Ventricular Volume Dynamics During the Development of Adult Chronic Communicating Hydrocephalus in a Rodent Model.

INTRODUCTION: The pathophysiology of normal-pressure hydrocephalus and the correlation with its symptomatology is not well understood. OBJECTIVE: To monitor and evaluate the enlargement patterns of the ventricular system for each ventricle and its correlation with the presenting symptoms. METHODS: Bilateral kaolin injection into the subarachnoid space overlying the cranial convexities was done in 18 adult rats. Magnetic resonance imaging was performed on an 11.7-T scanner 15, 60, 90, and 120 days after injection. Volumes of the ventricular system were measured for each ventricle and correlated with biweekly behavioral findings. RESULTS: There was a progressive increase in the ventricular volume for the lateral ventricles since day 15 in the kaolin-injected animals. There was a nonsignificant trend in volume growth for the third ventricle, but its enlargement was synchronous with the lateral ventricles. No significant change for the fourth ventricle. No symptoms were detected in the first 60 days. Association was found between the ventricular volume and locomotor changes. In addition, the odds of locomotor symptoms increased by 3% for every additional cubic millimeter of volume in the left (P < 0.001) and right (P = 0.023) ventricles, and for the total magnetic resonance imaging volume by 1% (P = 0.013). CONCLUSIONS: Expansion of the lateral ventricles maintained similar proportions over time, accompanied by a synchronous third ventricular expansion with less proportion and a nonsignificant fourth enlargement. Lateral ventricles enlarged most in those animals that were to develop late locomotor deterioration. Further research using this animal model combined with different radiologic imaging techniques, such as diffusion tensor imaging and perfusion studies, is recommended.

Improved rate of substrate oxidation catalyzed by genetically-engineered myoglobin.

This study showcases the potential of unnatural amino acids to enable non-natural functions when incorporated in the protein scaffold of heme metalloproteins. For this purpose, a genetically-engineered myoglobin (Mb) mutant was created by incorporating redox-active 3-amino-l-tyrosine (NH2Tyr) into its active site, replacing the distal histidine (H64) with NH2Tyr. In peroxide-shunt assays, this variant exhibits an increased rate of turnover for thioanisole and benzaldehyde oxidation as compared to the wild-type (WT) Mb. Indeed, in the presence of excess hydrogen peroxide (H2O2), a 9-fold and 81-fold increase in activity was observed over multiple turnovers for thioanisole sulfoxidation and benzoic acid formation, respectively. The increased oxidation activity in the H64NH2Tyr Mb mutant underlined the role of NH2Tyr in the distal active-site scaffold in peroxide activation. Kinetic, electrochemical, and EPR spectroscopic experiments were performed. On the basis of these studies, it is argued that the single NH2Tyr residue within the Mb variant simultaneously serves the role of the conserved His/Arg-pair within the distal pocket of horseradish peroxidase.

A Novel Experimental Animal Model of Adult Chronic Hydrocephalus.

BACKGROUND: The pathogenesis of adult chronic hydrocephalus is not fully understood, and the temporal relationship between development of the radiological changes and neurological deterioration is unknown. OBJECTIVE: To clarify the progression of radiological-histological changes and subsequent clinical manifestations of adult chronic hydrocephalus. METHODS: Kaolin was injected bilaterally into the subarachnoid space overlying the cranial convexities in 20 adult rats. Magnetic resonance imaging (MRI) was obtained by using an 11.7 T scanner at 14, 60, 90, and 120 days after kaolin injection. Locomotor, gait, and cognitive evaluations were performed independently. Kaolin distribution and the associated inflammatory and fibrotic responses were histologically analyzed. RESULTS: Evans index of ventriculomegaly showed significant progressive growth in ventricular size over all time points examined. The greatest enlargement occurred within the first 2 months. Evans index also correlated with the extent of kaolin distribution by MRI and by pathological examination at all time points. First gait changes occurred at 69 days, anxiety at 80, cognitive impairment at 81, and locomotor difficulties after 120 days. Only locomotor deterioration was associated with Evans index or the radiological evaluation of kaolin extension. Inflammatory/fibrotic response was histologically confirmed over the cranial convexities in all rats, and its extension was associated with ventricular size and with the rate of ventricular enlargement. CONCLUSION: Kaolin injected into the subarachnoid space over the cerebral hemispheres of adult rats produces an inflammatory/fibrotic response leading in a slow-onset communicating hydrocephalus that is initially asymptomatic. Increased ventricular size eventually leads to gait, memory, and locomotor impairment closely resembling the course of human adult chronic hydrocephalus. ABBREVIATION: NPH, normal pressure hydrocephalus.

Anti-NMDA receptor autoantibodies and associated neurobehavioral pathology in mice are dependent on age of first exposure to Toxoplasma gondii.

BACKGROUND: Toxoplasma gondii is a pathogen implicated in psychiatric disorders. As elevated antibodies to T. gondii are also present in non-symptomatic individuals, we hypothesized that the age during first exposure to the pathogen may affect symptom manifestation. We tested this hypothesis by evaluating neurobehavioral abnormalities and the immune response in mice following adolescent or adult T. gondii infection. METHODS: Mice were infected with T. gondii at postnatal day 33 (adolescent/juvenile) or 61 (adult). At 8weeks post-infection (wpi), pre-pulse inhibition of the acoustic startle (PPI) in mice administered MK-801 (0.1 and 0.3mg/kg) and amphetamine (5 and 10mg/kg) was assessed. Peripheral (anti-T. gondii, C1q-associated IgG and anti-GLUN2 antibodies) and central (C1q and Iba1) markers of the immune response were also evaluated. In addition, regional brain expression of N-methyl-d-aspartate receptor (NMDAR) subunits (GLUN1 and GLUN2A), glutamatergic (vGLUT1, PSD95) and GABAergic (GAD67) markers, and monoamines (DA, NE, 5-HT) and their metabolites were measured. RESULTS: Juvenile and adult infected mice exhibited opposite effects of MK-801 on PPI, with decreased PPI in juveniles and increased PPI in adults. There was a significantly greater elevation of GLUN2 autoantibodies in juvenile-compared to adult-infected mice. In addition, age-dependent differences were found in regional expression of NMDAR subunits and markers of glutamatergic, GABAergic, and monoaminergic systems. Activated microglia and C1q elevations were found in both juvenile- and adult-T. gondii infected mice. CONCLUSIONS: Our study demonstrates that the age at first exposure to T. gondii is an important factor in shaping distinct behavioral and neurobiological abnormalities. Elevation in GLUN2 autoantibodies or complement protein C1q may be a potential underlying mechanism. A better understanding of these age-related differences may lead to more efficient treatments of behavioral disorders associated with T. gondii infection.

Inositol Hexakisphosphate Kinase-3 Regulates the Morphology and Synapse Formation of Cerebellar Purkinje Cells via Spectrin/Adducin.

The inositol hexakisphosphate kinases (IP6Ks) are the principal enzymes that generate inositol pyrophosphates. There are three IP6Ks (IP6K1, 2, and 3). Functions of IP6K1 and IP6K2 have been substantially delineated, but little is known of IP6K3's role in normal physiology, especially in the brain. To elucidate functions of IP6K3, we generated mice with targeted deletion of IP6K3. We demonstrate that IP6K3 is highly concentrated in the brain in cerebellar Purkinje cells. IP6K3 physiologically binds to the cytoskeletal proteins adducin and spectrin, whose mutual interactions are perturbed in IP6K3-null mutants. Consequently, IP6K3 knock-out cerebella manifest abnormalities in Purkinje cell structure and synapse number, and the mutant mice display deficits in motor learning and coordination. Thus, IP6K3 is a major determinant of cytoskeletal disposition and function of cerebellar Purkinje cells. SIGNIFICANCE STATEMENT: We identified and cloned a family of three inositol hexakisphosphate kinases (IP6Ks) that generate the inositol pyrophosphates, most notably 5-diphosphoinositol pentakisphosphate (IP7). Of these, IP6K3 has been least characterized. In the present study we generated IP6K3 knock-out mice and show that IP6K3 is highly expressed in cerebellar Purkinje cells. IP6K3-deleted mice display defects of motor learning and coordination. IP6K3-null mice manifest aberrations of Purkinje cells with a diminished number of synapses. IP6K3 interacts with the cytoskeletal proteins spectrin and adducin whose altered disposition in IP6K3 knock-out mice may mediate phenotypic features of the mutant mice. These findings afford molecular/cytoskeletal mechanisms by which the inositol polyphosphate system impacts brain function.

Single turnover of substrate-bound ferric cysteine dioxygenase with superoxide anion: enzymatic reactivation, product formation, and a transient intermediate.

Cysteine dioxygenase (CDO) is a non-heme mononuclear iron enzyme that catalyzes the O(2)-dependent oxidation of L-cysteine (Cys) to produce cysteine sulfinic acid (CSA). In this study we demonstrate that the catalytic cycle of CDO can be "primed" by one electron through chemical oxidation to produce CDO with ferric iron in the active site (Fe(III)-CDO, termed 2). While catalytically inactive, the substrate-bound form of Fe(III)-CDO (2a) is more amenable to interrogation by UV-vis and EPR spectroscopy than the 'as-isolated' Fe(II)-CDO enzyme (1). Chemical-rescue experiments were performed in which superoxide (O(2)(•-)) anions were introduced to 2a to explore the possibility that a Fe(III)-superoxide species represents the first intermediate within the catalytic pathway of CDO. In principle, O(2)(•-) can serve as a suitable acceptor for the remaining 3-electrons necessary for CSA formation and regeneration of the active Fe(II)-CDO enzyme (1). Indeed, addition of O(2)(•-) to 2a resulted in the rapid formation of a transient species (termed 3a) observable at 565 nm by UV-vis spectroscopy. The subsequent decay of 3a is kinetically matched to CSA formation. Moreover, a signal attributed to 3a was also identified using parallel mode X-band EPR spectroscopy (g ~ 11). Spectroscopic simulations, observed temperature dependence, and the microwave power saturation behavior of 3a are consistent with a ground state S = 3 from a ferromagnetically coupled (J ~ -8 cm(-1)) high-spin ferric iron (S(A) = 5/2) with a bound radical (S(B) = 1/2), presumably O(2)(•-). Following treatment with O(2)(•-), the specific activity of recovered CDO increased to ~60% relative to untreated enzyme.

Synthesis and characterization of sterically encumbered ß-ketoiminate complexes of iron(II) and zinc(II).

The synthesis, structure, and spectroscopic signatures of a series of four-coordinate iron(II) complexes of ß-ketoiminates and their zinc(II) analogues are presented. An unusual five-coordinate iron(II) triflate with three oxygen bound protonated ß-ketoimines is also synthesized and structurally characterized. Single-crystal X-ray crystallographic analysis reveals that the deprotonated bis(chelate)metal complexes are four-coordinate with various degrees of distortion depending on the degree of steric bulk and the electronics of the metal center. Each of the high-spin iron(II) centers exhibits multiple electronic transitions including ligand π to π*, metal-to-ligand charge transfer, and spin-forbidden d-d bands. The (1)H NMR spectra of the paramagnetic high-spin iron(II) centers are assigned on the basis of chemical shifts, longitudinal relaxation times (T(1)), relative integrations, and substitution of the ligands. The electrochemical studies support variations in the ligand strength. Parallel mode EPR measurements for the isopropyl substituted ligand complex of iron(II) show low-field resonances (g > 9.5) indicative of complex aggregation or crystallite formation. No suitable solvent system or glassing mixture was found to remedy this phenomenon. However, the bulkier diisopropylphenyl substituted ligand exhibits an integer spin signal consistent with an isolated iron(ii) center [S = 2; D = -7.1 ± 0.8 cm(-1); E/D = 0.1]. A tentative molecular orbital diagram is assembled.

Recombinant expression, purification, and characterization of ThmD, the oxidoreductase component of tetrahydrofuran monooxygenase.

Tetrahydrofuran monooxygenase (Thm) catalyzes the NADH-and oxygen-dependent hydroxylation of tetrahydrofuran to 2-hydroxytetrahydrofuran. Thm is composed of a hydroxylase enzyme, a regulatory subunit, and an oxidoreductase named ThmD. ThmD was expressed in Escherichia coli as a fusion to maltose-binding protein (MBP) and isolated to homogeneity after removal of the MBP. Purified ThmD contains covalently bound FAD, [2Fe-2S] center, and was shown to use ferricyanide, cytochrome c, 2,6-dichloroindophenol, and to a lesser extent, oxygen as surrogate electron acceptors. ThmD displays 160-fold preference for NADH over NADPH and functions as a monomer. The flavin-binding domain of ThmD (ThmD-FD) was purified and characterized. ThmD-FD displayed similar activity as the full-length ThmD and showed a unique flavin spectrum with a major peak at 463nm and a small peak at 396 nm. Computational modeling and mutagenesis analyses suggest a novel three-dimensional fold or covalent flavin attachment in ThmD.