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1.
Sci Rep ; 14(1): 19, 2024 01 02.
Article in English | MEDLINE | ID: mdl-38167670

ABSTRACT

Long-chain acyl-CoAs (LC-acyl-CoAs) are important intermediary metabolites and are also thought to function as intracellular signaling molecules; however, the direct effects of LC-acyl-CoAs have been difficult to determine in real-time and dissociate from Protein Kinase A (PKA) signaling. Here, we examined the direct role of lipolysis in generating intracellular LC-acyl-CoAs and activating AMPK in white adipocytes by pharmacological activation of ABHD5 (also known as CGI-58), a lipase co-activator. Activation of lipolysis in 3T3-L1 adipocytes independent of PKA with synthetic ABHD5 ligands, resulted in greater activation of AMPK compared to receptor-mediated activation with isoproterenol, a ß-adrenergic receptor agonist. Importantly, the effect of pharmacological activation of ABHD5 on AMPK activation was blocked by inhibiting ATGL, the rate-limiting enzyme for triacylglycerol hydrolysis. Utilizing a novel FRET sensor to detect intracellular LC-acyl-CoAs, we demonstrate that stimulation of lipolysis in 3T3-L1 adipocytes increased the production of LC-acyl-CoAs, an effect which was blocked by inhibition of ATGL. Moreover, ATGL inhibition blocked AMPKß1 S108 phosphorylation, a site required for allosteric regulation. Increasing intracellular LC-acyl-CoAs by removal of BSA in the media and pharmacological inhibition of DGAT1 and 2 resulted in greater activation of AMPK. Finally, inhibiting LC-acyl-CoA generation reduced activation of AMPK; however, did not lower energy charge. Overall, results demonstrate that lipolysis in white adipocytes directly results in allosteric activation of AMPK through the generation of LC-acyl-CoAs.


Subject(s)
Acyl Coenzyme A , Lipolysis , Mice , Animals , Acyl Coenzyme A/metabolism , AMP-Activated Protein Kinases/metabolism , Signal Transduction , Adipocytes, White/metabolism , 3T3-L1 Cells
2.
Transl Stroke Res ; 12(1): 1-14, 2021 02.
Article in English | MEDLINE | ID: mdl-32862401

ABSTRACT

Stroke is a major cause of death and disability worldwide. Yet therapeutic strategies available to treat stroke are very limited. There is an urgent need to develop novel therapeutics that can effectively facilitate functional recovery. The injury that results from stroke is known to induce neurogenesis in penumbra of the infarct region. There is considerable interest in harnessing this response for therapeutic purposes. This review summarizes what is currently known about stroke-induced neurogenesis and the factors that have been identified to regulate it. Additionally, some key studies in this field have been highlighted and their implications on future of stroke therapy have been discussed. There is a complex interplay between neuroinflammation and neurogenesis that dictates stroke outcome and possibly recovery. This highlights the need for a better understanding of the neuroinflammatory process and how it affects neurogenesis, as well as the need to identify new mechanisms and potential modulators. Neuroinflammatory processes and their impact on post-stroke repair have therefore also been discussed.


Subject(s)
Brain Ischemia/pathology , Neurogenesis/physiology , Recovery of Function/physiology , Stroke/pathology , Animals , Brain/cytology , Brain/pathology , Brain/physiology , Brain Ischemia/therapy , Humans , Neurogenesis/drug effects , Neuroprotective Agents/administration & dosage , Stem Cell Transplantation/trends , Stroke/therapy
3.
J Neuroimmunol ; 346: 577318, 2020 Jul 09.
Article in English | MEDLINE | ID: mdl-32682140

ABSTRACT

Stroke is the leading cause of death and the main cause of disability in surviving patients. The detrimental interaction between immune cells, glial cells, and matrix components in stroke pathology results in persistent inflammation that progresses to fibrosis. A substantial effort is being directed toward understanding the exact neuroinflammatory events that take place as a result of stroke. The initiation of a potent cytokine response, along with immune cell activation and infiltration in the ischemic core, has massive acute deleterious effects, generally exacerbated by comorbid inflammatory conditions. There is secondary neuroinflammation that promotes further injury, resulting in cell death, but conversely plays a beneficial role, by promoting recovery. This highlights the need for a better understanding of the neuroinflammatory and fibrotic processes, as well as the need to identify new mechanisms and potential modulators. In this review, we summarize several aspects of stroke-induced inflammation, fibrosis, and include a discussion of cytokine inhibitors/inducers, immune cells, and fibro-inflammation signaling inhibitors in order to identify new pharmacological means of intervention.

4.
Neuroscience ; 441: 33-45, 2020 08 10.
Article in English | MEDLINE | ID: mdl-32540366

ABSTRACT

The motor features of Parkinson's disease (PD) result from the loss of dopaminergic (DA) neurons in the substantia nigra with autophagy dysfunction being closely linked to this disease. A PD-causing familial mutation in VPS35 (D620N) has been reported to inhibit autophagy. In order to identify signaling pathways responsible for this autophagy defect, we performed an unbiased screen using RNA sequencing (RNA-Seq) of wild-type or VPS35 D620N-expressing retinoic acid-differentiated SH-SY5Y cells. We report that VPS35 D620N-expressing cells exhibit transcriptome changes indicative of alterations in extracellular matrix (ECM)-receptor interaction as well as PI3K-AKT signaling, a pathway known to regulate autophagy. Hyaluronan (HA) is a major component of brain ECM and signals via the ECM receptors CD44, a top RNA-Seq hit, and HA-mediated motility receptor (HMMR) to the autophagy-regulating PI3K-AKT pathway. We find that high (>950 kDa), but not low (15-40 kDa), molecular weight HA treatment inhibits autophagy. In addition, VPS35 D620N facilitated enhanced HA-AKT signaling. Transcriptomic assessment and validation of protein levels identified the differential expression of CD44 and HMMR isoforms in VPS35 D620N mutant cells. We report that knockdown of HMMR or CD44 results in upregulated autophagy in cells expressing wild-type VPS35. However, only HMMR knockdown resulted in rescue of autophagy dysfunction by VPS35 D620N indicating a potential pathogenic role for this receptor and HA signaling in Parkinson's disease.


Subject(s)
Parkinson Disease , Vesicular Transport Proteins , Autophagy , Humans , Hyaluronan Receptors/genetics , Hyaluronic Acid , Phosphatidylinositol 3-Kinases , Vesicular Transport Proteins/genetics
5.
J Neuroinflammation ; 16(1): 222, 2019 Nov 14.
Article in English | MEDLINE | ID: mdl-31727174

ABSTRACT

BACKGROUND: Stroke remains a leading cause of death and disability worldwide despite recent treatment breakthroughs. A primary event in stroke pathogenesis is the development of a potent and deleterious local and peripheral inflammatory response regulated by the pro-inflammatory cytokine interleukin-1 (IL-1). While the role of IL-1ß (main released isoform) has been well studied in stroke, the role of the IL-1α isoform remains largely unknown. With increasing utilization of intravenous tissue plasminogen activator (t-PA) or thrombectomy to pharmacologically or mechanically remove ischemic stroke causing blood clots, respectively, there is interest in pairing successful cerebrovascular recanalization with neurotherapeutic pharmacological interventions (Fraser et al., J Cereb Blood Flow Metab 37:3531-3543, 2017; Hill et al., Lancet Neurol 11:942-950, 2012; Amaro et al., Stroke 47:2874-2876, 2016). METHODS: Transient stroke was induced in mice via one of two methods. One group of mice were subjected to tandem ipsilateral common carotid artery and middle cerebral artery occlusion, while another group underwent the filament-based middle cerebral artery occlusion. We have recently developed an animal model of intra-arterial (IA) drug administration after recanalization (Maniskas et al., J Neurosci Met 240:22-27, 2015). Sub groups of the mice were treated with either saline or Il-1α, wherein the drug was administered either acutely (immediately after surgery) or subacutely (on the third day after stroke). This was followed by behavioral and histological analyses. RESULTS: We now show in the above-mentioned mouse stroke models (transient tandem ipsilateral common carotid artery (CCA) and middle cerebral artery occlusion (MCA) occlusion, MCA suture occlusion) that IL-1α is neuroprotective when acutely given either intravenously (IV) or IA at low sub-pathologic doses. Furthermore, while IV administration induces transient hemodynamic side effects without affecting systemic markers of inflammation, IA delivery further improves overall outcomes while eliminating these side effects. Additionally, we show that delayed/subacute IV IL-1α administration ameliorates functional deficit and promotes neurorepair. CONCLUSIONS: Taken together, our present study suggests for the first time that IL-1α could, unexpectedly, be an effective ischemic stroke therapy with a broad therapeutic window.


Subject(s)
Brain Ischemia/drug therapy , Interleukin-1alpha/therapeutic use , Neuroprotective Agents/therapeutic use , Stroke/drug therapy , Animals , Behavior, Animal/drug effects , Brain/drug effects , Brain/pathology , Brain Ischemia/pathology , Disease Models, Animal , Interleukin-1alpha/pharmacology , Male , Mice , Neurons/drug effects , Neurons/pathology , Neuroprotective Agents/pharmacology , Recovery of Function/drug effects , Stroke/pathology
6.
Neuroscience ; 401: 1-10, 2019 03 01.
Article in English | MEDLINE | ID: mdl-30660673

ABSTRACT

Parkinson's Disease (PD) is a multi-system neurodegenerative disease where approximately 90% of cases are idiopathic. The remaining 10% of the cases can be traced to a genetic origin and research has largely focused on these associated genes to gain a better understanding of the molecular and cellular pathogenesis for PD. The gene encoding vacuolar protein sorting protein 35 (VPS35) has been definitively linked to late onset familial PD following the identification of a point mutation (D620N) as the causal agent in a Swiss family. Since its discovery, numerous studies have been undertaken to characterize the role of VPS35 in cellular processes and efforts have been directed toward understanding the perturbations caused by the D620N mutation. In this review, we examine what is currently known about VPS35, which has pleiotropic effects, as well as proposed mechanisms of pathogenesis by the D620N mutation. A brief survey of other VPS35 polymorphisms is also provided. Lastly, model systems that are being utilized for these investigations and possible directions for future research are discussed.


Subject(s)
Mutation , Parkinson Disease/genetics , Vesicular Transport Proteins/genetics , Animals , Humans , Parkinson Disease/metabolism , Protein Transport , Vesicular Transport Proteins/metabolism
7.
Neural Regen Res ; 12(11): 1865-1869, 2017 Nov.
Article in English | MEDLINE | ID: mdl-29239333

ABSTRACT

The function of dopaminergic neurons in the substantia nigra is of central importance to the coordination of movement by the brain's basal ganglia circuitry. This is evidenced by the loss of these neurons, resulting in the cardinal motor deficits associated with Parkinson's disease. In order to fully understand the physiology of these key neurons and develop potential therapies for their loss, it is essential to determine if and how dopaminergic neurons are replenished in the adult brain. Recent work has presented evidence for adult neurogenesis of these neurons by Nestin+/Sox2- neural progenitor cells. We sought to further validate this finding and explore a potential atypical origin for these progenitor cells. Since neural progenitor cells have a proximal association with the vasculature of the brain and subsets of endothelial cells are Nestin+, we hypothesized that dopaminergic neural progenitors might share a common cell lineage. Therefore, we employed a VE-cadherin promoter-driven CREERT2:THlox/THlox transgenic mouse line to ablate the tyrosine hydroxylase gene from endothelial cells in adult animals. After 26 weeks, but not 13 weeks, following the genetic blockade of tyrosine hydroxylase expression in VE-cadherin+ cells, we observed a significant reduction in tyrosine hydroxylase+ neurons in the substantia nigra. The results from this genetic lineage tracing study suggest that dopaminergic neurons are replenished in adult mice by a VE-cadherin+ progenitor cell population potentially arising from an endothelial lineage.

8.
Article in English | MEDLINE | ID: mdl-28533891

ABSTRACT

Although harboring the apolipoprotein E4 (APOE4) allele is a well known risk factor in Alzheimer's disease (AD), the mechanism by which it contributes to disease risk remains elusive. To investigate the role of proteolysis of apoE4 as a potential mechanism, we designed and characterized a site-directed cleavage antibody directed at position D151 of the mature form of apoE4 and E3. Characterization of this antibody indicated a high specificity for detecting synthesized recombinant proteins corresponding to the amino acid sequences 1-151 of apoE3 and E4 that would generate the 17 kDa (p17) fragment. In addition, this antibody also detected a ~17 kDa amino-terminal fragment of apoE4 following incubation with collagenase and matrix metalloproteinase-9 (MMP-9), but did not react with full-length apoE4. Application of this amino-terminal apoE cleavage-fragment (nApoECFp17) antibody, revealed nuclear labeling within glial cells and labeling of a subset of neurofibrillary tangles in the human AD brain. A quantitative analysis indicated that roughly 80% of labeled nuclei were microglia. To confirm these findings, cultured BV2 microglia cells were incubated with the amino-terminal fragment of apoE4 corresponding to the cleavage site at D151. The results indicated efficient uptake of this fragment and trafficking to the nucleus that also resulted in significant cell death. In contrast, a similarly designed apoE3 fragment showed no toxicity and primarily localized within the cytoplasm. These data suggest a novel cleavage event by which apoE4 is cleaved by the extracellular proteases, collagenase and MMP-9, generating an amino-terminal fragment that is then taken up by microglia, traffics to the nucleus and promotes cell death. Collectively, these findings provide important mechanistic insights into the mechanism by which harboring the APOE4 allele may elevate dementia risk observed in AD.

9.
Neurosci Lett ; 615: 50-4, 2016 Feb 26.
Article in English | MEDLINE | ID: mdl-26806039

ABSTRACT

The primary clinical motor symptoms of Parkinson's disease (PD) result from loss of dopaminergic (DA) neurons in the substantia nigra (SN). Consequently, neurogenesis of this group of neurons in the adult brain has drawn considerable interest for the purpose of harnessing endogenous neurogenerative potential as well as devising better strategies for stem cell therapy for PD. However, the existence of adult neurogenesis for DA neurons within the SN remains controversial. To overcome technical and design limitations associated with previous studies, our group has developed a novel genetic mouse model for assessing adult nigral DA neurogenesis. This system utilizes transgenic mice that express a tamoxifen-activatable Cre recombinase (Cre(ERT2)) under the control of the neuronal progenitor cell promoters nestin or Sox2 leading to suppression of the DA neuron marker tyrosine hydroxylase (TH) via excision of exon 1 by flanking loxP sites in adult animals. This study reports that six months following initiation of a six week treatment with tamoxifen mice with nestin-mediated Th excision displayed a significant reduction in TH+ neurons in the SN. This finding indicates that nestin-expressing cells regenerate DA neurons within the SN of adult animals. Interestingly, no reduction was observed in TH+ cells following Sox2-mediated Th excision suggesting that a nestin+/SOX2- precursor cell population drives DA neurogenesis in the adult SN. This information represents a substantial leap in current knowledge of adult DA neurogenesis, will enable improved in vitro and in vivo modeling, as well as facilitate the harnessing of this process for therapeutic intervention for PD.


Subject(s)
Dopaminergic Neurons/physiology , Nestin/metabolism , SOXB1 Transcription Factors/metabolism , Substantia Nigra/cytology , Animals , Mice, Transgenic , Neurogenesis , Substantia Nigra/metabolism , Tyrosine 3-Monooxygenase/genetics
10.
Infect Genet Evol ; 12(5): 978-86, 2012 Jul.
Article in English | MEDLINE | ID: mdl-22414917

ABSTRACT

Evidence for natural selection, positive or negative, on gene encoding antigens may indicate variation or functional constraints that are immunologically relevant. Most malaria surface antigens with high genetic diversity have been reported to be under positive-diversifying selection. However, antigens with limited genetic variation are usually ignored in terms of the role that natural selection may have in generating such patterns. We investigated orthologous genes encoding two merozoite proteins, MSP8 and MSP10, among several mammalian Plasmodium spp. These antigens, together with MSP1, are among the few MSPs that have two epidermal growth factor-like domains (EGF) at the C-terminal. Those EGF are relatively conserved (low levels of genetic polymorphism) and have been proposed to act as ligands during the invasion of RBCs. We use several evolutionary genetic methods to detect patterns consistent with natural selection acting on MSP8 and MSP10 orthologs in the human parasites Plasmodium falciparum and P. vivax, as well as closely related malarial species found in non-human primates (NHPs). Overall, these antigens have low polymorphism in the human parasites in comparison with the orthologs from other Plasmodium spp. We found that the MSP10 gene polymorphism in P. falciparum only harbor non-synonymous substitutions, a pattern consistent with a gene under positive selection. Evidence of purifying selection was found on the polymorphism observed in both orthologs from P. cynomolgi, a non-human primate parasite closely related to P. vivax, but it was not conclusive in the human parasite. Yet, using phylogenetic base approaches, we found evidence for purifying selection on both MSP8 and MSP10 in the lineage leading to P. vivax. Such antigens evolving under strong functional constraints could become valuable vaccine candidates. We discuss how comparative approaches could allow detecting patterns consistent with negative selection even when there is low polymorphism in the extant populations.


Subject(s)
Antigens, Protozoan/genetics , Plasmodium/genetics , Polymorphism, Genetic/genetics , Protozoan Proteins/genetics , Amino Acid Sequence , Antigens, Protozoan/chemistry , Bayes Theorem , Binding Sites/genetics , Epidermal Growth Factor/chemistry , Evolution, Molecular , Genes, Protozoan/genetics , Humans , Malaria/parasitology , Molecular Sequence Data , Phylogeny , Plasmodium/chemistry , Protein Structure, Tertiary/genetics , Protozoan Proteins/chemistry , Selection, Genetic , Sequence Alignment
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