Aviva Katzav

CNS dysfunction in the antiphospholipid syndrome

Though many neurological deficits have been described in the antiphospholipid syndrome (APS), only stroke is well establishedand accepted as a diagnosticcriterion in this disease. We review clinical data obtainedfrom a large series of cases regardingstroke, dementia, epilepsy, chorea, migraine, white matter disease and behavioralchangesin APS or linked to laboratory criteria such as antiphospholipid antibodies (aPL). The contribution of animal models to our understanding of these manifestations of APS is stressed, especially regarding the cognitive and behavioral aspects for which we have established model systems in the mouse. These models utilize immunization of mice with b2-glycoprotein I, a central autoantigen in APS, which induces persistent high levels of aPL. These mice develop hyperactive behavior after a period of four to five months as well as deficits in learning and memory and are potentiallyvaluableas a system in which to study the pathogenesisand treatment of cognitive and behavioral aspects of APS. Another model we have developed, in which IgG from APS patients induce depolarization of brain synaptoneurosomes, may serve as a model for the pathogenesis of epilepsy in APS.

Behavioral and cognitive deficits occur only after prolonged exposure of mice to antiphospholipid antibodies

The antiphospholipid (Hughes) syndrome (APS) includes systemic and central nervous system (CNS) pathology associated with antibodies to a complex of phospholipids and β2-glycoprotein I (β2-GPI). β2-GPI immunized mice develop systemic manifestations of APS and we presently examined CNS manifestations in this APS model. Female BALB/c mice were immunized once with β2-GPI in complete Freunds adjuvant (CFA) or with CFA alone (controls). A staircase test and a T-maze alternation test were performed to test behavior and cognition in independent groups of mice 6, 12 and 18 weeks following the immunization. The APS mice developed elevated levels of antibodies against negatively charged phospholipids and β2-GPI. Neurological impairment was detected only 18 weeks after the induction of the APS and consisted of both cognitive (53± 4 71± 3% correct choices in the T-maze alternation for APS behavioral changes (higher number of rears (18± 2 vs 7±1, P < 0.02). This is the first report of cognitive deficits in this APS model and demonstrates the time course for the development of previously described behavioral changes. The mechanism involved in these CNS manifestations remains to be elucidated.

Anti-P ribosomal antibodies induce defect in smell capability in a model of CNS -SLE (depression)

UNLABELLED Systemic lupus erythematosus (SLE) is an autoimmune disease associated with more than 100 different autoantibodies, some of which may be associated with specific neuropsychiatric (NPSLE) manifestations. Injection of anti-P ribosomal antibodies (anti-P) directly to the brain ventricles of mice induces depression manifested by increased immobility time in the forced swim test (FST). METHODS Mice were injected intracerebroventricularily (ICV) with affinity-purified human anti-P antibodies or normal commercial IgG as control. Mice were examined for depression by the forced swimming test (FST) and for olfactory function by the smell threshold test. Treatments included the antidepressant drug fluoxetine or aroma therapy by exposure to lemon or cinnamon odor. RESULTS Mice injected with anti-P developed depression-like behavior, which improved significantly upon treatment with fluoxetine. Depressed mice had a significant deficit in olfactory function which was not reversed by fluoxetine. Exposure of anti-P-injected mice to lemon odor was associated with some improvement of the immobility time, a measure of depression. CONCLUSIONS ICV injection of anti-P induces both depression-like behavior and impaired olfactory function in mice. Fluoxetine and possibly lemon odor exposure improve depressive behavior in these mice.

Hyperactivity in a mouse model of the antiphospholipid syndrome

In the antiphospholipid syndrome (APS), antibodies to a complex of phospholipids and beta2-glycoprotein I (β2-GPI) are associated with recurrent thromboembolic events, spontaneous abortions, thrombocytopenia and central nervous system (CNS) disturbances. Animals immunized with b2-GPI develop the systemic manifestations of APS but the involvement of the (CNS) in these animals has not been studied. The objective of the present study was to examine mice with induced experimental APS for behavioral changes. Female Balb/C mice were immunized once with β2-GPI in complete Freunds adjuvant (CFA) or with CFA alone. Four months after immunization the mice were tested in the staircase apparatus and the following two variables were measured: (1) number of rears; and (2) number of stairs climbed by the mice. Immunization with β2-GPI resulted in elevated levels of circulating anti-negatively charged phospholipids and anti-β2-GPI antibodies. The APS mice exhibited hyperactive behavior as reflected by more frequent rears (P < 0.023) and higher number of stairs climbed (P < 0.019) by the mice in 3 min. This simple test demonstrated that experimental APS animals are significantly hyperactive and may serve as a marker for CNS involvement in this model.

Passive transfer of narcolepsy: Anti-TRIB2 autoantibody positive patient IgG causes hypothalamic orexin neuron loss and sleep attacks in mice

Narcolepsy is a sleep disorder characterized by excessive daytime sleepiness and cataplexy (a sudden weakening of posture muscle tone usually triggered by emotion) caused by the loss of orexin neurons in the hypothalamus. Autoimmune mechanisms are implicated in narcolepsy by increased frequency of specific HLA alleles and the presence of specific autoantibody (anti-Tribbles homolog 2 (TRIB2) antibodies) in the sera of patients with narcolepsy. Presently, we passively transferred narcolepsy to naïve mice by injecting intra-cerebra-ventricularly (ICV) pooled IgG positive for anti-TRIB2 antibodies. Narcolepsy-IgG-injected mice had a loss of the NeuN (neuronal marker), synaptophysin (synaptic marker) and orexin-positive neurons in the lateral hypothalamus area in narcolepsy compared to control-IgG-injected mice and these changes were associated with narcolepsy-like immobility attacks at four weeks post injection and with hyperactivity and long term memory deficits in the staircase and novel object recognition tests. Similar behavioral and cognitive deficits are observed in narcoleptic patients. This is the first report of passive transfer of experimental narcolepsy to naïve mice induced by autoantibodies and supports the autoimmune pathogenesis in narcolepsy.

Calpastatin levels affect calpain activation and calpain proteolytic activity in APP transgenic mouse model of Alzheimer's disease.

The intracellular Ca(2+)-dependent protease calpain and the specific calpain endogenous inhibitor calpastatin are widely distributed, with the calpastatin/calpain ratio varying among tissues and species. Increased Ca(2+) and calpain activation have been implicated in Alzheimers disease (AD), with scant data available on calpastatin/calpain ratio in AD. Information is lacking on calpain activation and calpastatin levels in transgenic mice that exhibit AD-like pathology. We studied calpain and calpastatin in Tg2576 mice and in their wild type littermates (control mice). We found that in control mice calpastatin level varies among brain regions; it is significantly higher in the cerebellum than in the hippocampus, frontal and temporal cortex, whereas calpain levels are similar in all these regions. In the Tg2576 mice, calpain is activated, calpastatin is diminished, and calpain-dependent proteolysis is observed in brain regions affected in AD and in transgenic mice (especially hippocampus). In contrast, no differences are observed between the Tg2576 and the control mice in the cerebellum, which does not exhibit AD-like pathology. The results are consistent with the notion that a high level of calpastatin in the cerebellum renders the calpain in this brain region less liable to be activated; in the other brain parts, in which calpastatin is low, calpain is more easily activated in the presence of increased Ca(2+), and in turn the activated calpain leads to further diminution in calpastatin (a known calpain substrate). The results indicate that calpastatin is an important factor in the regulation of calpain-induced protein degradation in the brains of the affected mice, and imply a role for calpastatin in attenuating AD pathology. Promoting calpastatin expression may be used to ameliorate some manifestations of AD.

Interaction of inflammation, thrombosis, aspirin and enoxaparin in CNS experimental antiphospholipid syndrome.

Experimental antiphospholipid syndrome (eAPS) induced by immunization with beta(2)-glycoprotein I (beta(2)-GPI) causes behavioral hyperactivity. We assessed the role of thrombotic and inflammatory perivascular factors and standard APS therapies for CNS manifestations. Groups of mice (n=10 per group) were immunized once with beta(2)-GPI (eAPS) or adjuvant (controls) and treated daily from 1 month after immunization with either sham injections, aspirin (1.2 mg/kg) or enoxaparin (1 mg/kg) for 3 months. Serum antiphospholipid antibodies (aPL) and brain levels of tissue necrosis factor-alpha (TNF-alpha) and prostaglandin E (PGE) were then measured by ELISA and thrombin inhibitors by immunoblot. Behavioral hyperactivity was assessed by the staircase test. The eAPS mice had higher levels of aPL than adjuvant immunized controls. Inflammatory markers were found to be twofold higher and intrinsic brain thrombin inhibitors 50% lower in eAPS brains compared to controls. aPL titers were unaffected by treatment. Both aspirin and enoxaparin normalized brain concentrations of PGE and TNF-alpha and elevated thrombin inhibitors, the latter effect being more pronounced for enoxaparin. The increased activity and rearing exploratory behavior in eAPS (138.6+/-13.6 and 141.9+/-13.9% of controls, respectively) were attenuated significantly more by treatment with enoxaparin (91.5+/-12.3 and 95.0+/-9.8%) than by aspirin (167.0+/-18.4 and 114.7+/-13.1%, p<0.01, ANOVA). Together, these results demonstrate that eAPS is associated with significant brain inflammation and thrombosis that are well treated with both standard therapies for human APS. Enoxaparin attenuates behavior better than aspirin possibly by reducing thrombosis or stabilization of the blood brain barrier, suggesting an advantage for similar drugs in CNS APS.

The Pathogenesis of Neural Injury in Animal Models of the Antiphospholipid Syndrome

Circulating antiphospholipid antibodies (aPL) are associated with central nervous system dysfunction in antiphospholipid syndrome (APS) patients and in a mouse model of APS. We propose a logical pathway of how experimental APS (eAPS) causes brain dysfunction: binding of the antibodies to the brain endothelium evoking microthrombosis, endothelial dysfunction, and IgG leakage through the blood–brain barrier (BBB), then secondary inflammatory cell spread around blood vessels and production of cytokines by these inflammatory cells leading to further disruption of the BBB. The diffuse brain endothelial dysfunction would result in extravasation of serum proteins including APS IgG and activated thrombin, which may induce the behavioral changes observed in the APS mice. We have collected data from the mouse eAPS model which supports this hypothesis. Elucidating the mechanism of the pathogenicity of aPL in vitro and in vivo will serve as a much needed basis for developing new therapeutic modalities in this important disorder.

Adjuvant immunization induces high levels of pathogenic antiphospholipid antibodies in genetically prone mice: another facet of the ASIA syndrome

Adjuvants may induce autoimmune diseases in susceptible individuals, a phenomenon recently defined as autoimmune/inflammatory syndrome induced by adjuvants (ASIA). Patients with both antiphospholipid antibodies (aPL) and the genetic coagulopathy factor V Leiden (FVL) are frequently found. We therefore evaluated whether adjuvant can induce aPL in heterozygous FVL mice. aPL were measured in naïve mice and at 1 and 5 months after immunization with either complete or incomplete Freund’s adjuvant (CFA, IFA) in FVL and control C57/B6 background mice. We defined antibody levels 3 SD above the mean of C57/B6 mice immunized with adjuvant as positive (specificity of 99%). For β2GPI-dependent aPL, 28.6% (6/21) of FVL mice 5 months after immunization with adjuvant (both IFA and CFA) were positive compared with 4.8% (1/22) of FVL mice 1 month after adjuvant and 0% of naïve FVL and C57/B6 mice (0/16, p < 0.001). aPL levels correlated with behavioral hyperactivity in the staircase test. FVL mice immunized with adjuvant did not develop β2GPI-independent aPL. We hypothesize that the FVL aPL association is not a coincidence, but that chronic coagulation defects combined with external inflammatory stimuli analogous to adjuvant may induce aPL and also antiphospholipid syndrome, thus supporting the notion of ASIA.

Genetic and immunological factors interact in a mouse model of CNS antiphospholipid syndrome

The antiphospholipid syndrome (APS) includes systemic and central nervous system (CNS) pathology associated with antibodies to a complex of phospholipids and beta(2)-glycoprotein I (beta(2)-GPI). We have recently reported the induction of APS associated with behavioral and cognitive deficits in BALB/c female mice that developed 4-5 months after immunization with beta(2)-GPI. In the present study, we examined the influence of genetic factors on the ability to induce experimental APS with CNS involvement by testing several mouse strains immunized with beta(2)-GPI. Female mice from five strains were immunized once with beta(2)-GPI in complete Freunds adjuvant (CFA) or with CFA alone (controls). Autoantibody levels were examined at 1 and 5 months after immunization. Neurological assessment in a staircase test was performed 4-5 months following the immunization. Induction of APS resulted in elevated levels of antibodies against negatively charged phospholipids and beta(2)-GPI in all five mouse strains. Autoantibody levels were significantly higher in Balb/c, ICR, and C57BL/6 mouse strains compared to AKR and C3H. aPL levels dropped significantly more in the C57BL/6 compared to Balb/c mice over a period of 4 months. Hyperactivity reflected by higher number of stairs climbed in 3 min, was induced by APS in the Balb/c and ICR, mouse strains. Exploratory behavior reflected by more frequent rears, was seen in the APS-Balb/c and AKR mice. Hypoactivity and less exploration were seen in the APS-C57BL/6 and C3H mice. The study supports a link between high levels of aPL and behavioral changes in a mouse APS model. Qualitative differences in behavioral patterns may be due to nervous system as well as immune genetic factors. The minimal effect of APS in C57BL/6 mice may provide a suitable background for the study of transgenes in these mice.