Significance Alzheimer’s disease (AD) is the most common cause of age-related neurodegeneration. Damage initially occurs in the hippocampus, a neurogenic brain region essential in forming memories. Since there is no cure for AD, therapeutic strategies that may aid to slow hippocampal dysfunction are necessary. We describe the precocious hippocampal stem cell loss of a mouse model that mimics the onset of pathological AD-like neurodegeneration. The loss is due to an increase in BMP6 that limits neurogenesis. We demonstrate that blocking BMP signaling by means of Noggin administration is beneficial to the hippocampal microenvironment, restoring stem cell numbers, neurogenesis, and behavior. Our findings support further development of BMP antagonists into translatable molecules for the rescue of stem cells and neurogenesis in neurodegeneration/aging.

Abstract Increasing age is the greatest known risk factor for the sporadic late-onset forms of neurodegenerative disorders such as Alzheimer’s disease (AD). One of the brain regions most severely affected in AD is the hippocampus, a privileged structure that contains adult neural stem cells (NSCs) with neurogenic capacity. Hippocampal neurogenesis decreases during aging and the decrease is exacerbated in AD, but the mechanistic causes underlying this progressive decline remain largely unexplored. We here investigated the effect of age on NSCs and neurogenesis by analyzing the senescence accelerated mouse prone 8 (SAMP8) strain, a nontransgenic short-lived strain that spontaneously develops a pathological profile similar to that of AD and that has been employed as a model system to study the transition from healthy aging to neurodegeneration. We show that SAMP8 mice display an accelerated loss of the NSC pool that coincides with an aberrant rise in BMP6 protein, enhanced canonical BMP signaling, and increased astroglial differentiation. In vitro assays demonstrate that BMP6 severely impairs NSC expansion and promotes NSC differentiation into postmitotic astrocytes. Blocking the dysregulation of the BMP pathway and its progliogenic effect in vivo by intracranial delivery of the antagonist Noggin restores hippocampal NSC numbers, neurogenesis, and behavior in SAMP8 mice. Thus, manipulating the local microenvironment of the NSC pool counteracts hippocampal dysfunction in pathological aging. Our results shed light on interventions that may allow taking advantage of the brain’s natural plastic capacity to enhance cognitive function in late adulthood and in chronic neurodegenerative diseases such as AD.

Decades of research have firmly established that new neurons are produced in the postnatal and adult hippocampus of most mammals, including humans, due to the existence of a stem cell reservoir that confers regenerative capacity to this brain area essential in forming memories (1, 2). Despite the long-lasting plastic potential of the hippocampus, defects in hippocampal neurogenesis emerge during aging (3⇓⇓–6) and are exacerbated in pathological conditions such as Alzheimer’s disease (AD) (7⇓–9). Previous studies indicated that the newly generated cells in aged mice develop at a slower pace (6), have a reduced survival (10, 11), and differentiate less into neurons and more into astrocytes (10⇓⇓–13), but why this defective fate choice occurs and how we might manipulate stem cells or their niche to effectively counteract hippocampal dysfunction upon healthy and pathological aging remains largely unknown.

The neurogenic process involves several sequential steps. First, quiescent neural stem cells (NSCs) with radial morphology located in the subgranular zone (SGZ) of the dentate gyrus (DG) are activated and enter the cell cycle; next, the NSCs asymmetrically divide and produce nonradial (NR) proliferative progenitors that generate the new neurons. Finally, the young neurons mature and integrate into preexisting networks. Extracellular short-range niche signals regulate neurogenesis at all these different stages (14⇓–16) and alterations in the niche microenvironment may be linked to the age-related neurogenic decline (17). A relevant family of secreted factors whose function in the aged SGZ remains poorly defined is the bone morphogenetic protein (BMP) family (18). The expression of several BMPs is dysregulated in the hippocampus of old mice, in transgenic mouse models of familial AD, and in the brains of patients with early and severe AD compared with nondemented controls (19⇓⇓⇓⇓–24), pointing to a causative role of BMP signaling in the neurogenic defects found during aging and in AD.

We here analyze the causes underlying the decline in adult NSCs and neurogenesis during pathological aging, taking advantage of the senescence-accelerated mouse strain SAMP8. This nontransgenic short-lived strain precociously and progressively develops learning and memory deficits and a multisystemic aging phenotype (25⇓–27). SAMP8 also exhibits increased amyloid precursor protein (APP) and amyloid β (Aβ) levels, oxidative stress, tau phosphorylation, astrogliosis, and many of the biochemical findings of AD starting at the age of 6 mo (28⇓–30). Due to the spontaneous onset of pathology similar to that of human disease, SAMP8 is considered a good animal model for research into the transition from healthy aging to the onset phase of sporadic AD (28⇓⇓–31). SAMP8 is evaluated in reference to SAMR1, a genetically related strain that is resistant to accelerated senescence (25). We herein show that SAMP8 mice display an accelerated depletion of the hippocampal NSC pool compared with SAMR1 that coincides in space and time with an increase in astroglial differentiation, a reduction in neurogenesis, an increase in BMP6 protein level, and a dysregulation of the BMP signaling pathway. We also demonstrate that blocking the enhanced BMP signaling of the SAMP8 strain restores normal NSC numbers by counteracting the BMP-mediated NSC astroglial differentiation and simultaneously ameliorates the neurogenic and behavioral deficits of the senescent strain.

Discussion Age-related neurodegenerative disorders such as AD slowly undermine cognitive function and behavioral abilities. Although AD is not a part of normal healthy aging, the rate of the disease doubles every decade after the age of 60. Alterations in hippocampal neurogenesis, which have been extensively documented both during normal aging and in AD (7⇓–9), possibly contribute to the age- and AD-related hippocampal dysfunction, but the mechanistic causes underlying this phenomenon remain poorly understood. Hence, unraveling the changes affecting the hippocampal neurogenic niche and the hippocampal stem cell dynamics during aging and, most importantly, at early presymptomatic AD stages, may provide new insights into the progression of the disease. We herein show that BMP6 accumulates very early in the hippocampal niche of SAMP8 animals, a senescent strain that has been used to model some age-related aspects of the onset of sporadic AD. BMP6 is also significantly increased at the mRNA and protein levels in the hippocampus of patients with early and severe AD compared with nondemented controls and in a transgenic mouse model of familial AD (22). BMP6 accumulates around Aβ plaques in the hippocampus and, as in SAMP8, the accumulation in human tissue is accompanied by reduced hippocampal neurogenesis and SOX2+ cells. Moreover, at least in vitro, hippocampal stem cells exposed to Aβ overexpress BMP6 (22). An increase in BMP6 has been also reported during healthy aging in wild-type mice, possibly associated to microglial cells, the resident immune cells of the brain (23). Thus, although the available data suggest that BMP6 accumulates during healthy aging, the process may be exacerbated in pathological conditions such as AD that lead to increased Aβ levels, the activation of microglia, and an unfavorable inflammatory microenvironment (38, 39). Increased Aβ levels and inflammation have been detected in the SAMP8 brain (40⇓–42) and could therefore contribute to the rise of BMP6 in this mouse model. Our results indicate that the increase in BMP6 and in canonical BMP signaling mainly results in the precocious depletion of the adult hippocampal NSCs due to their BMP-mediated differentiation into astroglial cells. Interestingly, in 2-mo SAMP8 mice the remaining hippocampal NSCs proliferate and continue to generate new immature neurons, but these cells have a reduced survival and display an aberrant delay in differentiation, leading to a reduction in the number of NeuN+ granule neurons reaching maturity, as reported elsewhere (43). A similar aberrant hindrance to the normal development of the immature neuronal population has been described in the aging brain of wild-type mice (6), in the brain of AD patients, and in some transgenic AD models of the familial early-onset forms of the disease (7⇓–9, 22, 44). Increasing evidence suggests that the aged and AD neurogenic niche is no longer optimal for neurogenesis (7⇓–9); however, taking advantage of the SAMP8 model and employing the BMP antagonist Noggin we demonstrate that it is still possible to influence the niche microenvironment, to rescue stem cell numbers, and to revert the pathological neurogenic phenotype by means of blocking the aberrant BMP signaling. The treatment with Noggin also normalized the behavioral phenotype of SAMP8 animals, making the scores similar to those of control SAMR1 animals. The SAMP8 anxiety phenotype, the spatial learning defects, and the short-term memory retention capacity were improved by Noggin. In line with our results, hippocampal neurogenesis has been also rescued in transgenic AD models with Noggin (20) and it has been shown that increased BMP signaling results in cognitive impairments, while its reduction ameliorates hippocampus-dependent cognitive function in young and aged mice (24, 45, 46). In summary, age-related stem cell depletion and dysfunction may result from a combination of several factors, both intrinsic and extrinsic to the stem cell compartment, that are dysregulated with increasing age and that may be exacerbated in pathological conditions such as AD. Our data show that the temporal increase of BMP6 expression in the DG may be one of such altered extrinsic factors. The cell cycle lengthening of NSPCs may be rather a cell intrinsic feature (35). The loss of the stem cell population in SAMP8 mice is due to their differentiation into mature astrocytic cells, which is in accordance with previous findings in aged animals (19⇓⇓⇓⇓–24). We have uncovered why this defective fate choice occurs and how we might mitigate it by manipulating the stem cell niche. The increased glial differentiation of the NSCs can further help to explain the hippocampal astrocytosis that is detected in the SAMP8 strain starting at 6 mo of age (26) and that characterizes the late stages of AD (47). Our findings, combined with previous reports in which the in vivo delivery of lentiviral shRNA against downstream effectors of the BMP pathway partially rejuvenated NSC function in the aged brain (23, 24), support further development of BMP antagonists into translatable molecules for the rescue of stem cell depletion and neurogenesis during healthy and pathological aging.

Materials and Methods Animals. SAMP8 and SAMR1 males were used at 1, 2, 6, and 10 mo. Crl:CD1 males were used at 2 mo (SI Appendix). Mice were maintained under specific-pathogen-free conditions and all manipulations were approved by the Animal Care Committee of the Instituto de Salud Carlos III. Immunostaining. Antibody stainings were performed as described in SI Appendix. DG NSPC Culture. NSPCs were isolated from dissected hippocampi of 2-mo-old Crl:CD1, SAMR1, and SAMP8 mice and cultured in FGF2 and EGF (SI Appendix). Intracerebroventricular Infusion. Alzet osmotic minipumps were implanted in SAMR1 and SAMP8 mice for Noggin or vehicle solution infusion. Different experimental paradigms of infusion and BrdU administration were applied (SI Appendix). Lentiviral Injections. Five-month-old SAMP8 and SAMR1 males underwent stereotaxic surgery, and a 1.5-μL suspension of lentiviral ORF particles expressing Noggin or GFP (Origene Technologies) was injected into both hemispheres at DG coordinates (SI Appendix). Behavioral Assessments. Animals’ general activity was tested with a VersaMax Animal Activity Monitoring System. For anxiety measurement and spatial learning and memory tests, elevated plus maze and Morris water maze were performed as described in SI Appendix.

Acknowledgments We thank Prof. Gerd Kempermann and members of his laboratory for technical guidance in the NSPC isolation and the ISCIII Confocal Microscopy Core and of Lucía Casares-Crespo at the Instituto de Biomedicina de Valencia for assistance. This work was supported by a predoctoral fellowship from the Spanish Ministerio de Educación (to M.D.-M.), Spanish Ministerio de Economía y Competitividad Grant BFU2013-48907-R (to J.L.T.), and Grants PI12/101 and SAF2015-70433-R (to H.M.). We acknowledge support of the publication fee by the CSIC Open Access Publication Support Initiative through its Unit of Information Resources for Research (URICI).

Footnotes Author contributions: J.L.T. and H.M. designed research; M.D.-M., T.A., S.G., R.H., L.G.-C., Á.F.-L., J.L.T., and H.M. performed research; M.D.-M., T.A., S.G., J.L.T., and H.M. analyzed data; and J.L.T. and H.M. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

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