Quantification of total apolipoprotein E and its isoforms in cerebrospinal fluid from patients with neurodegenerative diseases.

BACKGROUND: The human APOE gene, which codes for apolipoprotein E (apoE), has three major polymorphic alleles: ε2, ε3, and ε4 that give rise to amino acid substitutions. APOE-ε4 is a strong risk factor of sporadic Alzheimer's disease (AD) but the reason why is still unknown despite intense research for more than 20 years. The aim of the study was to investigate if the concentrations of total apoE and the specific apoE isoforms in cerebrospinal fluid (CSF) differ between various neurodegenerative diseases and control individuals, as well as among the APOE genotypes. METHODS: Quantification of total apoE and specific apoE isoforms (E2, E3, and E4) in CSF was performed using high-resolution parallel reaction monitoring mass spectrometry. In total, 1820 individuals were involved in the study including clinically diagnosed AD patients (n = 228), cognitively unimpaired (CU) patients (n = 896), and patients with other neurodegenerative disorders (n = 696). Follow-up data was available for 100 individuals, assessed at two time points. Subjects were dichotomized based on an Aß42/40 CSF concentration ratio cut-off into Aß positive (Aß+, < 0.091) and Aß negative (Aß-, > 0.091) groups. RESULTS: Even though there was a significant increase of total apoE in the amyloid ß-positive (Aß+) group compared with amyloid ß-negative (Aß-) individuals (p < 0.001), the magnitude of the effect was very small (AUC = 0.55). Moreover, CSF total apoE concentrations did not differ between Aß- CU controls and clinically diagnosed AD patients. There was a difference in concentration between isoforms in heterozygous individuals in an isoform-dependent manner (E2 < E3 < E4) (p < 0.001, AUC = 0.64-0.69), and these associations remained when dichotomizing the samples into Aß+ and Aß- groups (p < 0.01, AUC = 0.63-0.74). In the cohort with follow-up samples, neither total apoE nor isoform-specific apoE concentrations differed between the two time points (p > 0.05). CONCLUSIONS: The results indicate that neither the concentrations of total apoE nor the different apoE isoforms in CSF are associated with APOE-ε4 carrier status, Aß status, or clinical dementia diagnoses.

CD44 Deficiency Is Associated with Increased Susceptibility to Stress-Induced Anxiety-like Behavior in Mice.

CD44 is a cell surface adhesion molecule and its principal ligand is hyaluronic acid (HA), a key component of the brain's extracellular matrix. CD44 levels are decreased in the cerebrospinal fluid (CSF) of depressed individuals, and the CD44 gene has been identified in genome wide association study as a possible risk gene in suicidal behavior. In order to define the pathobiological mechanisms by which CD44 may affect behavior, we investigated the role of CD44 using male CD44 knockout (CD44KO) and wild-type mice that underwent chronic mild stress (CMS). Behavior was characterized using the sucrose preference and forced swim tests, open field, novel object recognition, social preference, and the elevated plus maze tests. Gene expression in hippocampus was evaluated using quantitative real-time PCR. Brain monoamines and their metabolites were assessed by high-performance liquid chromatography and serum HA and IL-1ß levels were measured using ELISA and electrochemiluminescence assays. CD44KO mice were more susceptible to stress-induced anxiety-like behavior and displayed increased anhedonia and despair than the wild-type controls. The behavioral phenotype of stressed CD44KO mice was associated with reduced cortical serotonergic and striatal dopaminergic turnover. The hippocampal expression of the receptor for HA-mediated motility (RHAMM) was reduced in the non- stressed CD44KO mice compared with WT mice, in a value similar to that observed in WT mice following exposure to stress. Taken together, our experiments suggest that CD44 plays a key role in stress response in mice.

The CD44 ligand hyaluronic acid is elevated in the cerebrospinal fluid of suicide attempters and is associated with increased blood-brain barrier permeability.

BACKGROUND: The glycosaminoglycan hyaluronic acid (HA) is an important component of the extracellular matrix (ECM) in the brain. CD44 is a cell adhesion molecule that binds to HA in the ECM and is present on astrocytes, microglia and certain neurons. Cell adhesion molecules have been reported to be involved in anxiety and mood disorders. CD44 levels are decreased in the cerebrospinal fluid (CSF) of depressed individuals, and the CD44 gene has been identified in brain GWAS studies as a possible risk gene for suicidal behavior. METHOD: We measured the CSF levels of HA and the soluble CD44 (sCD44) in suicide attempters (n=94) and in healthy controls (n=45) using ELISA and electrochemiluminescence assays. We also investigated other proteins known to interact with CD44, such as osteopontin and the matrix metalloproteinases MMP1, MMP3 and MMP9. RESULTS: The suicide attempters had higher CSF levels of HA (p=.003) and MMP9 (p=.004). The CSF levels of HA correlated with BBB-permeability (rho=0.410, p<.001) and MMP9 correlated with sCD44 levels (rho=0.260, p=.005). LIMITATIONS: Other relevant biological contributors to suicidal behavior is not addressed in parallel to the specific role of CD44-HA signaling. The gender distribution of the patients from whom CSF was analyzed was uneven. CONCLUSIONS: Increased BBB-permeability and HA levels might be a results of increased neuroinflammation and can play a role in the pathobiology of suicidal behavior. The CD44 signaling pathway might be considered a novel target for intervention in mood disorders.

Low IL-8 is associated with anxiety in suicidal patients: genetic variation and decreased protein levels.

OBJECTIVE: Recent studies indicate that inflammation may play a role in the pathophysiology of suicidality. Interleukin-8 (IL-8) is a chemokine that in addition to its function in the immune system also exert neuroprotective properties. The involvement of this chemokine in neuropsychiatric conditions is incompletely known. METHOD: We measured plasma and cerebrospinal fluid (CSF) IL-8, as well as the genotype frequency of a single nucleotide polymorphism (-251A/T, rs4073) in the promoter region of the IL8 gene, in suicide attempters (n=206) and healthy controls (n=578). RESULTS: Plasma and CSF levels of IL-8 were significantly lower in suicide attempters with anxiety than in healthy controls. IL-8 in both plasma and CSF correlated negatively with symptoms of anxiety. Compared with the population-based cohort, the IL-8-251T allele was more prevalent among female suicide attempters. Furthermore, suicide attempters carrying this allele showed more severe anxiety. This correlative study warrants further mechanistic studies on the effects of IL-8 in the central nervous system. CONCLUSION: We suggest that IL-8 might be involved in the biological mechanisms mediating resilience to anxiety. Thus, our findings highlight the chemokine IL-8 as a potential target for future development of anti-anxiety treatments and suicide prevention.

CSF biomarkers in suicide attempters--a principal component analysis.

OBJECTIVE: The objective of the present study was to identify biological patterns (factors) among 20 cerebrospinal fluid (CSF) biomarkers in suicide attempters and subsequently analyse their association with suicidal behaviour. METHOD: We measured kynurenic acid, orexin, homovanillic acid (HVA), 5-hydroxyindoleacetic acid (5-HIAA), 3-methoxy-4-hydroxyphenylglycol, chemokines, matrix metalloproteases and cytokines in the CSF of 124 drug-free suicide attempters. Patients were evaluated for suicidality and psychiatric symptoms using well-defined psychiatric rating scales and followed-up regarding future suicide. We used principal component analysis to identify factors among the biological substances. RESULTS: Four factors were extracted from the 20 biomarkers, explaining 52.4% of the total variance. Factors 1 and 2 were characterized by high loadings of chemokines and cytokines respectively. They were both associated with severe depressive symptoms. Factor 2 was also associated with a high suicidal intent. Factor 4 was characterized by strong loadings of the monoamine metabolites 5-HIAA and HVA, as well as orexin and interleukin-6. High scores on this factor were found in patients who performed a violent suicide attempt and in patients who subsequently completed suicide. CONCLUSION: Our results suggest that specific combinations of CSF biomarkers may discriminate between types of suicidal behaviour and indicate increased risk for future suicide.

Inhibition of inducible nitric oxide synthase enhances anti-tumour immune responses in rats immunized with IFN-gamma-secreting glioma cells.

Interferon gamma (IFN-gamma) has successfully been used in immunotherapy of different experimental tumours. Mechanistically, IFN-gamma has extensive effects on the immune system including release of nitric oxide (NO) by upregulation of the inducible nitric oxide synthase (iNOS). NO has putative immunosuppressive effects but could also play a role in killing of tumour cells. Therefore, the aim of the present study was to clarify whether inhibition of iNOS in rats immunized with glioma cells (N32) producing IFN-gamma (N32-IFN-gamma), could enhance the anti-tumour immune response. Initially, both a selective iNOS, l-N(6)-(1-Iminoethyl)-l-lysine (l-NIL), and non-selective, N-nitro-l-arginine methyl ester (l-NAME), inhibitor of NOS were tested in vitro. After polyclonal stimulation with LPS and SEA, both l-NIL and l-NAME enhanced proliferation and production of IFN-gamma from activated rat splenocytes and this effect was inversely correlated to the production of NO. However, l-NIL had a broader window of efficacy and a lower minimal effective dose. When rats were immunized with N32-IFN-gamma, and administered NOS inhibitors by intraperitoneal (i.p.) mini-osmotic pumps, only splenocytes of rats treated with l-NIL, but not l-NAME, displayed an enhanced proliferation and production of IFN-gamma when re-stimulated with N32 tumour cells. Based on these findings, l-NIL was administered concurrently with N32-IFN-gamma cells to rats with intracerebral (i.c.) tumours resulting in a prolonged survival. These results show that inhibition of iNOS can enhance an IFN-gamma-based immunotherapy of experimental i.c. tumours implying that NO released after immunization has mainly immunosuppressive net effects.

Protein aggregation after focal brain ischemia and reperfusion.

Two hours of transient focal brain ischemia causes acute neuronal death in the striatal core region and a somewhat more delayed type of neuronal death in neocortex. The objective of the current study was to investigate protein aggregation and neuronal death after focal brain ischemia in rats. Brain ischemia was induced by 2 hours of middle cerebral artery occlusion. Protein aggregation was analyzed by electron microscopy, laser-scanning confocal microscopy, and Western blotting. Two hours of focal brain ischemia induced protein aggregation in ischemic neocortical neurons at 1 hour of reperfusion, and protein aggregation persisted until neuronal death at 24 hours of reperfusion. Protein aggregates were found in the neuronal soma, dendrites, and axons, and they were associated with intracellular membranous structures during the postischemic phase. High-resolution confocal microscopy showed that clumped protein aggregates surrounding nuclei and along dendrites were formed after brain ischemia. On Western blots, ubiquitinated proteins (ubi-proteins) were dramatically increased in neocortical tissues in the postischemic phase. The ubi-proteins were Triton-insoluble, indicating that they might be irreversibly aggregated. The formation of ubi-protein aggregates after ischemia correlated well with the observed decrease in free ubiquitin and neuronal death. The authors concluded that proteins are severely damaged and aggregated in neurons after focal ischemia. The authors propose that protein damage or aggregation may contribute to ischemic neuronal death.

Alterations of Akt1 (PKBalpha) and p70(S6K) in transient focal ischemia.

The serine-threonine kinase Akt1 promotes cell survival through inhibition of apoptosis. One of the potential downstream targets of Akt1 is p70 S6 kinase, p70(S6K), an enzyme implicated in the regulation of protein synthesis. In this study, we investigated the changes in total and phosphorylated levels of Akt1 and p70(S6K) during transient focal ischemia. Male Wistar rats were subjected to 2 h of middle cerebral artery occlusion followed by 1, 4, and 24 h of reperfusion. The expression of total and phosphorylated forms of Akt1 and p70(S6K) were examined by Western blot analysis. Phosphorylation of Akt1 on Ser473 transiently increased at 1 and 4 h of reperfusion, whereas phosphorylation of Akt1 on Thr308 was reduced during reperfusion. The levels of total Akt1 remained unchanged at 1 and 4 h of reperfusion, but decreased significantly at 24 h of reperfusion. Phosphorylation of p70(S6K) on Thr389 decreased at 1, 4, and 24 h of reperfusion, while the levels of total p70(S6K) protein remained unchanged at 1 and 4 h of reperfusion but decreased at 24 h of reperfusion. The results show that cell survival pathways, such as Akt1 and p70(S6K) signaling, are suppressed after transient focal ischemia, which may contribute to the development of neuronal cell death after an ischemic insult.

Is neuronal injury caused by hypoglycemic coma of the necrotic or apoptotic type?

In this study, we explored if a 30 minute period of hypoglycemic coma yields damage which shows some features associated with apoptosis. To that end, we induced insulin-hypoglycemic coma of 30 min duration, and studied brain tissues after the coma period, and after recovery period of 30 min, 3 h, and 6 h. Histopathological data confirmed neuronal damage in all of the vulnerable neuronal populations. Release of cytochrome c (cyt c), assessed by Western Blot, was observed in the neocortex and caudoputamen after 3 and 6 h of recovery. In these regions, the caspase-like activity increased above control after 6 h of recovery. By laser-scanning confocal microscopy, a clear expression of Bax was observed after 30 min of coma in the superficial layers of the neocortex, reaching a peak after 30 min of recovery. Punctuate immunolabeling surrounding nuclei in soma and dendrites in cortical pyramidal neurons likely represents mitochondria, which suggests that Bax protein assembled at the surface of mitochondria in vulnerable neocortical neurons. It is concluded that although previous morphological data have suggested that cells die by necrosis, neuronal damage after hypoglycemic coma shows some features of apoptosis.

Role and mechanisms of secondary mitochondrial failure.

Ischemia is accompanied by mitochondrial dysfunction, as assessed by measurements of mitochondrial respiratory activities in vitro. Following brief periods of ischemia, mitochondrial function is usually normalized during reperfusion. However, particularly after ischemia of longer duration, reperfusion may be accompanied by secondary mitochondrial failure. After short periods of ischemia this is observed in selectively vulnerable areas and, after intermediate to long periods of ischemia, in other areas as well. However, it has remained unsettled if the mitochondrial dysfunction is the result or the cause of cell death. Although it has been commonly assumed that such failure is secondary to cell injury by other mechanisms, recent results suggest that mitochondrial dysfunction may be the cause of cell death. Indirect evidence for this postulate is provided by experiments showing that cyclosporin A (CsA), when allowed to cross the blood-brain barrier, is a potent neuroprotectant. CsA is a virtually specific blocker of the mitochondrial permeability transition (MPT) pore, a voltage-gated channel allowing molecules and ions with a mass < 1500 Daltons to pass the inner mitochondrial membrane. Experiments on isolated cells in vitro demonstrate that cell calcium accumulation or oxidative stress triggers the assembly of an MPT pore, which leads to collapse of the mitochondrial membrane potential, to ATP hydrolysis, to enhanced production of reactive oxygen species (ROS), and to cell death. The beneficial effect of CsA could thus be related to its ability to block the MPT pore. Longer periods of ischemia, such as occurs after transient middle cerebral artery (MCA) occlusion, lead to pan-necrotic lesions (infarction). In the rat, recirculation following 2 h of MCA occlusion leads to partial normalization of the bioenergetic state but this is followed within 4-6 h by secondary bioenergetic failure. The latter seems unrelated to blockade of the microcirculation, but correlates to secondary mitochondrial failure. The brain damage incurred is ameliorated by the spin trap alpha-phenyl-N-butyl nitrone (PBN) and by the immunosuppressant FK506 even when given 1-3 h after the start of recirculation. The two drugs also prevent the secondary mitochondrial failure during early recirculation, suggesting that such failure is pathogenetically important. Probably, though, the mitochondrial dysfunction involves not only the assembly of an MPT pore but also other mechanisms. Since recirculation is associated with release of mitochondrial proteins it is not unlikely that such proteins, e.g. cytochrome c, trigger cascades of events leading to cell death.6.

The immunosuppressants cyclosporin A and FK506 equally ameliorate brain damage due to 30-min middle cerebral artery occlusion in hyperglycemic rats.

In the present experiments, we compared the anti-ischemic effects of the immunosuppressants cyclosporin A (CsA) and FK506 in hyperglycemic animals subjected to 30 min of middle cerebral artery (MCA) occlusion. Both immunosuppressants were given as pre-treatment, the effect of treatment being evaluated by 2,3, 5-triphenyltetrazolium (TTC) staining after 3 days of recovery. Both FK506 and CsA reduced the infarct volume to less than 1/3 of control. In spite of CsA's known effect as a blocker of the mitochondrial transition (MPT) pore, it failed to give a more robust effect than FK506. If anything, FK506, which lacks an effect on the MPT pore, had a more pronounced anti-ischemic effect. We conclude that, in this model of infarction, an MPT may not play a major pathogenetic role.