Felipe Salech

Age-Dependent Increases in Apoptosis/Necrosis Ratios in Human Lymphocytes Exposed to Oxidative Stress

Unlike apoptosis, mechanisms leading to necrosis are less well understood. Moreover, changes in necrosis as a function of age have not been studied in human lymphocytes. H(2)O(2)-induced death of peripheral lymphocytes (56 healthy donors, 24-95 years) was evaluated by flow cytometry and propidium iodide staining, caspase activation, DNA laddering, and electron microscopy. H(2)O(2)-induced stress was associated with high levels of necrosis in young individuals (≤30 years), whereas progressively enhanced apoptotic death was observed in older donors, without changes in overall lymphocyte survival. Thus, apoptosis/necrosis ratios were inverted in young versus elderly (≥65 years) donors. Death was not accompanied by increased caspase activity and, accordingly, unaffected by caspase inhibition; however, it was almost completely prevented by poly ADP ribose polymerase inhibition. In summary, aging was associated with changes in the apoptosis/necrosis ratios, rather than susceptibility per se to H(2)O(2)-induced death, which was caspase independent but poly ADP ribose polymerase dependent. Understanding this switch in death modes may aid in understanding age-related disorders.

Inverse Susceptibility to Oxidative Death of Lymphocytes Obtained From Alzheimer's Patients and Skin Cancer Survivors: Increased Apoptosis in Alzheimer's and Reduced Necrosis in Cancer

A paucity of cancer in individuals with Alzheimers disease (AD) and low rates of AD in cancer survivors has been reported in epidemiological studies. Deregulation in opposite directions of biological mechanisms, such as susceptibility to cell death, might be shared in the two disorders. We analyzed lymphocytes from AD and skin cancer patients as well as healthy controls and found significantly increased vulnerability of AD lymphocytes to H(2)O(2)-induced apoptotic death and higher resistance to death of skin cancer lymphocytes, due to reduced necrosis, as compared with healthy controls by pairwise comparisons adjusted for age and sex. H(2)O(2)-induced death in lymphocytes was caspase independent and significantly reduced by PARP-1 inhibition in all three groups. These differences in the susceptibility to cell death observed for lymphocytes from AD and skin cancer patients may be one of the mechanisms that help explain the inverse correlation detected between these diseases in epidemiological studies.

Frizzled-1 receptor regulates adult hippocampal neurogenesis

BackgroundIn the adult hippocampus new neurons are continuously generated from neural stem cells (NSCs) present at the subgranular zone of the dentate gyrus. This process is controlled by Wnt signaling, which plays a complex role in regulating multiple steps of neurogenesis including maintenance, proliferation and differentiation of progenitor cells and the development of newborn neurons. Differential effects of Wnt signaling during progression of neurogenesis could be mediated by cell-type specific expression of Wnt receptors. Here we studied the potential role of Frizzled-1 (FZD1) receptor in adult hippocampal neurogenesis.ResultsIn the adult dentate gyrus, we determined that FZD1 is highly expressed in NSCs, neural progenitors and immature neurons. Accordingly, FZD1 is expressed in cultured adult hippocampal progenitors isolated from mouse brain. To evaluate the role of this receptor in vivo we targeted FZD1 in newborn cells using retroviral-mediated RNA interference. FZD1 knockdown resulted in a marked decrease in the differentiation of newborn cells into neurons and increased the generation of astrocytes, suggesting a regulatory role for the receptor in cell fate commitment. In addition, FZD1 knockdown induced an extended migration of adult-born neurons within the granule cell layer. However, no differences were observed in total dendritic length and dendritic arbor complexity between control and FZD1-deficient newborn neurons.ConclusionsOur results show that FZD1 regulates specific stages of adult hippocampal neurogenesis, being required for neuronal differentiation and positioning of newborn neurons into the granule cell layer, but not for morphological development of adult-born granule neurons.

Increased Susceptibility to Oxidative Death of Lymphocytes from Alzheimer Patients Correlates with Dementia Severity

We previously reported on enhanced susceptibility to death of lymphocytes from Alzheimers disease (AD) patients when exposed to hydrogen peroxide (H2O2)-induced oxidative stress and an increased resistance to death in those of patients with a history of skin cancer. This is consistent with our hypothesis proposing that the cellular machinery controlling cell death is deregulated in opposite directions in Alzheimers disease (AD) and cancer, to explain the inverse association observed in epidemiological studies. Here we investigated whether the observed increased susceptibility correlates with the degree of dementia severity. Peripheral lymphocytes from 23 AD patients, classified using the Clinical Dementia Rating (CDR) into severe dementia (CDR 3, n=10) and mild-to-moderate dementia (CDR 1- 2, n=13), and 15 healthy controls (HC) (CDR 0), were exposed to H2O2 for 20 hours. Lymphocyte death was determined by flow cytometry and propidium iodide staining. The greatest susceptibility to H2O2-induced death was observed for lymphocytes from severe dementia patients, whereas those with mild-to-moderate dementia exhibited intermediate values, compared to healthy controls. A significant increase in the apoptosis/necrosis ratio was found in AD patients. Poly (ADP-ribosyl) polymerase-1 (PARP-1) inhibition significantly protected from H2O2-induced death of lymphocytes, whereby a lower degree of protection was observed in severe AD patients. Moreover, inhibition of PARP-1 abolished the differences in apoptosis/necrosis ratios observed between the three groups of patients. These results support the notion that AD is a systemic disorder, whereby enhanced susceptibility to H2O2-induced death in peripheral lymphocytes correlates with dementia severity and enhanced death in AD patients is attributable to a PARP-dependent increase in the apoptosis/necrosis ratio.

Local Klotho Enhances Neuronal Progenitor Proliferation in the Adult Hippocampus

Klotho is an aging-related protein associated with hippocampal cognitive performance in mammals. Klotho regulates progenitor cell proliferation in non-neuronal tissues, but its role in adult hippocampal neurogenesis (AHN) has not been explored. Klotho expression in the adult mouse hippocampus was examined by immunofluorescence and polymerase chain reaction. AHN was evaluated in the hippocampus of klotho knock-out mice (KO), klotho KO/vitamin D-receptor mutant mice, and in a model of local klotho hippocampal knockdown. The recombinant Klotho effect on proliferation was measured in mouse-derived hippocampal neural progenitor cells. Hippocampal-dependent memory was assessed by a dry-land version of the Morris water maze. Klotho was expressed in the granular cell layer of the adult Dentate Gyrus. AHN was increased in klotho KO mice, but not in klotho KO/vitamin D-receptor mutant mice. Inversely, local downregulation of hippocampal Klotho diminished AHN. Recombinant Klotho increased the proliferation rate of neural progenitors. Downregulation of hippocampal Klotho correlated with a decreased performance in hippocampal-dependent memory. These results suggest that Klotho directly participates in regulating AHN. Our observations indicate that Klotho promotes proliferation, AHN and hippocampal-dependent cognition. Increased neurogenesis in klotho KO mice may be secondary to the activation of other pathways altered in the model, such as vitamin D.

PARP-1 and p53 Regulate the Increased Susceptibility to Oxidative Death of Lymphocytes from MCI and AD Patients

Mild cognitive impairment (MCI) is a clinically detectable initial stage of cognitive deterioration with a high conversion rate to dementia. There is increasing evidence that some of the cerebral alterations present in Alzheimer type dementia can be found in peripheral tissues. We have previously shown that lymphocytes from Alzheimer’s disease (AD) patients have increased susceptibility to hydrogen peroxide (H2O2)-induced death that depends on dementia severity. We here investigated whether lymphocytes from MCI patients show increased vulnerability to death, and explored the involvement of Poly [ADP-ribose] polymerase (PARP-1) and p53 in the regulation of this process. Lymphocytes from 16 MCI and 10 AD patients, and 15 healthy controls (HCs) were submitted to increasing concentrations of H2O2 for 20 h. Cell death was determined by flow cytometry, in the presence or absence of PARP-1 inhibitors (3-aminobenzamide (3-ABA) or Nicotinamide (NAM)), or the p53 inhibitor (nutlin-3) or stabilizer (pifithrin-α). PARP-1 and p53 mRNA levels were determined by quantitative PCR (qPCR). Lymphocytes from MCI patients showed increased susceptibility to death, attaining intermediate values between AD and controls. PARP inhibitors -3-ABA and NAM- markedly protected from H2O2-induced death, making the difference between MCI and controls disappear, but not the difference between AD and controls. PARP-1 mRNA expression was increased in MCI lymphocytes. Modulation of p53 with Nutlin-3 or pifithrin-α did not modify the H2O2-induced death of lymphocytes from MCI or AD patients, but augmented the death in control lymphocytes attaining levels similar to MCI and AD. Accordingly, p53 mRNA expression was increased in AD and MCI lymphocytes compared to controls. In all, these results show that increased oxidative death is present in lymphocytes at the MCI stage. PARP-1 has a preponderant role, with complete death protection achieved with PARP inhibition in MCI lymphocytes, but not in AD, suggesting that PARP-1 might have a protective role. In addition, deregulations of the p53 pathway seem to contribute to the H2O2-induced death in MCI and AD lymphocytes, which show increased p53 expression. The results showing a prominent protective role of PARP inhibitors opens the door to study the use of these agents to prevent oxidative death in MCI patients.

Vitamin D Increases Aβ140 Plasma Levels and Protects Lymphocytes from Oxidative Death in Mild Cognitive Impairment Patients

BACKGROUND Mild cognitive impairment (MCI) has an increased rate of progression to dementia. Alterations of some metabolic factors, such as deficiency of vitamin D, are a risk factor for cognitive deterioration. Vitamin D is involved in the clearance of β-amyloid (Aβ) from the brain. We have reported that lymphocytes from Alzheimers disease (AD) patients have an increased susceptibility to oxidative death by H2O2 exposure, but currently it is unknown if this characteristic is modifiable in vivo. OBJECTIVE To determine if correction of low vitamin D levels protects lymphocytes from oxidative death and increases Aβ1-40 plasma levels in MCI and very early AD (VEAD) patients. METHOD Sixteen MCI, 11 VEAD and 25 healthy control (HC) voluntaries were evaluated with the Clinical Dementia Rating (CDR), Montreal Cognitive assessment (MoCA), and Memory Index score (MIS). Lymphocyte death was measured by flow cytometry after 20h exposure to H2O2. In patients with low levels of vitamin D -11 MCI, 9 VEAD and 20 HC- lymphocyte H2O2-death, plasma Aβ1-40 levels and cognitive status were evaluated pre- and post-vitamin D supplementation for 6 months. RESULTS Lymphocytes from MCI and VEAD patients showed increased susceptibility to oxidative death at study entry. In MCI, but not VEAD patients, lymphocyte susceptibility to death and Aβ1-40 levels plasma levels improved after 6 months of vitamin D supplementation. In addition, cognitive status on follow-up (18 months) improved in MCI patients after vitamin D supplementation. CONCLUSION Vitamin D supplementation may be beneficial in MCI. The lack of effect in VEAD may be due to a more advanced stage or different characteristics of the neurodegenerative process.

Cancer imprints an increased PARP-1 and p53-Dependent resistance to oxidative stress on lymphocytes of patients that later develop Alzheimer's disease

We have proposed that a common biological mechanism deregulated in opposite directions might explain the inverse epidemiological association observed between Alzheimers disease (AD) and cancer. Accordingly, we showed that lymphocytes from AD patients have an increased susceptibility, whereas those from survivors of a skin cancer, an increased resistance to oxidative death induced by hydrogen peroxide (H2O2), compared to healthy controls (HC). We investigated the susceptibility to H2O2-induced death of lymphocytes in survivors of any type of cancer and in cancer survivors who later developed AD (Ca&AD). We also explored the involvement of Poly [ADP-ribose] polymerase-1 (PARP-1) and p53 pathways in the process, since both are involved in the increased susceptibility to death of AD lymphocytes. Lymphocytes from 11 cancer and 13 Ca&AD patients, and 12 HC were submitted to increasing concentrations of H2O2 for 20 h. Cell death was determined by flow cytometry, in the presence or absence of PARP-1 inhibition (3-aminobenzamide, 3-ABA), or p53 inhibition (pifithrin-α) or stabilization (Nut-3). PARP-1 and p53 mRNA levels were determined by Real-Time PCR. Lymphocytes from cancer and Ca&AD patients showed increased survival compared to HC, without differences between them, opposite to the increased susceptibility to death previously shown in AD. PARP-1 inhibition provided marked protection from H2O2-induced death in the two groups of patients, significantly greater than in HC. Pharmacological inhibition of p53 increased lymphocyte survival in Ca&AD patients, contrary to the effect previously reported in HC and AD. PARP-1 and p53 mRNA levels were elevated in Ca&AD lymphocytes compared with controls. In all, these results show that cancer imprints an increased resistance to H2O2-induced death in lymphocytes that persists after AD development, and is dependent on both PARP-1 and p53. p53 inhibition showed a differential role in cancer and Ca&AD compared to HC and AD lymphocytes, that could explain the inverse susceptibility to oxidative death in cancer and AD. These results are in agreement with the hypothesis of a common biological mechanism in AD and cancer. The similar cell death susceptibility and cell death pattern observed in cancer and Ca&AD lymphocytes suggests that cancer history leaves long term effects on lymphocyte cell death susceptibility.

Advances in Alzheimer’s Research

The research team of Dr. Caghan Kizil at the DFG-Center for Regenerative Therapies Dresden (CRTD) Cluster of Excellence at the TU Dresden, achieved a major advance in Alzheimer’s research. They showed how a diseased vertebrate brain can naturally react to Alzheimer’s pathology by forming more neurons. Two proteins (Interleukin-4 and STAT6) have been identified to be relevant for this process. This is a big step towards the understanding, prevention or even healing of Alzheimer’s disease – a disease with about 170,000 new cases diagnosed every year in Germany. The results have been published in the scientific journal Cell Reports this week.