L-Norvaline, a new therapeutic agent against Alzheimer’s disease

Growing evidence highlights the role of arginase activity in the manifestation of Alzheimer’s disease (AD). Upregulation of arginase was shown to contribute to neurodegeneration. Regulation of arginase activity appears to be a promising approach for interfering with the pathogenesis of AD. Therefore, the enzyme represents a novel therapeutic target. In this study, we administered an arginase inhibitor, L-norvaline (250 mg/L), for 2.5 months to a triple-transgenic model (3×Tg-AD) harboring PS1M146V, APPSwe, and tauP301L transgenes. Then, the neuroprotective effects of L-norvaline were evaluated using immunohistochemistry, proteomics, and quantitative polymerase chain reaction assays. Finally, we identified the biological pathways activated by the treatment. Remarkably, L-norvaline treatment reverses the cognitive decline in AD mice. The treatment is neuroprotective as indicated by reduced beta-amyloidosis, alleviated microgliosis, and reduced tumor necrosis factor transcription levels. Moreover, elevated levels of neuroplasticity related postsynaptic density protein 95 were detected in the hippocampi of mice treated with L-norvaline. Furthermore, we disclosed several biological pathways, which were involved in cell survival and neuroplasticity and were activated by the treatment. Through these modes of action, L-norvaline has the potential to improve the symptoms of AD and even interferes with its pathogenesis. As such, L-norvaline is a promising neuroprotective molecule that might be tailored for the treatment of a range of neurodegenerative disorders. The study was approved by the Bar-Ilan University Animal Care and Use Committee (approval No. 82-10-2017) on October 1, 2017

Arginase Inhibition Supports Survival and Differentiation of Neuronal Precursors in Adult Alzheimer’s Disease Mice

Adult neurogenesis is a complex physiological process, which plays a central role in maintaining cognitive functions, and consists of progenitor cell proliferation, newborn cell migration, and cell maturation. Adult neurogenesis is susceptible to alterations under various physiological and pathological conditions. A substantial decay of neurogenesis has been documented in Alzheimer’s disease (AD) patients and animal AD models; however, several treatment strategies can halt any further decline and even induce neurogenesis. Our previous results indicated a potential effect of arginase inhibition, with norvaline, on various aspects of neurogenesis in triple-transgenic mice. To better evaluate this effect, we chronically administered an arginase inhibitor, norvaline, to triple-transgenic and wild-type mice, and applied an advanced immunohistochemistry approach with several biomarkers and bright-field microscopy. Remarkably, we evidenced a significant reduction in the density of neuronal progenitors, which demonstrate a different phenotype in the hippocampi of triple-transgenic mice as compared to wild-type animals. However, norvaline showed no significant effect upon the progenitor cell number and constitution. We demonstrated that norvaline treatment leads to an escalation of the polysialylated neuronal cell adhesion molecule immunopositivity, which suggests an improvement in the newborn neuron survival rate. Additionally, we identified a significant increase in the hippocampal microtubule-associated protein 2 stain intensity. We also explore the molecular mechanisms underlying the effects of norvaline on adult mice neurogenesis and provide insights into their machinery

FAK-Mediated Signaling Controls Amyloid Beta Overload, Learning and Memory Deficits in a Mouse Model of Alzheimer’s Disease

The non-receptor focal adhesion kinase (FAK) is highly expressed in the central nervous system during development, where it regulates neurite outgrowth and axon guidance, but its role in the adult healthy and diseased brain, specifically in Alzheimer’s disease (AD), is largely unknown. Using the 3xTg-AD mouse model, which carries three mutations associated with familial Alzheimer’s disease (APP KM670/671NL Swedish, PSEN1 M146V, MAPT P301L) and develops age-related progressive neuropathology including amyloid plaques and Tau tangles, we describe here, for the first time, the in vivo role of FAK in AD pathology. Our data demonstrate that while site-specific knockdown in the hippocampi of 3xTg-AD mice has no effect on learning and memory, hippocampal overexpression of the protein leads to a significant decrease in learning and memory capabilities, which is accompanied by a significant increase in amyloid β (Aβ) load. Furthermore, neuronal morphology is altered following hippocampal overexpression of FAK in these mice. High-throughput proteomics analysis of total and phosphorylated proteins in the hippocampi of FAK overexpressing mice indicates that FAK controls AD-like phenotypes by inhibiting cytoskeletal remodeling in neurons which results in morphological changes, by increasing Tau hyperphosphorylation, and by blocking astrocyte differentiation. FAK activates cell cycle re-entry and consequent cell death while downregulating insulin signaling, thereby increasing insulin resistance and leading to oxidative stress. Our data provide an overview of the signaling networks by which FAK regulates AD pathology and identify FAK as a novel therapeutic target for treating AD

Insights into the Functional Roles of N-Terminal and C-Terminal Domains of Helicobacter pylori DprA

DNA processing protein A (DprA) plays a crucial role in the process of natural transformation. This is accomplished through binding and subsequent protection of incoming foreign DNA during the process of internalization. DprA along with Single stranded DNA binding protein A (SsbA) acts as an accessory factor for RecA mediated DNA strand exchange. H. pylori DprA (HpDprA) is divided into an N-terminal domain and a C-terminal domain. In the present study, individual domains of HpDprA have been characterized for their ability to bind single stranded (ssDNA) and double stranded DNA (dsDNA). Oligomeric studies revealed that HpDprA possesses two sites for dimerization which enables HpDprA to form large and tightly packed complexes with ss and dsDNA. While the N-terminal domain was found to be sufficient for binding with ss or ds DNA, C-terminal domain has an important role in the assembly of poly-nucleoprotein complex. Using site directed mutagenesis approach, we show that a pocket comprising positively charged amino acids in the N-terminal domain has an important role in the binding of ss and dsDNA. Together, a functional cross talk between the two domains of HpDprA facilitating the binding and formation of higher order complex with DNA is discussed

Determination of oligomeric status of HpDprA and its domains.

<p>Elution chromatograms of purified HpDprA at 2 mg/ml and 1 mg/ml <b>(A)</b> concentrations are depicted. Similarly, the size exclusion profiles of HpRF at 2 mg/ml at 1 mg/ml <b>(B)</b> and HpDML1 at 8 mg/ml at 2 mg/ml <b>(C)</b> are depicted. <b>(D)</b> Table shows elution volume (V_e) and corresponding molecular weight of the elution peaks shown in (<b>A, B</b> and <b>C</b>). Elution was monitored using UV absorbance at 230 nm wavelength. The column was equilibrated in the same buffer as the protein samples (1X PBS). Total 500 µl sample was injected for each chromatophic experiment. The apparent molecular mass was calculated from elution volume using molecular weight standards as described in materials and methods.</p

Secondary structure composition of full length HpDprA and its individual domains.

<p>The secondary structure of individual proteins have been calculated using K2D2, a web server to estimate the α helix and β strand content of a protein from its circular dichroism spectrum and compared with the % α –helix and % β sheet calculated from HpDprA structure published by Wang et. al., (2014). RMSD values depicting goodness of fit has been shown in bracket<b>.</b></p><p>Secondary structure composition of full length HpDprA and its individual domains.</p

Nuclease protection assay.

<p><b>(A)</b> Schematic illustration of nuclease protection assay. ³²P-labeled dsDNA (0.5 nM) either alone (lane 2) or pre-bound with increasing concentrations of HpDprA <b>(B)</b> or HpRF<b>(C)</b> {100, 200, 400, 600, 800, 1000 (nM), lanes 3–8} was incubated for 30 min with 1 unit of DNaseI. Similarly, ³²P-labeled ssDNA (0.5 nM) either alone (lane 2) or pre-bound with above mentioned concentrations of HpDprA <b>(D)</b> or HpRF<b>(E)</b>was incubated for 30 min with 1 unit of mung bean endonuclease. Lane 1: DNAalone.</p

Surface plasmon resonance analysis of HpDprA mutants interaction with DNA.

<p>Different concentrations of <b>(A)</b>HpDprA^R48A/R49Aand <b>(C)</b>HpDprA^{R48A/R49A/K133A} were injected on the single stranded DNA surface in standard buffer containing 150 mM NaCl. Both sample injection and buffer injection were carried out as described in materials and methods. Representative sensorgrams illustrating changes in the response units (the y—axis) as a function of time (the x-axis) are shown. Similarly, binding sensorgrams were obtained for the interaction of dsDNA with <b>(B)</b>HpDprA^R48A/R49Aand <b>(D)</b>HpDprA^{R48A/R49A/K133A}. The concentrations of soluble analytes and affinity constant (K_d) values are indicated in the inset to the figures. The K_d values are determined as described in materials and methods.</p

Surface plasmon resonance analysis of HpDprA, HpRF and HpDML1 interaction with DNA.

<p>Different concentrations of full length HpDprA <b>(A)</b>HpRF<b>(C)</b> and HpDML1<b>(E)</b> were injected on to the immobilized single stranded DNA (90 mer, ~1000 RU) surface in buffer containing 150 mM NaCl. Both sample injection and buffer injection were carried out as described in materials and methods. Similarly, binding sensorgrams were obtained for the interaction of dsDNA (90 bp, ~1000 RU) with full length HpDprA <b>(B)</b>HpRF<b>(D)</b> and HpDML1<b>(F)</b>. Sensorgrams depicting changes in response unit (the y—axis) as a function of time (the x—axis) are shown. The concentrations of soluble analytes and affinity constant (K_d) values are indicated in the inset to the figures.</p

Sequences of the oligonucleotides used in this study.

<p>Sequences of the oligonucleotides used in this study.</p