11 research outputs found
Induction of Neuron-Specific Degradation of Coenzyme A Models Pantothenate Kinase-Associated Neurodegeneration by Reducing Motor Coordination in Mice
<div><p>Background</p><p>Pantothenate kinase-associated neurodegeneration, PKAN, is an inherited disorder characterized by progressive impairment in motor coordination and caused by mutations in <i>PANK2</i>, a human gene that encodes one of four pantothenate kinase (PanK) isoforms. PanK initiates the synthesis of coenzyme A (CoA), an essential cofactor that plays a key role in energy metabolism and lipid synthesis. Most of the mutations in <i>PANK2</i> reduce or abolish the activity of the enzyme. This evidence has led to the hypothesis that lower CoA might be the underlying cause of the neurodegeneration in PKAN patients; however, no mouse model of the disease is currently available to investigate the connection between neuronal CoA levels and neurodegeneration. Indeed, genetic and/or dietary manipulations aimed at reducing whole-body CoA synthesis have not produced a desirable PKAN model, and this has greatly hindered the discovery of a treatment for the disease.</p><p>Objective, Methods, Results and Conclusions</p><p>Cellular CoA levels are tightly regulated by a balance between synthesis and degradation. CoA degradation is catalyzed by two peroxisomal nudix hydrolases, Nudt7 and Nudt19. In this study we sought to reduce neuronal CoA in mice through the alternative approach of increasing Nudt7-mediated CoA degradation. This was achieved by combining the use of an adeno-associated virus-based expression system with the synapsin (Syn) promoter. We show that mice with neuronal overexpression of a cytosolic version of Nudt7 (scAAV9-Syn-Nudt7cyt) exhibit a significant decrease in brain CoA levels in conjunction with a reduction in motor coordination. These results strongly support the existence of a link between CoA levels and neuronal function and show that scAAV9-Syn-Nudt7cyt mice can be used to model PKAN.</p></div
GFP expression driven by the CMV or Syn promoter.
<p>(A) Western blotting analysis of GFP in tissues from animals injected with AAV9-Syn-GFP. GAPDH was used as the loading control. Immunohistochemistry of (B) brown adipose tissue and paraspinal muscle and (C) central nervous system regions. GFP expression (brown) was driven by the CMV or the Syn promoter. AAV particles were injected as described in ‘Material and Methods’ and mice were analyzed 4 weeks thereafter. The results are representative of 2 or more animals. The magnifications are indicated.</p
Nudt7 and Nudt7cyt activity and localization.
<p>(A) Nudt7 hydrolyzes the phosphodiester bond in acyl-CoA species producing acyl-phosphopantetheine and 3’,5’-ADP. (B) Enzymatic activities of purified mouse Nudt7 (closed circles) and Nudt7cyt (open circles). Data are reported as the mean ± the range. (C-H) HEK293 cells were transfected with expression plasmids encoding mCherry fused to the N-terminus of Nudt7 (C-E) or Nudt7cyt (F-H) as described in ‘Material and Methods’. mCherry-Nudt7 and mCherry-Nudt7cyt proteins are shown in magenta (C, F) and cell nuclei are stained with Hoechst 33342 in blue (D, E, G, H). Co-transfection with a plasmid encoding green fluorescent protein (GFP) with a C-terminal SKL (GFP-SKL) (D, G) designates the peroxisomes shown in green. GFP-SKL colocalizes with Nudt7 (E, shown in white) but not with Nudt7cyt (H). The mCherry-Nudt7, mCherry-Nudt7cyt and GFP-SKL proteins were visualized by live-cell confocal fluorescent microscopy. Scale bar, 10 μm.</p
Antibody-mediated neutralization and Hemagglutination Inhibition from CHIKV-infected patient serum.
<p>nAb titers (A) and Hemagglutination Inhibition (HI) antibody responses (B) in patient sera (SRMC-1 to SRMC-30) to CHIKV. Similar results were observed in 2 independent experiments. There is a positive correlation exists between nAb and HI on CHIKV infected patients (C). These relationships were evaluated using the Spearman correlation test using the Prism 5 Graph Pad software. Neutralization of CHIKV infectivity with patient serum (D). The IC50 is defined as the reciprocal of the antiserum dilution at which CHIKV virus entry is 50% inhibited (dashed line). Similar results were observed in 2 independent experiments.</p
CHIKV DNA vaccination induces strong immunity in mice.
<p>BALB/c mice were immunized three times, each 2 weeks apart, with 25 µg pVax1 vector or pMCE321-Env and sacrificed 1 week after the 3<sup>rd</sup> immunization. (A) Splenocytes from immunized animals were harvested and cultured overnight in the presence of peptide pool matrix spanning the Envelope protein (pool-1 & pool-2) and the IFN-γ response to each pool was measured by ELISpot as described in the <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0000928#s2" target="_blank">Materials and Methods</a>. Values represent the mean and standard deviation of triplicate wells and are representative of three independent experiments. (B) Systemic anti-Env IgG levels after DNA immunization. Each group of inbred BALB/c mice (<i>n = 4</i>) was immunized with indicated vaccines. Mice were bled 1 week after each immunization, and then sera were diluted to 1/100 for reaction with CHIKV-Env. OD was measured at 450 nm. Values and bars represent mean (<i>n = 4</i>) and the SEM. (C and D) Quantification of CHIKV specific neutralizing and HI titer in sera from DNA immunized mice (pVax1/pCHIKV-E1/pCHIKV-E2 and pMCE321) to CHIKV. The nAb titers are plotted as the highest dilution of serum that resulted in at least 50% inhibition of CPE. The highest dilution of the serum that inhibited hemagglutination was recorded as the HI titer. Similar results were observed in three independent experiments with at least <i>n</i> = 4 per group for each experiment.</p
CHIKV DNA vaccination and infection.
<p>(A&B) Analysis of proinflammatory cytokines (TNF-α and IL-6) in CHIKV vaccinated and infected mice. Cytokine levels (pg/ml) were assayed by ELISA from the cell free sera from 10 days post infection. These data represent the average 3 wells/mouse and standard deviations of 4 mice. (C) Percent survival in CHIKV-challenged mice. Similar results were observed in 2 independent experiments with at least <i>n</i> = 10 per group for each experiment. (D) The viremia, 5 day after challenge, as measured by a plaque assay. Mice immunized with pVax1 (control) or immunized pMCE321 (vaccine) were challenged with the PC-08 CHIKV strain at a dose of 7 log10 PFU by the intranasal route. Data are mean ± SEM of 5 animals.</p
Construction and characterization of CHIKV DNA vaccine.
<p>(A) Schematic representation of pMCE321 construct. The flanking enzyme sites used for cloning, Kozak expression element, CMV promoter, human IgE-leader, CHIKV fusion gene (E3-E2-E1), and cleavage sites (CS) are indicated and were cloned into the pVax1 vector. (B) Expression of pMCE321 constructs was confirmed <i>in vitro</i> using Envelope-E1 antiserum for the Western blot of CHIKV envelope antigens expressed in Vero and BHK-21 cells by Western blotting. Arrows indicate the positions of E1 protein expression. (C) Immunofluorescent assay showing staining of Vero cells transfected with pCHIKV-E1, pCHIKV-E2, or pMCE321 constructs and transient expression of the envelope proteins. (D). FACS analysis of envelope expression in transfected cells (0.5×10<sup>6</sup> cells). Vero cells were transfected with indicated constructs and stained with anti-Env sera raised in mice, followed by staining with secondary PE-conjugated anti-mouse IgG antibody as indicated. Two representative FACS histograms are shown.</p
Isolation and identification of CHIKV.
<p>The microphotographs show normal uninfected Vero cells (A) and the Vero cells infected with CHIKV virus isolate. CHIKV infection in Vero cells causes characteristic foamy cytopathic effect (CPE) 48 hours p.i. as seen with the isolate. (B) RT-PCR analysis of CHIKV viral isolates. Agarose gel photograph showing the RT-PCR amplified product (305 bp) of the CHIKV positive patient isolates (Lane 1&2). The uninfected negative control (Lane 3) shows no amplification. (C) Electron micrographs of CHIKV viral isolates (D) Phylogenetic Tree generated with E2 amplicon from CHIKV Isolate. * Indicates the PC-08 CHIKV strain.</p
Histopathology analysis of CHIKV challenged mice.
<p>(A) H&E stained sections of Brain. pMCE321 vaccinated mice showed no severe pathological changes and showed only minimal microglial formation. Capsid immunized mice group showed severe hemorrhage and microglia formation similar to the naïve group. (B) H&E stained sections of Heart, Liver, Kidney and Lungs. Envelope vaccinated group showed minimal or no pathological changes in the organs. The naïve group showed severe pathological changes indicative of virus infection and the Capsid DNA immunized group showed similar pathological changes to the naïve group. Representative data are shown from 2 mice/group.</p
Clinical observation in Chikungunya patients.
<p>Clinical observation in Chikungunya patients.</p