Publications

The hippocampus of cases with neurofibrillary tangles (NFT) pathology classified as stages I–II, III–IV, and V–VI without comorbidities, and middle-aged (MA) individuals with no NFT pathology, were examined to learn about the composition of granulovacuolar degeneration (GVD). Our results confirm the presence of CK1-?, p38-P Thr180/Tyr182, SAPK/JNK-P Thr183/Thr185, GSK-3?/?-P Tyr279/Tyr216, and GSK-3? Ser9 in the cytoplasmic granules in a subset of neurons of the CA1 and CA2 subfields of the hippocampus. Also, we identify the presence of PKA ?/?-P Thr197, SRC-P Tyr416, PAK1-P Ser199/Ser204, CAMK2A-P Tyr197, and PKCG-P Thr655 in cytoplasmic granules in cases with NFT pathology, but not in MA cases. Our results also confirm the presence of ?-catenin-P Ser45/Thr41, IRE?-P Ser274, eIF2?-P Ser51, TDP-43-P Ser403-404 (but absent TDP-43), and ubiquitin in cytoplasmic granules. Other components of the cytoplasmic granules are MAP2-P Thr1620/1623, MAP1B-P Thr1265, ADD1-P Ser726, and ADD1/ADD1-P Ser726/Ser713, in addition to several tau species including 3Rtau, 4Rtau, and tau-P Ser262. The analysis of GVD at progressive stages of NFT pathology reveals the early appearance of phosphorylated kinases and proteins in cytoplasmic granules at stages I–II, before the appearance of pre-tangles and NFTs. Most of these granules are not surrounded by LAMP1-positive membranes. Markers of impaired ubiquitin-protesome system, abnormal reticulum stress response, and altered endocytic and autophagic pathways occur in a subpopulation of neurons containing cytoplasmic granules, and they appear later. These observations suggest early phosphorylation of kinases leading to their activation, and resulting in the abnormal phosphorylation of various substrates, including tau, as a main alteration at the first stages of GVD. © 2021 The Author(s)

JTD Keywords: alzheimer's disease, alzheimers association guidelines, alzheimer’s disease, brain aging, cyclin-dependent kinase-5, granulovacuolar degeneration, kinases, national institute, neuropathologic assessment, p38 kinase, progressive supranuclear palsy, protein phosphorylation, tau, tau pathology, up-regulation, upstream activator, Alzheimer's disease, Brain aging, Glycogen-synthase kinase-3, Granulovacuolar degeneration, Kinases, Protein phosphorylation, Tau

Many lines of evidence demonstrate a correlation between liver glycogen content and food intake. We previously demonstrated that mice overexpressing protein targeting to glycogen (PTG) specifically in the liver—which have increased glycogen content in this organ—are protected from high-fat diet (HFD)-induced obesity by reduced food intake. However, the use of PTG to increase liver glycogen implies certain limitations. PTG stimulates glycogen synthesis but also inhibits the enzyme responsible for glycogen degradation. Furthermore, as PTG is a regulatory subunit of protein phosphatase 1 (PP1), which regulates many cellular functions, its overexpression could have side effects beyond the regulation of glycogen metabolism. Therefore, it is necessary to determine whether the direct activation of glycogen synthesis, without affecting its degradation or other cellular functions, has the same effects. To this end, we generated mice overexpressing a non-inactivatable form of glycogen synthase (GS) specifically in the liver (9A-MGSAlb mice). Control and 9a-MGSAlb mice were fed a standard diet (SD) or HFD for 16 weeks. Glucose tolerance and feeding behavior were analyzed. 9A-MGSAlb mice showed an increase in hepatic glycogen in fed and fasting conditions. When fed an HFD, these animals preserved their hepatic energy state, had a reduced food intake, and presented a lower body weight and fat mass than control animals, without changes in energy expenditure. Furthermore, 9A-MGSAlb animals showed improved glucose tolerance when fed an SD or HFD. Moreover, liver triacylglycerol levels that were increased after HFD feeding were lower in these mice. These results confirm that increased liver glycogen stores contribute to decreased appetite and improve glucose tolerance in mice fed an HFD. On the basis of our findings, strategies to preserve hepatic glycogen stores emerge as potential treatments for obesity and hyperglycemia.

JTD Keywords: accumulation, atp, attenuates obesity, expression, food intake, glucose, glycogen, glycogen synthase, high-fat diet, homeostasis, hyperglycemia, liver, mgat1, muscle, protein, ptg, Glycogen, Hepatic overexpression, Liver

The hallmark of Lafora disease, a fatal neurodegenerative disorder, is the accumulation of intracellular glycogen aggregates called Lafora bodies. Until recently, it was widely believed that brain Lafora bodies were present exclusively in neurons and thus that Lafora disease pathology derived from their accumulation in this cell population. However, recent evidence indicates that Lafora bodies are also present in astrocytes. To define the role of astrocytic Lafora bodies in Lafora disease pathology, we deleted glycogen synthase specifically from astrocytes in a mouse model of the disease (malin(KO)). Strikingly, blocking glycogen synthesis in astrocytes-thus impeding Lafora bodies accumulation in this cell type-prevented the increase in neurodegeneration markers, autophagy impairment, and metabolic changes characteristic of the malin(KO) model. Conversely, mice that over-accumulate glycogen in astrocytes showed an increase in these markers. These results unveil the deleterious consequences of the deregulation of glycogen metabolism in astrocytes and change the perspective that Lafora disease is caused solely by alterations in neurons.

JTD Keywords: Bodies, Deficient mice, Epilepsy, Glycogen, Impairment, Lafora disease, Malin, Modulation, Mouse model, Neurodegeneration, Neuroinflammation, Neurons, Progressive myoclonus epilepsy, Seizure susceptibility, Synthase

Tau protein is largely responsible for tauopathies, including Alzheimer’s disease (AD), where it accumulates in the brain as insoluble aggregates. Tau mRNA is regulated by alternative splicing, and inclusion or exclusion of exon 10 gives rise to the 3R and 4R isoforms respectively, whose balance is physiologically regulated. In this sense, one of the several factors that regulate alternative splicing of tau is GSK3?, whose activity is inhibited by the cellular prion protein (PrPC), which has different physiological functions in neuroprotection and neuronal differentiation. Moreover, a relationship between PrPC and tau expression levels has been reported during AD evolution. For this reason, in this study we aimed to analyze the role of PrPC and the implication of GSK3? in the regulation of tau exon 10 alternative splicing. We used AD human samples and mouse models of PrPC ablation and tau overexpression. In addition, we used primary neuronal cultures to develop functional studies. Our results revealed a paralleled association between PrPC expression and tau 4R isoforms in all models analyzed. In this sense, reduction or ablation of PrPC levels induces an increase in tau 3R/4R balance. More relevantly, our data points to GSK3? activity downstream from PrPC in this phenomenon. Our results indicate that PrPC plays a role in tau exon 10 inclusion through the inhibitory capacity of GSK3?. © 2021 by the authors. Licensee MDPI, Basel, Switzerland.

JTD Keywords: alternative splicing, alzheimer's disease, alzheimers-disease, alzheimer’s disease, amyloid-beta, cellular prion protein, frontotemporal dementia, glycogen-synthase kinase-3, gsk3 beta, gsk3?, gsk3β, messenger-rna, microtubule-associated protein tau, neurofibrillary tangles, progressive supranuclear palsy, promotes neuronal differentiation, stem-cells, tauopathies, Alternative splicing, Alzheimer’s disease, Cellular prion protein, Gsk3?, Microtubule-associated protein tau, Tauopathies

Aims Creutzfeldt-Jakob disease (CJD) is a rapid progressive neurological disease leading to dementia and death. Prion biomarkers are altered in the cerebrospinal fluid (CSF) of CJD patients, but the pathogenic mechanisms underlying these alterations are still unknown. The present study examined prion biomarker levels in the brain and CSF of sporadic CJD (sCJD) cases and their correlation with neuropathological lesion profiles. Methods The expression levels of 14-3-3, Tau, phospho-Tau and α-synuclein were measured in the CSF and brain of sCJD cases in a subtype- and region-specific manner. In addition, the activity of prion biomarker kinases, the expression levels of CJD hallmarks and the most frequent neuropathological sCJD findings were analysed. Results Prion biomarkers levels were increased in the CSF of sCJD patients; however, correlations between mRNA, total protein and their phosphorylated forms in brain were different. The observed downregulation of the main Tau kinase, GSK3, in sCJD brain samples may help to explain the differential phospho-Tau/Tau ratios between sCJD and other dementias in the CSF. Importantly, CSF biomarkers levels do not necessarily correlate with sCJD neuropathological findings. Interpretation Present findings indicate that prion biomarkers levels in sCJD tissues and their release into the CSF are differentially regulated following specific modulated responses, and suggest a functional role for these proteins in sCJD pathogenesis.

JTD Keywords: Creutzfeldt-Jakob disease, Prion Protein, Cerebrospinal fluid, Prion Biomarkers, disease subtype, Glycogen synthase kinase 3

Molecular machines and nanomotors are sophisticated biological assemblies that convert potential energy stored either in transmembrane ion gradients or in ATP into kinetic energy. Studying these highly dynamic biological devices by X-ray crystallography is challenging, as they are difficult to produce, purify, and crystallize. Phage display technology allows us to put a handle on these molecules in the form of highly specific antibody fragments that can also stabilize conformations and allow versatile labelling for electron microscopy, immunohistochemistry, and biophysics experiments. Here, we describe a widely applicable protocol for selecting high-affinity monoclonal antibody fragments against a complex molecular machine, the A-type ATPase from T. thermophilus that allows fast and simple purification of this transmembrane rotary motor from its wild-type source. The approach can be readily extended to other integral membrane proteins and protein complexes as well as to soluble molecular machines and nanomotors.

JTD Keywords: ATP synthase, Crystallization, Domain antibodies, Electron microscopy, Labelling, Membrane proteins, Monoclonal antibody fragments, Phage display, Protein purification, X-ray crystallography

An important goal of nanotechnology is the application of individual molecule handling techniques to the discovery of potential new therapeutic agents. Of particular interest is the search for new inhibitors of metabolic routes exclusive of human pathogens, such as the 2-C-methyl-D-erythritol-4-phosphate (MEP) pathway essential for the viability of most human pathogenic bacteria and of the malaria parasite. Using atomic force microscopy single-molecule force spectroscopy (SMFS), we have probed at the single-molecule level the interaction of 1-deoxy-D-xylulose 5-phosphate synthase (DXS), which catalyzes the first step of the MEP pathway, with its two substrates, pyruvate and glyceraldehyde-3-phosphate. The data obtained in this pioneering SMFS analysis of a bisubstrate enzymatic reaction illustrate the substrate sequentiality in DXS activity and allow for the calculation of catalytic parameters with single-molecule resolution. The DXS inhibitor fluoropyruvate has been detected in our SMFS competition experiments at a concentration of 10 mu M, improving by 2 orders of magnitude the sensitivity of conventional enzyme activity assays. The binding of DXS to pyruvate is a 2-step process with dissociation constants of k(off) = 6.1 x 10(-4) +/- 7.5 x 10(-3) and 1.3 x 10(-2) +/- 1.0 x 10(-2) s(-1), and reaction lengths of x(beta) = 3.98 +/- 0.33 and 0.52 +/- 0.23 angstrom. These results constitute the first quantitative report on the use of nanotechnology for the biodiscovery of new antimalarial enzyme inhibitors and open the field for the identification of compounds represented only by a few dozens of molecules in the sensor chamber.

JTD Keywords: Malaria, 2-C-methyl-D-erythritol-4-phosphate pathway, 1-deoxy-D-xylulose 5-phosphate synthase, Pyruvate, Glyceraldehyde-3-phosphate, Drug discovery