A Mouse Model of Heritable Cerebrovascular Disease

The study of animal models of heritable cerebrovascular diseases can improve our understanding of disease mechanisms, identify candidate genes for related human disorders, and provide experimental models for preclinical trials. Here we describe a spontaneous mouse mutation that results in reproducible, adult-onset, progressive, focal ischemia in the brain. The pathology is not the result of hemorrhage, embolism, or an anatomical abnormality in the cerebral vasculature. The mutation maps as a single site recessive locus to mouse Chromosome 9 at 105 Mb, a region of shared synteny with human chromosome 3q22. The genetic interval, defined by recombination mapping, contains seven protein-coding genes and one processed transcript, none of which are changed in their expression level, splicing, or sequence in affected mice. Targeted resequencing of the entire interval did not reveal any provocative changes; thus, the causative molecular lesion has not been identified

Increased RhoA prenylation in the loechrig (loe) mutant leads to progressive neurodegeneration.

The Drosophila mutant loechrig (loe) shows age-dependent degeneration of the nervous system and is caused by the loss of a neuronal isoform of the AMP-activated protein kinase (AMPK) γ-subunit (also known as SNF4Aγ). The trimeric AMPK complex is activated by low energy levels and metabolic insults and regulates multiple important signal pathways that control cell metabolism. A well-known downstream target of AMPK is hydroxyl-methylglutaryl-CoA reductase (HMGR), a key enzyme in isoprenoid synthesis, and we have previously shown that HMGR genetically interacts with loe and affects the severity of the degenerative phenotype. Prenylation of proteins like small G-proteins is an important posttranslational modification providing lipid moieties that allow the association of these proteins with membranes, thereby facilitating their subsequent activation. Rho proteins have been extensively studied in neuronal outgrowth, however, much less is known about their function in neuronal maintenance. Here we show that the loe mutation interferes with isoprenoid synthesis, leading to increased prenylation of the small GTPase Rho1, the fly orthologue of vertebrate RhoA. We also demonstrate that increased prenylation and Rho1 activity causes neurodegeneration and aggravates the behavioral and degenerative phenotypes of loe. Because we cannot detect defects in the development of the central nervous system in loe, this suggests that loe only interferes with the function of the RhoA pathway in maintaining neuronal integrity during adulthood. In addition, our results show that alterations in isoprenoids can result in progressive neurodegeneration, supporting findings in vertebrates that prenylation may play a role in neurodegenerative diseases like Alzheimer's Disease

Organophosphate-Induced Changes in the PKA Regulatory Function of Swiss Cheese/NTE Lead to Behavioral Deficits and Neurodegeneration

<div><p>Organophosphate-induced delayed neuropathy (OPIDN) is a Wallerian-type axonopathy that occurs weeks after exposure to certain organophosphates (OPs). OPs have been shown to bind to Neuropathy Target Esterase (NTE), thereby inhibiting its enzymatic activity. However, only OPs that also induce the so-called aging reaction cause OPIDN. This reaction results in the release and possible transfer of a side group from the bound OP to NTE and it has been suggested that this induces an unknown toxic function of NTE. To further investigate the mechanisms of aging OPs, we used <i>Drosophila</i>, which expresses a functionally conserved orthologue of NTE named Swiss Cheese (SWS). Treating flies with the organophosporous compound tri-ortho-cresyl phosphate (TOCP) resulted in behavioral deficits and neurodegeneration two weeks after exposure, symptoms similar to the delayed effects observed in other models. In addition, we found that primary neurons showed signs of axonal degeneration within an hour after treatment. Surprisingly, increasing the levels of SWS, and thereby its enzymatic activity after exposure, did not ameliorate these phenotypes. In contrast, reducing SWS levels protected from TOCP-induced degeneration and behavioral deficits but did not affect the axonopathy observed in cell culture. Besides its enzymatic activity as a phospholipase, SWS also acts as regulatory PKA subunit, binding and inhibiting the C3 catalytic subunit. Measuring PKA activity in TOCP treated flies revealed a significant decrease that was also confirmed in treated rat hippocampal neurons. Flies expressing additional PKA-C3 were protected from the behavioral and degenerative phenotypes caused by TOCP exposure whereas primary neurons were not. In addition, knocking-down PKA-C3 caused similar behavioral and degenerative phenotypes as TOCP treatment. We therefore propose a model in which OP-modified SWS cannot release PKA-C3 and that the resulting loss of PKA-C3 activity plays a crucial role in developing the delayed symptoms of OPIDN but not in the acute toxicity.</p></div

Effects of SWS levels on TOCP-induced behavioral deficits.

<p><b>A</b>. Wild type flies treated with 8 mg/ml or 16 mg/ml TOCP show a significantly reduced performance in the fast phototaxis assay. <b>B</b>. A similar deficit is detectable in 14 d old control flies (expressing lacZ pan-neuronally; <i>elav</i>>lacZ) treated with 8 mg/ml TOCP. Expressing additional SWS pan-neuronally (<i>elav</i>>SWS) does not protect 14 d old flies from behavioral deficits caused by TOCP however heterozygosity for <i>sws¹</i> (<i>sws</i>/WT) protects flies from the TOCP induced behavioral deficits (treated WT to <i>sws</i>/WT, *p<0.05). <b>C</b>. Comparing untreated flies in the phototaxis assay shows that overexpression of SWS alone results in less successful transitions already in 7 d old flies. This effect is even more severe when untreated 14 d old flies are tested. The analysis in <b>A</b> was done using one-way ANOVA with a Dunett's post test and the analyses in <b>B, C</b> with a Student's t-test, comparing treated and untreated flies of each genotype (in <b>B</b>) and control and SWS overexpressing flies of a given age (in C). n = is number of groups tested with 10–20 flies each. All flies were females. SEMs are indicated in all graphs. *p<0.05, ***p<0.001. (The was no significant difference in the variance in any of the comparisons).</p

TOCP induces neurite shortening in primary neuronal cultures.

<p><b>A</b>. Dose response curve showing that TOCP doses equal or higher than 7 µg/ml cause a significant reduction in neurite length. 30–56 neurons were measured for each condition. <b>B</b>. Live imaging of a neuron treated with 14 µg/ml TOCP reveals the formation of varicosities (arrows) and neurite degeneration (arrowheads) 50 min after the addition of TOCP. Another 50 mins later, these phenotypes are even more pronounced. <b>C</b>. The length of neurites is dramatically reduced in TOCP treated cells versus mock treated cells, but also significantly shorter in TOCP treated cells compared to cells that have been fixed at the time of treatment (left graph). The graph on the right shows the change in length between each condition. Analysis was done using one-way ANOVA with a Dunett's post test to compare to mock treated cells. n = number of cells measured and the SEMs are shown. **p<0.01, ***p<0.001. Scale bar in <b>B</b> = 2 µm. (The variances were significantly different between treated and untreated cells; p<0.001).</p

PKA-C3 and SWSR^133A expression prevent TOCP-induced reduction in PKA activity.

<p><b>A</b>. PKA-C3 overexpression increases PKA activity and this is not affected by TOCP treatment. <b>B</b>. Additional expression of SWS significantly reduces PKA activity whereas heterozygosity for <i>sws¹</i> does not. The values are shown in percent of the PKA activity in untreated wild type. n = number of independent measurement and the SEMs are indicated. Student's t-tests were used to compare activity in untreated and treated flies. One-way ANOVA with a Dunett's post test was used to compare the untreated flies in B. *p<0.05, ***p<0.001. (.The variances were not significantly different).</p

TOCP treatment inhibits SWS-esterase activity, but not AChE activity.

<p><b>A</b>. Esterase activity against phenyl valerate is significantly inhibited by TOCP treatment (16 mg/ml) in wild type whereas the residual activity in <i>sws¹</i> is not further reduced by TOCP. Flies overexpressing SWS using <i>elav</i>-GAL4 show approximately a 1.6 fold increase in activity in vehicle treated flies compared to wild type. Although this activity is significantly reduced by TOCP, the SWS overexpressing flies still show 50% of the activity of untreated wild type. Two independent measurements were done for treated flies and four for untreated flies. <b>B</b>. Neither TOCP treatment nor SWS levels have a significant effect on AChE activity. Two independent measurements were performed for each genotype and treatment. <b>A, B</b>. Flies were tested at the end of the 16 h treatment period. All values are shown relative to untreated wild type. A Student's t-test was used to compare each treated group to its corresponding untreated one. SEMs are indicated; **p<0.01. (The variances were not significantly different between treated and untreated flies for each genotype).</p

TOCP treatment induces lethality.

<p><b>A</b>. Wild type fly fed on glucose. <b>B</b>. A wild type fly fed with glucose containing TOCP and blue food coloring shows food uptake by the blue coloring of the abdomen and proboscis (arrows). <b>C</b>. Survival of flies treated with different concentrations of TOCP. n =  number of independent tests with 10–15 flies. Analysis was done using one-way ANOVA with a Dunett's post test to compare the treated flies to mock treated flies. The SEMs are indicated. *p<0.05, **p<0.0. (the variance was not significantly different with the exception of day 14 with p = 0.02)</p

PKA-C3 overexpression protects against TOCP-induced degeneration and behavioral deficits.

<p><b>A</b>. Flies expressing additional PKA-C3 in neurons via <i>elav</i>-GAL4 do not show the TOCP-induced reduction in performance seen in <i>elav</i>>lacZ control flies. Also flies expressing the PKA-C3 binding deficient SWS^R133A construct are protected against TOCP-induced behavioral deficits. In addition, these flies do not show the reduction in performance observed in untreated flies overexpressing the wild type SWS construct (<i>elav</i>>SWS). <b>B</b>. Although PKA-C3 overexpressing flies show a significant increase in vacuole formation when untreated, TOCP treatment does not enhance this phenotype, but significantly reduces vacuole formation. <b>C</b>. PKA-C3 overexpression has no effect on the neurite shortening observed after TOCP treatment of primary neurons. n = is number of groups tested with 10–20 female flies each in <b>A</b>, n = number of cells or head sections analyzed in <b>B</b> and <b>C</b>. Student's t-tests were used to compare treated and untreated flies and to compare untreated SWS and SWSR^133A overexpressing flies. A student's t-test was also used to compare vacuole size in untreated PKA-C3 overexpresing flies with controls in <b>B</b>. All flies used in the fast phototaxis assays were 14 d old females. SEMs are indicated in all graphs. *p<0.05, **p<0.01, ***p<0.001. (The variances were not significantly different in the tests done to compare vacuole size and behavioral deficits, but were different between treated and untreated cells: p<0.001).</p