Inhibiting CSF1R alleviates cerebrovascular white matter disease and cognitive impairment.

White matter abnormalities, related to poor cerebral perfusion, are a core feature of small vessel cerebrovascular disease, and critical determinants of vascular cognitive impairment and dementia. Despite this importance there is a lack of treatment options. Proliferation of microglia producing an expanded, reactive population and associated neuroinflammatory alterations have been implicated in the onset and progression of cerebrovascular white matter disease, in patients and in animal models, suggesting that targeting microglial proliferation may exert protection. Colony-stimulating factor-1 receptor (CSF1R) is a key regulator of microglial proliferation. We found that the expression of CSF1R/Csf1r and other markers indicative of increased microglial abundance are significantly elevated in damaged white matter in human cerebrovascular disease and in a clinically relevant mouse model of chronic cerebral hypoperfusion and vascular cognitive impairment. Using the mouse model, we investigated long-term pharmacological CSF1R inhibition, via GW2580, and demonstrated that the expansion of microglial numbers in chronic hypoperfused white matter is prevented. Transcriptomic analysis of hypoperfused white matter tissue showed enrichment of microglial and inflammatory gene sets, including phagocytic genes that were the predominant expression modules modified by CSF1R inhibition. Further, CSF1R inhibition attenuated hypoperfusion-induced white matter pathology and rescued spatial learning impairments and to a lesser extent cognitive flexibility. Overall, this work suggests that inhibition of CSF1R and microglial proliferation mediates protection against chronic cerebrovascular white matter pathology and cognitive deficits. Our study nominates CSF1R as a target for the treatment of vascular cognitive disorders with broader implications for treatment of other chronic white matter diseases.

Deficiency of Nrf2 exacerbates white matter damage and microglia/macrophage levels in a mouse model of vascular cognitive impairment.

BACKGROUND: Chronic cerebral hypoperfusion causes damage to the brain's white matter underpinning vascular cognitive impairment. Inflammation and oxidative stress have been proposed as key pathophysiological mechanisms of which the transcription factor Nrf2 is a master regulator. We hypothesised that white matter pathology, microgliosis, blood-brain barrier breakdown and behavioural deficits induced by chronic hypoperfusion would be exacerbated in mice deficient in the transcription factor Nrf2. METHODS: Mice deficient in Nrf2 (male heterozygote or homozygous for Nrf2 knockout) or wild-type littermates on a C57Bl6/J background underwent bilateral carotid artery stenosis (BCAS) to induce chronic cerebral hypoperfusion or sham surgery and survived for a further 6 weeks. White matter pathology was assessed with MAG immunohistochemistry as a marker of altered axon-glial integrity; alterations to astrocytes and microglia/macrophages were assessed with GFAP and Iba1 immunohistochemistry, and blood-brain barrier breakdown was assessed with IgG immunohistochemistry. Behavioural alterations were assessed using 8-arm radial arm maze, and alterations to Nrf2-related and inflammatory-related genes were assessed with qRT-PCR. RESULTS: Chronic cerebral hypoperfusion induced white matter pathology, elevated microglial/macrophage levels and blood-brain barrier breakdown in white matter tracts that were increased in Nrf2+/- mice and further exacerbated by the complete absence of Nrf2. Chronic hypoperfusion induced white matter astrogliosis and induced an impairment in behaviour assessed with radial arm maze; however, these measures were not affected by Nrf2 deficiency. Although Nrf2-related antioxidant gene expression was not altered by chronic cerebral hypoperfusion, there was evidence for elevated pro-inflammatory related gene expression following chronic hypoperfusion that was not affected by Nrf2 deficiency. CONCLUSIONS: The results demonstrate that the absence of Nrf2 exacerbates white matter pathology and microgliosis following cerebral hypoperfusion but does not affect behavioural impairment.

Astrocyte-specific overexpression of Nrf2 protects against optic tract damage and behavioural alterations in a mouse model of cerebral hypoperfusion.

Mouse models have shown that cerebral hypoperfusion causes white matter disruption and memory impairment relevant to the study of vascular cognitive impairment and dementia. The associated mechanisms include inflammation and oxidative stress are proposed to drive disruption of myelinated axons within hypoperfused white matter. The aim of this study was to determine if increased endogenous anti-oxidant and anti-inflammatory signalling in astrocytes was protective in a model of mild cerebral hypoperfusion. Transgenically altered mice overexpressing the transcription factor Nrf2 (GFAP-Nrf2) and wild type littermates were subjected to bilateral carotid artery stenosis or sham surgery. Behavioural alterations were assessed using the radial arm maze and tissue was collected for pathology and transcriptome analysis six weeks post-surgery. GFAP-Nrf2 mice showed less pronounced behavioural impairments compared to wild types following hypoperfusion, paralleled by reduced optic tract white matter disruption and astrogliosis. There was no effect of hypoperfusion on anti-oxidant gene alterations albeit the levels were increased in GFAP-Nrf2 mice. Instead, pro-inflammatory gene expression was determined to be significantly upregulated in the optic tract of hypoperfused wild type mice but differentially affected in GFAP-Nrf2 mice. In particular, complement components (C4 and C1q) were increased in wild type hypoperfused mice but expressed at levels similar to controls in hypoperfused GFAP-Nrf2 mice. This study provides evidence that overexpression of Nrf2 in astrocytes exerts beneficial effects through repression of inflammation and supports the potential use of Nrf2-activators in the amelioration of cerebrovascular-related inflammation and white matter degeneration.

On the role of the axial ligand in heme proteins: a theoretical study.

We present a systematic investigation of how the axial ligand in heme proteins influences the geometry, electronic structure, and spin states of the active site, and the energies of the reaction cycles. Using the density functional B3LYP method and medium-sized basis sets, we have compared models with His, His+Asp, Cys, Tyr, and Tyr+Arg as found in myoglobin and hemoglobin, peroxidases, cytochrome P450, and heme catalases, respectively. We have studied 12 reactants and intermediates of the reaction cycles of these enzymes, including complexes with H(2)O, OH(-), O(2-), CH(3)OH, O(2), H(2)O(2), and HO(2)(-) in various formal oxidation states of the iron ion (II to V). The results show that His gives ~0.6 V higher reduction potentials than the other ligands. In particular, it is harder to reduce and protonate the O(2) complex with His than with the other ligands, in accordance with the O(2) carrier function of globins and the oxidative chemistry of the other proteins. For most properties, the trend Cys

Metal binding to Bacillus subtilis ferrochelatase and interaction between metal sites.

Ferrochelatase, the terminal enzyme in heme biosynthesis, catalyses metal insertion into protoporphyrin IX. The location of the metal binding site with respect to the bound porphyrin substrate and the mode of metal binding are of central importance for understanding the mechanism of porphyrin metallation. In this work we demonstrate that Zn(2+), which is commonly used as substrate in assays of the ferrochelatase reaction, and Cd(2+), an inhibitor of the enzyme, bind to the invariant amino acids His183 and Glu264 and water molecules, all located within the porphyrin binding cleft. On the other hand, Mg(2+), which has been shown to bind close to the surface at 7 A from His183, was largely absent from its site. Activity measurements demonstrate that Mg(2+) has a stimulatory effect on the enzyme, lowering K(M) for Zn(2+) from 55 to 24 micro M. Changing one of the Mg(2+) binding residues, Glu272, to serine abolishes the effect of Mg(2+). It is proposed that prior to metal insertion the metal may form a sitting-atop (SAT) complex with the invariant His-Glu couple and the porphyrin. Metal binding to the Mg(2+) site may stimulate metal release from the protein ligands and its insertion into the porphyrin.

The importance of porphyrin distortions for the ferrochelatase reaction.

Ferrochelatase is the terminal enzyme in haem biosynthesis, i.e. the enzyme that inserts a ferrous ion into the porphyrin ring. Suggested reaction mechanisms for this enzyme involve a distortion of the porphyrin ring when it is bound to the enzyme. We have examined the energetics of such distortions using various theoretical calculations. With the density functional B3LYP method we calculate how much energy it costs to tilt one of the pyrrole rings out of the porphyrin plane for an isolated porphyrin molecule without or with a divalent metal ion in the centre of the ring. A tilt of 30 degrees costs 65-130 kJ/mol for most metal ions, but only approximately 48 kJ/mol for free-base (neutral) porphine. This indicates that once the metal is inserted, the porphyrin becomes stiffer and flatter, and therefore binds with lower affinity to a site designed to bind a distorted porphyrin. This would facilitate the release of the product from ferrochelatase. This proposal is strengthened by the fact that the only tested metal ion with a lower distortion energy than free-base porphyrin (Cd(2+)) is an inhibitor of ferrochelatase. Moreover, it costs even less energy to tilt a doubly deprotonated porphine(2-) molecule. This suggests that the protein may lower the acid constant of the pyrrole nitrogen atoms by deforming the porphyrin molecule. We have also estimated the structure of the protoporphyrin IX substrate bound to ferrochelatase using combined quantum chemical and molecular mechanics calculations. The result shows that the protein may distort the porphyrin by approximately 20 kJ/mol, leading to a distinctly non-planar structure. All four pyrrole rings are tilted out of the porphyrin mean plane (1-16 degrees ) but most towards the putative binding site of the metal ion. The predicted tilt is considerably smaller than that observed in the crystal structure of a porphyrin inhibitor.

An automatic method to generate force-field parameters for hetero-compounds.

A program, Hess2FF, has been developed that automatically constructs parameter and topology files to be used in crystallographic refinement for any molecule, based on a Hessian (force-constant) matrix estimated by any method. The program is tested by redefining hetero-compounds in five different proteins: the inhibitor N-methylmesoporphyrin bound to ferrochelatase, the haem group and its axial ligands in cyctochrome c(553), the active-site metal ion in iron superoxide dismutase, the catalytic zinc ion in alcohol dehydrogenase with a bound trifluoroethanol molecule and the 5'-deoxyadenosyl group in methylmalonyl coenzyme A mutase. It is shown that the resulting structures are improved in several aspects. In particular, the free R(free) factor always decreases and it is shown that a 1.70 A structure of cyctochrome c(553) becomes more similar to a high-resolution (0.97 A) structure of the same protein after re-refinement with Hess2FF. Thus, the force field used in crystallographic refinement significantly affects the final structure and therefore should be published together with the structure to ensure reproducibility. Various methods of obtaining the Hessian matrix employed by Hess2FF are discussed and some recommendations are given. Hess2FF allows the user to divide the atoms of the molecule into atom types that share the same force-field parameters. However, it seems to be favourable to assign a separate type to each atom, which can be performed automatically.

Theoretical study of the discrimination between O(2) and CO by myoglobin.

Combined quantum chemical and molecular mechanics geometry optimisations have been performed on myoglobin without or with O(2) or CO bound to the haem group. The results show that the distal histidine residue is protonated on the N(epsilon 2) atom and forms a hydrogen bond to the haem ligand both in the O(2) and the CO complexes. We have also re-refined the crystal structure of CO[bond]myoglobin by a combined quantum chemical and crystallographic refinement. Thereby, we probably obtain the most accurate available structure of the active site of this complex, showing a Fe[bond]C[bond]O angle of 171 degrees, and Fe[bond]C and C[bond]O bond lengths of 170-171 and 116-117 pm. The resulting structures have been used to calculate the strength of the hydrogen bond between the distal histidine residue and O(2) or CO in the protein. This amounts to 31-33 kJ/mol for O(2) and 2-3 kJ/mol for CO. The difference in hydrogen-bond strength is 21-22 kJ/mol when corrected for entropy effects. This is slightly larger than the observed discrimination between O(2) or CO by myoglobin, 17 kJ/mol. We have also estimated the strain of the active site inside the protein. It is 2-4 kJ/mol larger for the O(2) complex than for the CO complex, independent of which crystal structure the calculations are based on. Together, these results clearly show that myoglobin discriminates between O(2) and CO mainly by electrostatic interactions, rather than by steric strain.

Restrained point-charge models for disaccharides.

Various methods for deriving atomic partial charges from the quantum chemical electrostatic potential and moments have been tested for the sucrose molecule. We show that if no further information is used, the charges on some carbon atoms become large and charge patterns involving these atoms are badly determined and poorly transferable. Adding lone-pairs on the ether oxygen atoms or dividing the molecule into smaller fragments did not cure the instabilities. We develop a method, CHELP-BOW0, that restrains charges toward zero with different weights for different atoms. These harmonic restraints preserve the linear form of the least-squares equations, which are solved in a single step using singular-value decomposition. CHELP-BOW0 improves the chemical transferability of the charges compared to unrestrained methods, and slightly improves their conformational transferability. It introduces a modest degradation of the fit compared to unrestrained CHELP-BOW (mean average deviation of the potential 0.00016 vs. 0.00010 a.u.). A second new method, CHELP-BOWC, avoids the need for restraints by including several conformations in the fit, weighting each according to its estimated energy in solution. CHELP-BOWC charges are more transferable than CHELP-BOW or CHELP-BOW0 charges to conformations not included in the training set. Restraints to zero charge do not further improve transferability of the CHELP-BOWC charges. We, therefore, recommend CHELP-BOW charges for rigid molecules and CHELP-BOWC charges for flexible molecules.