Rafael Hortigüela

Signaling through BMPR-IA Regulates Quiescence and Long-Term Activity of Neural Stem Cells in the Adult Hippocampus

Neural stem cells (NSCs) in the adult hippocampus divide infrequently, and the molecules that modulate their quiescence are largely unknown. Here, we show that bone morphogenetic protein (BMP) signaling is active in hippocampal NSCs, downstream of BMPR-IA. BMPs reversibly diminish proliferation of cultured NSCs while maintaining their undifferentiated state. In vivo, acute blockade of BMP signaling in the hippocampus by intracerebral infusion of Noggin first recruits quiescent NSCs into the cycle and increases neurogenesis; subsequently, it leads to decreased stem cell division and depletion of precursors and newborn neurons. Consistently, selective ablation of Bmpr1a in hippocampal NSCs, or inactivation of BMP canonical signaling in conditional Smad4 knockout mice, transiently enhances proliferation but later leads to a reduced number of precursors, thereby limiting neuronal birth. BMPs are therefore required to balance NSC quiescence/proliferation and to prevent loss of the stem cell activity that supports continuous neurogenesis in the mature hippocampus.

Impact of the clinical context on the 14-3-3 test for the diagnosis of sporadic CJD.

BackgroundThe 14-3-3 test appears to be a valuable aid for the clinical diagnosis of sporadic Creutzfeldt-Jakob disease (sCJD) in selected populations. However, its usefulness in routine practice has been challenged. In this study, the influence of the clinical context on the performance of the 14-3-3 test for the diagnosis of sCJD is investigated through the analysis of a large prospective clinical series.MethodsSix hundred seventy-two Spanish patients with clinically suspected sCJD were analyzed. Clinical classification at sample reception according to the World Health Organizations (WHO) criteria (excluding the 14-3-3 test result) was used to explore the influence of the clinical context on the pre-test probabilities, and positive (PPV) and negative (NPV) predictive values of the 14-3-3 test.ResultsPredictive values of the test varied greatly according to the initial clinical classification: PPV of 98.8%, 96.5% and 45.0%, and NPV of 26.1%, 66.6% and 100% for probable sCJDi (n = 115), possible sCJDi (n = 73) and non-sCJDi (n = 484) cases, respectively. According to multivariate and Bayesian analyses, these values represent an improvement of diagnostic certainty compared to clinical data alone.ConclusionIn three different contexts of sCJD suspicion, the 14-3-3 assay provides useful information complementary to clinical and electroencephalographic (EEG) data. The test is most useful supporting a clinical impression, whilst it may show deceptive when it is not in agreement with clinical data.

The Cyclin‐Dependent Kinase Inhibitor p27kip1 Regulates Radial Stem Cell Quiescence and Neurogenesis in the Adult Hippocampus

Members of the cyclin‐dependent kinase (CDK)‐inhibitory protein (CIP)/kinase‐inhibitory protein (KIP) family of cyclin‐dependent kinase inhibitors regulate proliferation and cell cycle exit of mammalian cells. In the adult brain, the CIP/KIP protein p27kip1 has been related to the regulation of intermediate progenitor cells located in neurogenic niches. Here, we uncover a novel function of p27kip1 in the adult hippocampus as a dual regulator of stem cell quiescence and of cell‐cycle exit of immature neurons. In vivo, p27kip1 is detected in radial stem cells expressing SOX2 and in newborn neurons of the dentate gyrus. In vitro, the Cdkn1b gene encoding p27kip1 is transcriptionally upregulated by quiescence signals such as BMP4. The nuclear accumulation of p27kip1 protein in adult hippocampal stem cells encompasses the BMP4‐induced quiescent state and its overexpression is able to block proliferation. p27kip1 is also expressed in immature neurons upon differentiation of adult hippocampal stem cell cultures. Loss of p27kip1 leads to an increase in proliferation and neurogenesis in the adult dentate gyrus, which results from both a decrease in the percentage of radial stem cells that are quiescent and a delay in cell cycle exit of immature neurons. Analysis of animals carrying a disruption in the cyclin‐CDK interaction domain of p27kip1 indicates that the CDK inhibitory function of the protein is necessary to control the activity of radial stem cells. Thus, we report that p27kip1 acts as a central player of the molecular program that keeps adult hippocampal stem cells out of the cell cycle. Stem Cells 2015;33:219–229

Aβ increases neural stem cell activity in senescence-accelerated SAMP8 mice.

Neurogenesis persists in the adult brain as a form of plasticity due to the existence of neural stem cells (NSCs). Alterations in neurogenesis have been found in transgenic Alzheimers disease (AD) mouse models, but NSC activity and neurogenesis in sporadic AD models remains to be examined. We herein describe a remarkable increase in NSC proliferation in the forebrain of SAMP8, a non-transgenic mouse strain that recapitulates the transition from healthy aging to AD. The increase in proliferation is transient, precedes AD-like symptoms such as amyloid beta 1-42 [Aβ(1-42)] increase or gliosis, and is followed by a steep decline at later stages. Interestingly, in vitro studies indicate that secreted Aβ(1-42) and PI3K signaling may account for the early boost in NSC proliferation. Our results highlight the role of soluble Aβ(1-42) peptide and PI3K in the autocrine regulation of NSCs, and further suggest that over-proliferation of NSCs before the appearance of AD pathology may underlie neurogenic failure during the age-related progression of the disease. These findings have implications for therapeutic approaches based on neurogenesis in AD.

Symmetric Expansion of Neural Stem Cells from the Adult Olfactory Bulb Is Driven by Astrocytes Via WNT7A

Adult neural stem cells (NSCs) located in the subventricular zone (SVZ) persistently produce new neurons destined to the olfactory bulb (OB). Recent research suggests that the OB is also a source of NSCs that remains largely unexplored. Using single/dual‐labeling procedures, we address the existence of NSCs in the innermost layers of the OB. In vivo, these cells are more quiescent that their SVZ counterparts, but after in vitro expansion, they behave similarly. Self‐renewal and proliferation assays in co‐culture with niche astrocytes indicate that OB‐glia restricts NSC activity whereas SVZ‐glia has the opposite effect. Gene expression profiling identifies WNT7A as a key SVZ‐glial factor lacking in OB‐glia that enhances self‐renewal, thereby improving the propagation of OB‐NSC cultures. These data demonstrate that region‐specific glial factors account for in vivo differences in NSC activity and point to WNT7A as a tool that may be instrumental for the NSC expansion phase that precedes grafting. STEM CELLS 2012;30:2796–2809

Genetic variability of the gene cluster CALHM1–3 in sporadic Creutzfeldt-Jakob disease

Perturbations of calcium homeostasis have been associated with several neurodegenerative disorders. A common polymorphism (rs2986017) in the CALHM1 gene, coding for a regulator of calcium homeostasis, is a genetic risk factor for the development of Alzheimer disease (AD). Although some authors failed to confirm these results, a meta-analysis has shown that this polymorphism modulates the age at disease onset. Furthermore, a recent association study has explored the genetic variability of CALHM1 gene and two adjacent paralog genes (CALHM3 and CALHM2) in an Asian population. Since several lines of evidence suggest that AD and prion diseases share pathophysiologic mechanisms, we investigated for the first time the genetic variability of the gene cluster formed by CALHM1 and its paralogs in a series of 235 sporadic Creutzfeldt-Jakob disease (sCJD) patients, and compared the genotypic and allelic frequencies with those presented in 329 controls from the same ancestry. As such, this work also represents the first association analysis of CALHM genes in sCJD. Sequencing analysis of the complete coding regions of the genes demonstrated the presence of 10 single nucleotide polymorphisms (SNP) within the CALHM genes. We observed that rs4918016-rs2986017-rs2986018 and rs41287502-rs41287500 polymorphic sites at CALHM1 were in linkage disequilibrium. We found marginal associations for sCJD risk at CALHM1 polymorphic sites rs41287502 and rs41287500 [coding for two linked missense mutations (p.(Met323Ile); (Gly282Cys)], and rs2986017 [p.(Leu86Pro)]. Interestingly, a TGG haplotype defined by the rs4918016-rs2986017-rs2986018 block was associated with sCJD. These findings underscore the need of future multinational collaborative initiatives in order to corroborate these seminal data.

Allelic discrimination of genetic human prion diseases by real-time PCR genotyping

The complete molecular characterization of human genetic prion diseases from different backgrounds is important for clinical diagnosis and epidemiological classification. The characterization of the PRNP gene should always include the description of the pathogenic mutation, as well as the status at each allele of the polymorphic codon 129 (M129V), a well-established susceptibility marker and phenotypic variability factor for different types of human prion diseases. Indeed, the phenotypical expression of two of the most common mutations in the human PRNP gene associated with genetic prion diseases, D178N and E200K, is clearly modulated by the codon 129 polymorphism. Here, we describe two simple, fast, cost-effective and suited for high-throughput protocols to resolve cis-trans ambiguities between these mutations respect the M129V polymorphism. This methodology is based on differential amplification by allele-specific primers using Real Time PCR monitored by SYBR® Green dye. The main advantages of these protocols are their relative simplicity and the reduced cost compared to other methods such as cloning protocols, and that it may be readily applicable to the characterization of other mutations with codon 129-dependent expression, e.g. P102L.

Familial cerebral cavernous malformations associated with a splice-site CCM2 deletion

JO N 3070 an autosomal dominant pattern with variable penetrance, and it has been associated with mutations of three genes, CCM1, CCM2 and CCM3 [2]. We describe a novel CCM2 mutation detected in a Spanish pedigree with familial CCM. The proband is a 75-year-old man who complained of progressive gait instability in the last 2 weeks. He reported a past history of myocardial infarction at 60 years of age, and vertical diplopia initiated in his thirties. Brain MRI showed multiple intraparenchymal lesions consistent with cavernous malformations (Fig. 1). His family history was remarkable because of multiple cases of intracerebral hemorrhage (Fig. 2, II-7). His father died at 60 years of age from myocardial infarction, and his mother died in the ninth decade from unknown reasons. Neither of them had obvious neurological problems. Genetic analysis of the CCM1 gene did not show pathogenic mutations. Sequence analysis of the CCM2 gene (Chr. 7p15-p13) detected a 14 bp deletion, described as NM_001029835.1 : c.99211_994delGTCCCCCACTGCAG Adolfo Jiménez-Huete Rafael Hortigüela Elena Riva Juan Bernar Pedro Guardado Santervás Jesús Esteban Oriol Franch Miguel Calero

BMP and WNT signalling cooperate through LEF1 in the neuronal specification of adult hippocampal neural stem and progenitor cells

Neuronal production from neural stem cells persists during adulthood in the subgranular zone of the hippocampal dentate gyrus. Extracellular signals provided by the hippocampal microenvironment regulate the neuronal fate commitment of the stem cell progeny. To date, the identity of those signals and their crosstalk has been only partially resolved. Here we show that adult rat hippocampal neural stem and progenitor cells (AH-NSPCs) express receptors for bone morphogenetic proteins (BMPs) and that the BMP/P-Smad pathway is active in AH-NSPCs undergoing differentiation towards the neuronal lineage. In vitro, exposure to the BMP2 and BMP4 ligands is sufficient to increase neurogenesis from AH-NSPCs in a WNT dependent manner while decreasing oligodendrogenesis. Moreover, BMP2/4 and WNT3A, a key regulator of adult hippocampal neurogenesis, cooperate to further enhance neuronal production. Our data point to a mechanistic convergence of the BMP and WNT pathways at the level of the T-cell factor/lymphoid enhancer factor gene Lef1. Altogether, we provide evidence that BMP signalling is an important regulator for the neuronal fate specification of AH-NSPCs cultures and we show that it significantly cooperates with the previously described master regulator of adult hippocampal neurogenesis, the WNT signalling pathway.

Noggin rescues age-related stem cell loss in the brain of senescent mice with neurodegenerative pathology

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. 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.