experimental Design*

A longitudinal study divided into successive phases was performed, starting at 14 months of age, with the characterization of their physical and mental health status (behavioral phenotype and physical condition (weight, Mouse Clinical Frailty Index Assessment, and Survival) 441.7 ± 1.55 days (14.52 months). Thereafter, different physiological variables were evaluated: systolic blood pressure 470.9 ± 1.41 days (15.48 months); relative cerebral blood flow 477.9 ± 1.49 days (15.71 months); aortic function and angiogenesis 499.6 ± 2.14 days (16.43 month). Survival was continuously monitored, and glucocorticoid levels, as an indicator of HPA axis function, were analyzed from blood samples collected during the euthanasia.
