Hippocampal Deletion of CB1 Receptor Impairs Social Memory and Leads to Age-Related Changes in the Hippocampus of Adult Mice.

Endocannabinoid system activity declines with age in the hippocampus, along with the density of the cannabinoid receptor type-1 (CB1). This process might contribute to brain ageing, as previous studies showed that the constitutive deletion of the CB1 receptor in mice leads to early onset of memory deficits and histological signs of ageing in the hippocampus including enhanced pro-inflammatory glial activity and reduced neurogenesis. Here we asked whether the CB1 receptor exerts its activity locally, directly influencing hippocampal ageing or indirectly, accelerating systemic ageing. Thus, we deleted the CB1 receptor site-specifically in the hippocampus of 2-month-old CB1flox/flox mice using stereotaxic injections of rAAV-Cre-Venus viruses and assessed their social recognition memory four months later. Mice with hippocampus-specific deletion of the CB1 receptor displayed a memory impairment, similarly as observed in constitutive knockouts at the same age. We next analysed neuroinflammatory changes in the hippocampus, neuronal density and cell proliferation. Site-specific mutant mice had enhanced glial cell activity, up-regulated levels of TNFα in the hippocampus and decreased cell proliferation, specifically in the subgranular zone of the dentate gyrus. Our data indicate that a local activity of the CB1 receptor in the hippocampus is required to maintain neurogenesis and to prevent neuroinflammation and cognitive decline.

Local cannabinoid receptor type-1 regulates glial cell activity and insulin-like growth factor-1 receptor signaling in the mediobasal hypothalamus.

As organisms age, the activity of the endocannabinoid system in the brain declines, coinciding with increased neuroinflammation and disrupted hypothalamic functions. Notably, cannabinoid receptors type-1 (CB1) are highly expressed in the ventromedial hypothalamic nucleus (VMH) within the mediobasal hypothalamus, a central area of neuroendocrine regulation. This study investigates whether the CB1 receptor influences age-related changes in a brain region-dependent manner. Therefore, we performed stereotaxic injections of rAAV1/2 expressing Cre recombinase in 2-month-old CB1flox/flox male animals to delete the CB1 gene and in CB1-deficient (CB1-STOP) mice to induce its re-expression. The intensity of pro-inflammatory glial activity, gonadotropin-releasing hormone (GnRH) and insulin-like growth factor-1 receptor (IGF-1R) expression was assessed in the hypothalamus of mice at 18-19 months of age. Site-specific CB1 receptor deletion induced pro-inflammatory glial activity and increased hypothalamic Igf1r mRNA expression. Unexpectedly, GnRH levels remained unaltered. Importantly, rescuing the receptor in null mutant animals had the opposite effect: it reduced pro-inflammatory glial activation and decreased Igf1r mRNA expression without affecting GnRH production. Overall, the study highlights the important role of the CB1 receptor in the VMH in reducing age-related inflammation and modulating IGF-1R signaling.

Possible role of locus coeruleus neuronal loss in age-related memory and attention deficits.

Introduction: Aging is associated with a decline in cognitive abilities, including memory and attention. It is generally accepted that age-related histological changes such as increased neuroinflammatory glial activity and a reduction in the number of specific neuronal populations contribute to cognitive aging. Noradrenergic neurons in the locus coeruleus (LC) undergo an approximately 20 % loss during ageing both in humans and mice, but whether this change contributes to cognitive deficits is not known. To address this issue, we asked whether a similar loss of LC neurons in young animals as observed in aged animals impairs memory and attention, cognitive domains that are both influenced by the noradrenergic system and impaired in aging. Methods: For that, we treated young healthy mice with DSP-4, a toxin that specifically kills LC noradrenergic neurons. We compared the performance of DSP-4 treated young mice with the performance of aged mice in models of attention and memory. To do this, we first determined the dose of DSP-4, which causes a similar 20 % neuronal loss as is typical in aged animals. Results: Young mice treated with DSP-4 showed impaired attention in the presence of distractor and memory deficits in the 5-choice serial reaction time test (5-CSRTT). Old, untreated mice showed severe deficits in both the 5-CSRTT and in fear extinction tests. Discussion: Our data now suggest that a reduction in the number of LC neurons contributes to impaired working memory and greater distractibility in attentional tasks but not to deficits in fear extinction. We hypothesize that the moderate loss of LC noradrenergic neurons during aging contributes to attention deficits and working memory impairments.

Efficacy of Δ9 -Tetrahydrocannabinol (THC) Alone or in Combination With a 1:1 Ratio of Cannabidiol (CBD) in Reversing the Spatial Learning Deficits in Old Mice.

Decline in cognitive performance, an aspect of the normal aging process, is influenced by the endocannabinoid system (ECS). Cannabinoid receptor 1 (CB1) signaling diminishes with advancing age in specific brain regions that regulate learning and memory and abolishing CB1 receptor signaling accelerates cognitive aging in mice. We recently demonstrated that prolonged exposure to low dose (3 mg/kg/day) Δ9-tetrahydrocannabinol (THC) improved the cognitive performances in old mice on par with young untreated mice. Here we investigated the potential influence of cannabidiol (CBD) on this THC effect, because preclinical and clinical studies indicate that the combination of THC and CBD often exhibits an enhanced therapeutic effect compared to THC alone. We first tested the effectiveness of a lower dose (1 mg/kg/day) THC, and then the efficacy of the combination of THC and CBD in 1:1 ratio, same as in the clinically approved medicine Sativex®. Our findings reveal that a 1 mg/kg/day THC dose still effectively improved spatial learning in aged mice. However, a 1:1 combination of THC and CBD failed to do so. The presence of CBD induced temporal changes in THC metabolism ensuing in a transient elevation of blood THC levels. However, as CBD metabolizes, the inhibitory effect on THC metabolism was alleviated, causing a rapid clearance of THC. Thus, the beneficial effects of THC seemed to wane off more swiftly in the presence of CBD, due to these metabolic effects. The findings indicate that THC-treatment alone is more efficient to improve spatial learning in aged mice than the 1:1 combination of THC and CBD.

Depletion of TrkB Receptors From Adult Serotonergic Neurons Increases Brain Serotonin Levels, Enhances Energy Metabolism and Impairs Learning and Memory.

Neurotrophin brain-derived neurotrophic factor (BDNF) and neurotransmitter serotonin (5-HT) regulate each other and have been implicated in several neuronal mechanisms, including neuroplasticity. We have investigated the effects of BDNF on serotonergic neurons by deleting BDNF receptor TrkB from serotonergic neurons in the adult brain. The transgenic mice show increased 5-HT and Tph2 levels with abnormal behavioral phenotype. In spite of increased food intake, the transgenic mice are significantly leaner than their wildtype littermates, which may be due to increased metabolic activity. Consistent with increased 5-HT, the proliferation of hippocampal progenitors is significantly increased, however, long-term survival of newborn cells is unchanged. Our data indicates that BDNF-TrkB signaling regulates the functional phenotype of 5-HT neurons with long-term behavioral consequences.

NPM1 mutations are more stable than FLT3 mutations during the course of disease in patients with acute myeloid leukemia.

NPM1 mutations have been reported to be the most frequent mutations in acute myeloid leukemia (AML). They are associated with a wide spectrum of morphologic subtypes of AML, normal karyotype and FLT3 mutations. The high frequency of NPM1 mutations might provide a suitable marker for monitoring residual disease of AML.

An overview on the role of FLT3-tyrosine kinase receptor in acute myeloid leukemia: biology and treatment.

Hematopoiesis, the process by which the hematopoietic stem cells and progenitors differentiate into blood cells of various lineages, involves complex interactions of transcription factors that modulate the expression of downstream genes and mediate proliferation and differentiation signals. Despite the many controls that regulate hematopoiesis, mutations in the regulatory genes capable of promoting leukemogenesis may occur. The FLT3 gene encodes a tyrosine kinase receptor that plays a key role in controlling survival, proliferation and differentiation of hematopoietic cells. Mutations in this gene are critical in causing a deregulation of the delicate balance between cell proliferation and differentiation. In this review, we provide an update on the structure, synthesis and activation of the FLT3 receptor and the subsequent activation of multiple downstream signaling pathways. We also review activating FLT3 mutations that are frequently identified in acute myeloid leukemia, cause activation of more complex downstream signaling pathways and promote leukemogenesis. Finally, FLT3 has emerged as an important target for molecular therapy. We, therefore, report on some recent therapies directed against it.

Monitoring of FLT3 phosphorylation status and its response to drugs by flow cytometry in AML blast cells.

FLT3 mutation and overexpression in most acute myeloid leukaemia (AML) patients make this tyrosine kinase receptor an attractive therapeutic target. FLT3 kinase inhibitors are actually in clinical trials, thus it is critical to develop a reproducible and standardized method for screening of FLT3 activation and for monitoring its inhibition in response to drug in AML patients. We developed a flow cytometry method to analyse phosphorylated FLT3 (P-FLT3) in samples with <10(5) cells. The method was first validated in FLT3 wild-type (HL60/WT) and mutant (MV4-11/ITD(+)) as well as FLT3 negative (K562) cell lines. The method also proved to be reproducible in AML patient samples. Analysis was performed after exposure to drugs (CEP-701 and SU11657), in vitro and in vivo. In response to increasing drug concentrations, there was a linear reduction in P-FLT3. Intracellular flow cytometry analysis correlated with Western blot and XTT assays; flow cytometry data also correlated with FLT3 mutational status. The results highlight a rapid method to detect P-FLT3 protein at the single cell level by flow cytometry which enables an accurate assessment of FLT3 kinase activity in blast cells in response to novel tyrosine kinase inhibitors.

Molecular and chromosomal alterations: new therapies for relapsed acute myeloid leukemia.

Acute myeloid leukemia (AML) remains the most common form of leukemia and the most common cause of leukemia death. Although conventional chemotherapy can cure between 25 and 45% of AML patients, the majority of patients die after relapse or of complications associated with treatment. Thus, more specific and less toxic treatments for AML patients are needed, especially for elderly patients. An indispensable prerequisite to investigate tailored approaches for AML is the recent progress in the understanding the molecular features that distinguish leukemia progenitors from normal hematopoietic counterparts and the identification of a variety of dysregulated molecular pathways. This in turn would allow the identification of tumor-specific characteristics that provide a rational basis for the development of more tailored, and hence potentially more effective and less toxic, therapeutic approaches. In this review, we describe some of the signaling pathways that are aberrantly regulated in AML, with a specific focus on their pathogenetic and therapeutic significance, and we examine some recent therapies directed against these targets, used in clinical trial for relapsed patients or unfit for conventional chemotherapy.