Phosphorylation of collapsin response mediator protein-2 disrupts neuronal maturation in a model of adult neurogenesis: Implications for neurodegenerative disorders

<p>Abstract</p> <p>Background</p> <p>Recent studies suggest that the pathogenic process in neurodegenerative disorders may disrupt mature neuronal circuitries and neurogenesis in the adult brain. Abnormal activation of CDK5 is associated with neurodegenerative disorders, and recently a critical role for CDK5 in adult neurogenesis has been identified. We have developed an <it>in vitro </it>model of abnormal CDK5 activation during adult hippocampal neurogenesis, and here we used this model to investigate aberrantly phosphorylated downstream targets of CDK5.</p> <p>Results</p> <p>Abnormal CDK5 activation in an <it>in vitro </it>model of adult neurogenesis results in hyperphosphorylation of collapsin-response mediator protein-2 (CRMP2) and impaired neurite outgrowth. Inhibition of CDK5, or expression of a non-phosphorylatable (S522A) CRMP2 construct reduced CRMP2 hyperphosphorylation, and reversed neurite outgrowth deficits. CRMP2 plays a role in microtubule dynamics; therefore we examined the integrity of microtubules in this model using biochemical and electron microscopy techniques. We found that microtubule organization was disrupted under conditions of CDK5 activation. Finally, to study the relevance of these findings to neurogenesis in neurodegenerative conditions associated with HIV infection, we performed immunochemical analyses of the brains of patients with HIV and transgenic mice expressing HIV-gp120 protein. CDK5-mediated CRMP2 phosphorylation was significantly increased in the hippocampus of patients with HIV encephalitis and in gp120 transgenic mice, and this effect was rescued by genetic down-modulation of CDK5 in the mouse model.</p> <p>Conclusions</p> <p>These results reveal a functional mechanism involving microtubule destabilization through which abnormal CDK5 activation and CRMP2 hyperphosphorylation might contribute to defective neurogenesis in neurodegenerative disorders such as HIV encephalitis.</p

Long-term neprilysin gene transfer is associated with reduced levels of intracellular Abeta and behavioral improvement in APP transgenic mice

<p>Abstract</p> <p>Background</p> <p>Proteolytic degradation has emerged as a key pathway involved in controlling levels of the Alzheimer's disease (AD)-associated amyloid-β (Aβ) peptide in the brain. The endopeptidase, neprilysin, has been implicated as a major Aβ degrading enzyme in mice and humans. Previous short and intermediate term studies have shown the potential therapeutic application of neprilysin by delivering this enzyme into the brain of APP transgenic mice using gene transfer with viral vectors. However the effects of long-term neprilysin gene transfer on other aspects of Aβ associated pathology have not been explored yet in APP transgenic mice.</p> <p>Results</p> <p>We show that the sustained expression of neprilysin for up to 6 months lowered not only the amyloid plaque load but also reduced the levels of intracellular Aβ immunoreactivity. This was associated with improved behavioral performance in the water maze and ameliorated the dendritic and synaptic pathology in the APP transgenic mice.</p> <p>Conclusion</p> <p>These data support the possibility that long-term neprilysin gene therapy improves behavioral and neurodegenerative pathology by reducing intracellular Aβ.</p

An RNA editing fingerprint of cancer stem cell reprogramming

BackgroundDeregulation of RNA editing by adenosine deaminases acting on dsRNA (ADARs) has been implicated in the progression of diverse human cancers including hematopoietic malignancies such as chronic myeloid leukemia (CML). Inflammation-associated activation of ADAR1 occurs in leukemia stem cells specifically in the advanced, often drug-resistant stage of CML known as blast crisis. However, detection of cancer stem cell-associated RNA editing by RNA sequencing in these rare cell populations can be technically challenging, costly and requires PCR validation. The objectives of this study were to validate RNA editing of a subset of cancer stem cell-associated transcripts, and to develop a quantitative RNA editing fingerprint assay for rapid detection of aberrant RNA editing in human malignancies.MethodsTo facilitate quantification of cancer stem cell-associated RNA editing in exons and intronic or 3'UTR primate-specific Alu sequences using a sensitive, cost-effective method, we established an in vitro RNA editing model and developed a sensitive RNA editing fingerprint assay that employs a site-specific quantitative PCR (RESSq-PCR) strategy. This assay was validated in a stably-transduced human leukemia cell line, lentiviral-ADAR1 transduced primary hematopoietic stem and progenitor cells, and in primary human chronic myeloid leukemia stem cells.ResultsIn lentiviral ADAR1-expressing cells, increased RNA editing of MDM2, APOBEC3D, GLI1 and AZIN1 transcripts was detected by RESSq-PCR with improved sensitivity over sequencing chromatogram analysis. This method accurately detected cancer stem cell-associated RNA editing in primary chronic myeloid leukemia samples, establishing a cancer stem cell-specific RNA editing fingerprint of leukemic transformation that will support clinical development of novel diagnostic tools to predict and prevent cancer progression.ConclusionsRNA editing quantification enables rapid detection of malignant progenitors signifying cancer progression and therapeutic resistance, and will aid future RNA editing inhibitor development efforts

Passive Immunization Reduces Behavioral and Neuropathological Deficits in an Alpha-Synuclein Transgenic Model of Lewy Body Disease

Dementia with Lewy bodies (DLB) and Parkinson's Disease (PD) are common causes of motor and cognitive deficits and are associated with the abnormal accumulation of alpha-synuclein (α-syn). This study investigated whether passive immunization with a novel monoclonal α-syn antibody (9E4) against the C-terminus (CT) of α-syn was able to cross into the CNS and ameliorate the deficits associated with α-syn accumulation. In this study we demonstrate that 9E4 was effective at reducing behavioral deficits in the water maze, moreover, immunization with 9E4 reduced the accumulation of calpain-cleaved α-syn in axons and synapses and the associated neurodegenerative deficits. In vivo studies demonstrated that 9E4 traffics into the CNS, binds to cells that display α-syn accumulation and promotes α-syn clearance via the lysosomal pathway. These results suggest that passive immunization with monoclonal antibodies against the CT of α-syn may be of therapeutic relevance in patients with PD and DLB

Chronic Myeloid Leukemia Stem Cell Biology

Leukemia progression and relapse is fueled by leukemia stem cells (LSC) that are resistant to current treatments. In the progression of chronic myeloid leukemia (CML), blast crisis progenitors are capable of adopting more primitive but deregulated stem cell features with acquired resistance to targeted therapies. This in turn promotes LSC behavior characterized by aberrant self-renewal, differentiation, and survival capacity. Multiple reports suggest that cell cycle alterations, activation of critical signaling pathways, aberrant microenvironmental cues from the hematopoietic niche, and aberrant epigenetic events and deregulation of RNA processing may facilitate the enhanced survival and malignant transformation of CML progenitors. Here we review the molecular evolution of CML LSC that promotes CML progression and relapse. Recent advances in these areas have identified novel targets that represent important avenues for future therapeutic approaches aimed at selectively eradicating the LSC population while sparing normal hematopoietic progenitors in patients suffering from chronic myeloid malignancies

Molecular Pathology of Neuro-AIDS (CNS-HIV)

The cognitive deficits in patients with HIV profoundly affect the quality of life of people living with this disease and have often been linked to the neuro-inflammatory condition known as HIV encephalitis (HIVE). With the advent of more effective anti-retroviral therapies, HIVE has shifted from a sub-acute to a chronic condition. The neurodegenerative process in patients with HIVE is characterized by synaptic and dendritic damage to pyramidal neurons, loss of calbindin-immunoreactive interneurons and myelin loss. The mechanisms leading to neurodegeneration in HIVE might involve a variety of pathways, and several lines of investigation have found that interference with signaling factors mediating neuroprotection might play an important role. These signaling pathways include, among others, the GSK3β, CDK5, ERK, Pyk2, p38 and JNK cascades. Of these, GSK3β has been a primary focus of many previous studies showing that in infected patients, HIV proteins and neurotoxins secreted by immune-activated cells in the brain abnormally activate this pathway, which is otherwise regulated by growth factors such as FGF. Interestingly, modulation of the GSK3β signaling pathway by FGF1 or GSK3β inhibitors (lithium, valproic acid) is protective against HIV neurotoxicity, and several pilot clinical trials have demonstrated cognitive improvements in HIV patients treated with GSK3β inhibitors. In addition to the GSK3β pathway, the CDK5 pathway has recently been implicated as a mediator of neurotoxicity in HIV, and HIV proteins might activate this pathway and subsequently disrupt the diverse processes that CDK5 regulates, including synapse formation and plasticity and neurogenesis. Taken together, the GSK3β and CDK5 signaling pathways are important regulators of neurotoxicity in HIV, and modulation of these factors might have therapeutic potential in the treatment of patients suffering from HIVE. In this context, the subsequent sections will focus on reviewing the involvement of the GSK3β and CDK5 pathways in neurodegeneration in HIV

Selective Molecular Alterations in the Autophagy Pathway in Patients with Lewy Body Disease and in Models of α-Synucleinopathy

Lewy body disease is a heterogeneous group of neurodegenerative disorders characterized by α-synuclein accumulation that includes dementia with Lewy bodies (DLB) and Parkinson's Disease (PD). Recent evidence suggests that impairment of lysosomal pathways (i.e. autophagy) involved in α-synuclein clearance might play an important role. For this reason, we sought to examine the expression levels of members of the autophagy pathway in brains of patients with DLB and Alzheimer's Disease (AD) and in α-synuclein transgenic mice.By immunoblot analysis, compared to controls and AD, in DLB cases levels of mTor were elevated and Atg7 were reduced. Levels of other components of the autophagy pathway such as Atg5, Atg10, Atg12 and Beclin-1 were not different in DLB compared to controls. In DLB brains, mTor was more abundant in neurons displaying α-synuclein accumulation. These neurons also showed abnormal expression of lysosomal markers such as LC3, and ultrastructural analysis revealed the presence of abundant and abnormal autophagosomes. Similar alterations were observed in the brains of α-synuclein transgenic mice. Intra-cerebral infusion of rapamycin, an inhibitor of mTor, or injection of a lentiviral vector expressing Atg7 resulted in reduced accumulation of α-synuclein in transgenic mice and amelioration of associated neurodegenerative alterations.This study supports the notion that defects in the autophagy pathway and more specifically in mTor and Atg7 are associated with neurodegeneration in DLB cases and α-synuclein transgenic models and supports the possibility that modulators of the autophagy pathway might have potential therapeutic effects

NOTCH1 Signaling Promotes Human T-Cell Acute Lymphoblastic Leukemia Initiating Cell Regeneration in Supportive Niches

Leukemia initiating cells (LIC) contribute to therapeutic resistance through acquisition of mutations in signaling pathways, such as NOTCH1, that promote self-renewal and survival within supportive niches. Activating mutations in NOTCH1 occur commonly in T cell acute lymphoblastic leukemia (T-ALL) and have been implicated in therapeutic resistance. However, the cell type and context specific consequences of NOTCH1 activation, its role in human LIC regeneration, and sensitivity to NOTCH1 inhibition in hematopoietic microenvironments had not been elucidated.We established humanized bioluminescent T-ALL LIC mouse models transplanted with pediatric T-ALL samples that were sequenced for NOTCH1 and other common T-ALL mutations. In this study, CD34(+) cells from NOTCH1(Mutated) T-ALL samples had higher leukemic engraftment and serial transplantation capacity than NOTCH1(Wild-type) CD34(+) cells in hematopoietic niches, suggesting that self-renewing LIC were enriched within the NOTCH1(Mutated) CD34(+) fraction. Humanized NOTCH1 monoclonal antibody treatment reduced LIC survival and self-renewal in NOTCH1(Mutated) T-ALL LIC-engrafted mice and resulted in depletion of CD34(+)CD2(+)CD7(+) cells that harbor serial transplantation capacity.These results reveal a functional hierarchy within the LIC population based on NOTCH1 activation, which renders LIC susceptible to targeted NOTCH1 inhibition and highlights the utility of NOTCH1 antibody targeting as a key component of malignant stem cell eradication strategies