Bafilomycin 1A Affects p62/SQSTM1 Autophagy Marker Protein Level and Autophagosome Puncta Formation Oppositely under Various Inflammatory Conditions in Cultured Rat Microglial Cells.

Regulation of autophagy through the 62 kDa ubiquitin-binding protein/autophagosome cargo protein sequestosome 1 (p62/SQSTM1), whose level is generally inversely proportional to autophagy, is crucial in microglial functions. Since autophagy is involved in inflammatory mechanisms, we investigated the actions of pro-inflammatory lipopolysaccharide (LPS) and anti-inflammatory rosuvastatin (RST) in secondary microglial cultures with or without bafilomycin A1 (BAF) pretreatment, an antibiotic that potently inhibits autophagosome fusion with lysosomes. The levels of the microglia marker protein Iba1 and the autophagosome marker protein p62/SQSTM1 were quantified by Western blots, while the number of p62/SQSTM1 immunoreactive puncta was quantitatively analyzed using fluorescent immunocytochemistry. BAF pretreatment hampered microglial survival and decreased Iba1 protein level under all culturing conditions. Cytoplasmic p62/SQSTM1 level was increased in cultures treated with LPS+RST but reversed markedly when BAF+LPS+RST were applied together. Furthermore, the number of p62/SQSTM1 immunoreactive autophagosome puncta was significantly reduced when RST was used but increased significantly in BAF+RST-treated cultures, indicating a modulation of autophagic flux through reduction in p62/SQSTM1 degradation. These findings collectively indicate that the cytoplasmic level of p62/SQSTM1 protein and autophagocytotic flux are differentially regulated, regardless of pro- or anti-inflammatory state, and provide context for understanding the role of autophagy in microglial function in various inflammatory settings.

Opposite and Differently Altered Postmortem Changes in H3 and H3K9me3 Patterns in the Rat Frontal Cortex and Hippocampus.

Temporal and spatial epigenetic modifications in the brain occur during ontogenetic development, pathophysiological disorders, and aging. When epigenetic marks, such as histone methylations, in brain autopsies or biopsy samples are studied, it is critical to understand their postmortem/surgical stability. For this study, the frontal cortex and hippocampus of adult rats were removed immediately (controls) or after a postmortem delay of 15, 30, 60, 90, 120, or 150 min. The patterns of unmodified H3 and its trimethylated form H3K9me3 were analyzed in frozen samples for Western blot analysis and in formalin-fixed tissues embedded in paraffin for confocal microscopy. We found that both the unmodified H3 and H3K9me3 showed time-dependent but opposite changes and were altered differently in the frontal cortex and hippocampus with respect to postmortem delay. In the frontal cortex, the H3K9me3 marks increased approximately 450% with a slow parallel 20% decrease in the unmodified H3 histones after 150 min. In the hippocampus, the change was opposite, since H3K9me3 marks decreased steadily by approximately 65% after 150 min with a concomitant rapid increase of 20-25% in H3 histones at the same time. Confocal microscopy located H3K9me3 marks in the heterochromatic regions of the nuclei of all major cell types in the control brains: oligodendrocytes, astrocytes, neurons, and microglia. Therefore, epigenetic marks could be affected differently by postmortem delay in different parts of the brain.

The kynurenic acid analog SZR104 induces cytomorphological changes associated with the anti-inflammatory phenotype in cultured microglia.

We previously showed the anti-inflammatory effects of kynurenic acid (KYNA) and its brain-penetrable analog N-(2-(dimethylamino)ethyl)-3-(morpholinomethyl)-4-hydroxyquinoline-2-carboxamide (SZR104) both in vivo and in vitro. Here, we identified the cytomorphological effects of KYNA and SZR104 in secondary microglial cultures established from newborn rat forebrains. We quantitatively analyzed selected morphological aspects of microglia in control (unchallenged), lipopolysaccharide (LPS)-treated (challenged), KYNA- or SZR104-treated, and LPS + KYNA or LPS + SZR104-treated cultures. Multicolor immunofluorescence labeling followed by morphometric analysis (area, perimeter, transformation index, lacunarity, density, span ratio, maximum span across the convex hull, hull circularity, hull area, hull perimeter, max/min radii, mean radius, diameter of bounding circle, fractal dimension, roughness, circularity) on binary (digital) silhouettes of the microglia revealed their morphological plasticity under experimental conditions. SZR104 and, to a lesser degree, KYNA inhibited proinflammatory phenotypic changes. For example, SZR104 treatment resulted in hypertrophied microglia characterized by a swollen cell body, enlarged perimeter, increased transformation index/decreased circularity, increased convex hull values (area, perimeter, mean radius, maximum span, diameter of the bounding circle and hull circularity), altered box-counting parameters (such as fractal dimension), and increased roughness/decreased density. Taken together, analysis of cytomorphological features could contribute to the characterization of the anti-inflammatory activity of SZR104 on cultured microglia.

A conserved MTMR lipid phosphatase increasingly suppresses autophagy in brain neurons during aging.

Ageing is driven by the progressive, lifelong accumulation of cellular damage. Autophagy (cellular self-eating) functions as a major cell clearance mechanism to degrade such damages, and its capacity declines with age. Despite its physiological and medical significance, it remains largely unknown why autophagy becomes incapable of effectively eliminating harmful cellular materials in many cells at advanced ages. Here we show that age-associated defects in autophagic degradation occur at both the early and late stages of the process. Furthermore, in the fruit fly Drosophila melanogaster, the myotubularin-related (MTMR) lipid phosphatase egg-derived tyrosine phosphatase (EDTP) known as an autophagy repressor gradually accumulates in brain neurons during the adult lifespan. The age-related increase in EDTP activity is associated with a growing DNA N6-adenine methylation at EDTP locus. MTMR14, the human counterpart of EDTP, also tends to accumulate with age in brain neurons. Thus, EDTP, and presumably MTMR14, promotes brain ageing by increasingly suppressing autophagy throughout adulthood. We propose that EDTP and MTMR14 phosphatases operate as endogenous pro-ageing factors setting the rate at which neurons age largely independently of environmental factors, and that autophagy is influenced by DNA N6-methyladenine levels in insects.

Kynurenic Acid and Its Analog SZR104 Exhibit Strong Antiinflammatory Effects and Alter the Intracellular Distribution and Methylation Patterns of H3 Histones in Immunochallenged Microglia-Enriched Cultures of Newborn Rat Brains.

Kynurenic acid (KYNA) is implicated in antiinflammatory processes in the brain through several cellular and molecular targets, among which microglia-related mechanisms are of paramount importance. In this study, we describe the effects of KYNA and one of its analogs, the brain-penetrable SZR104 (N-(2-(dimethylamino)ethyl)-3-(morpholinomethyl)-4-hydroxyquinoline-2-carboxamide), on the intracellular distribution and methylation patterns of histone H3 in immunochallenged microglia cultures. Microglia-enriched secondary cultures made from newborn rat forebrains were immunochallenged with lipopolysaccharide (LPS). The protein levels of selected inflammatory markers C-X-C motif chemokine ligand 10 (CXCL10) and C-C motif chemokine receptor 1 (CCR1), histone H3, and posttranslational modifications of histone H3 lys methylation sites (H3K9me3 and H3K36me2, marks typically associated with opposite effects on gene expression) were analyzed using quantitative fluorescent immunocytochemistry and western blots in control or LPS-treated cultures with or without KYNA or SZR104. KYNA and SZR104 reduced levels of the inflammatory marker proteins CXCL10 and CCR1 after LPS-treatment. Moreover, KYNA and SZR104 favorably affected histone methylation patterns as H3K9me3 and H3K36me2 immunoreactivities, and histone H3 protein levels returned toward control values after LPS treatment. The cytoplasmic translocation of H3K9me3 from the nucleus indicated inflammatory distress, a process that could be inhibited by KYNA and SZR104. Thus, KYNA signaling and metabolism, and especially brain-penetrable KYNA analogs such as SZR104, could be key targets in the pathway that connects chromatin structure and epigenetic mechanisms with functional consequences that affect neuroinflammation and perhaps neurodegeneration.

Cellular and Molecular Effects of SARS-CoV-2 Linking Lung Infection to the Brain.

In December 2019, a new viral disease emerged and quickly spread all around the world. In March 2020, the COVID-19 outbreak was classified as a global pandemic and by June 2021, the number of infected people grew to over 170 million. Along with the patients' mild-to-severe respiratory symptoms, reports on probable central nervous system (CNS) effects appeared shortly, raising concerns about the possible long-term detrimental effects on human cognition. It remains unresolved whether the neurological symptoms are caused directly by the SARS-CoV-2 infiltration in the brain, indirectly by secondary immune effects of a cytokine storm and antibody overproduction, or as a consequence of systemic hypoxia-mediated microglia activation. In severe COVID-19 cases with impaired lung capacity, hypoxia is an anticipated subsidiary event that can cause progressive and irreversible damage to neurons. To resolve this problem, intensive research is currently ongoing, which seeks to evaluate the SARS-CoV-2 virus' neuroinvasive potential and the examination of the antibody and autoantibody generation upon infection, as well as the effects of prolonged systemic hypoxia on the CNS. In this review, we summarize the current research on the possible interplay of the SARS-CoV-2 effects on the lung, especially on alveolar macrophages and direct and indirect effects on the brain, with special emphasis on microglia, as a possible culprit of neurological manifestation during COVID-19.

Quantitative morphometric and cell-type-specific population analysis of microglia-enriched cultures subcloned to high purity from newborn rat brains.

Morphological and functional characterizations of cultured microglia are essential for the improved understanding of their roles in neuronal health and disease. Although some studies (phenotype analysis, phagocytosis) can be carried out in mixed or microglia-enriched cultures, in others (gene expression) pure microglia must be used. If the use of genetically modified microglial cells is not feasible, isolation of resident microglia from nervous tissue must be carried out. In this study, mixed primary cultures were established from the forebrains of newborn rats. Secondary microglia-enriched cultures were then prepared by shaking off these cells from the primary cultures, which were subsequently used to establish tertiary cultures by further shaking off the easily detachable microglia. The composition of these cultures was quantitatively analyzed by immunocytochemistry of microglia-, astrocyte-, oligodendrocyte- and neuron-specific markers to determine yield and purity. Microglia were quantitatively characterized regarding morphological and proliferation aspects. Secondary and tertiary cultures typically exhibited 73.3% ± 17.8% and 93.1% ± 6.0% purity for microglia, respectively, although the total number of microglia in the latter was much smaller. One in seven attempts of culturing the tertiary cultures had ~99% purity for microglia. The overall yield from the number of cells plated at DIV0 to the Iba1-positive microglia in tertiary cultures was ~1%. Astrocytic and neuronal contamination progressively decreased during subcloning, while oligodendrocytes were found sporadically throughout culturing. Although the tertiary microglia cultures had a low yield, they produced consistently high purity for microglia; after validation, such cultures are suitable for purity-sensitive functional screenings (gene/protein expression).

Epigenetic Consequences of in Utero Exposure to Rosuvastatin: Alteration of Histone Methylation Patterns in Newborn Rat Brains.

Rosuvastatin (RST) is primarily used to treat high cholesterol levels. As it has potentially harmful but not well-documented effects on embryos, RST is contraindicated during pregnancy. To demonstrate whether RST could induce molecular epigenetic events in the brains of newborn rats, pregnant mothers were treated daily with oral RST from the 11th day of pregnancy for 10 days (or until delivery). On postnatal day 1, the brains of the control and RST-treated rats were removed for Western blot or immunohistochemical analyses. Several antibodies that recognize different methylation sites for H2A, H2B, H3, and H4 histones were quantified. Analyses of cell-type-specific markers in the newborn brains demonstrated that prenatal RST administration did not affect the composition and cell type ratios as compared to the controls. Prenatal RST administration did, however, induce a general, nonsignificant increase in H2AK118me1, H2BK5me1, H3, H3K9me3, H3K27me3, H3K36me2, H4, H4K20me2, and H4K20me3 levels, compared to the controls. Moreover, significant changes were detected in the number of H3K4me1 and H3K4me3 sites (134.3% ± 19.2% and 127.8% ± 8.5% of the controls, respectively), which are generally recognized as transcriptional activators. Fluorescent/confocal immunohistochemistry for cell-type-specific markers and histone methylation marks on tissue sections indicated that most of the increase at these sites belonged to neuronal cell nuclei. Thus, prenatal RST treatment induces epigenetic changes that could affect neuronal differentiation and development.

Sensitivity of Rodent Microglia to Kynurenines in Models of Epilepsy and Inflammation In Vivo and In Vitro: Microglia Activation is Inhibited by Kynurenic Acid and the Synthetic Analogue SZR104.

Kynurenic acid is an endogenous modulator of ionotropic glutamate receptors and a suppressor of the immune system. Since glutamate and microglia are important in the pathogenesis of epilepsy, we investigated the possible action of the synthetic kynurenic acid analogue, SZR104, in epileptic mice and the action of kynurenic acid and SZR104 on the phagocytotic activity of cultured microglia cells. Pilocarpine epilepsy was used to test the effects of SZR104 on morphological microglia transformation, as evaluated through ionized calcium-binding adaptor molecule 1 (Iba1) immunohistochemistry. Microglia-enriched rat secondary cultures were used to investigate phagocytosis of fluorescent microbeads and Iba1 protein synthesis in control and lipopolysaccharide-challenged cultures. SZR104 inhibited microglia transformation following status epilepticus. Kynurenic acid and SZR104 inhibited lipopolysaccharide-stimulated phagocytotic activity of microglia cells. Although kynurenic acid and its analogues proved to be glutamate receptor antagonists, their immunosuppressive action was dominant in epilepsy. The inhibition of phagocytosis in vitro raised the possibility of the inhibition of genes encoding inflammatory cytokines in microglial cells.

Orofacial skin inflammation increases the number of macrophages in the maxillary subregion of the rat trigeminal ganglion in a corticosteroid-reversible manner.

Inflammation of the cutaneous orofacial tissue can lead to a prolonged alteration of neuronal and nonneuronal cellular functions in trigeminal nociceptive pathways. In this study, we investigated the effects of experimentally induced skin inflammation by dithranol (anthralin) on macrophage activation in the rat trigeminal ganglion. Tissue localization and protein expression levels of ionized calcium-binding adaptor molecule 1 (Iba1), a macrophage/microglia-specific marker, and proliferation/mitotic marker antigen identified by the monoclonal antibody Ki67 (Ki67), were quantitatively analyzed using immunohistochemistry and western blots in control, dithranol-treated, dithranol- and corticosteroid-treated, and corticosteroid-treated trigeminal ganglia. Chronic orofacial dithranol treatment elicited a strong pro-inflammatory effect in the ipsilateral trigeminal ganglion. Indeed, daily dithranol treatment of the orofacial skin for 3-5 days increased the number of macrophages and Iba1 protein expression in the maxillary subregion of the ipsilateral ganglion. In the affected ganglia, none of the Iba1-positive cells expressed Ki67. This absence of mitotically active cells suggested that the accumulation of macrophages in the ganglion was not the result of resident microglia proliferation but rather the extravasation of hematogenous monocytes from the periphery. Subsequently, when a 5-day-long anti-inflammatory corticosteroid therapy was employed on the previously dithranol-treated orofacial skin, Iba1 immunoreactivity was substantially reduced in the ipsilateral ganglion. Collectively, our findings indicate that both peripheral inflammation and subsequent anti-inflammatory therapy affect macrophage activity and thus interfere with the functioning of the affected sensory ganglion neurons.

A novel pleiotropic effect of aspirin: Beneficial regulation of pro- and anti-inflammatory mechanisms in microglial cells.

Aspirin, one of the most widely used non-steroidal anti-inflammatory drugs, has extensively studied effects on the cardiovascular system. To reveal further pleiotropic, beneficial effects of aspirin on a number of pro- and anti-inflammatory microglial mechanisms, we performed morphometric and functional studies relating to phagocytosis, pro- and anti-inflammatory cytokine production (IL-1ß, tumor necrosis factor-α (TNF-α) and IL-10, respectively) and analyzed the expression of a number of inflammation-related genes, including those related to the above functions, in pure microglial cells. We examined the effects of aspirin (0.1mM and 1mM) in unchallenged (control) and bacterial lipopolysaccharide (LPS)-challenged secondary microglial cultures. Aspirin affected microglial morphology and functions in a dose-dependent manner as it inhibited LPS-elicited microglial activation by promoting ramification and the inhibition of phagocytosis in both concentrations. Remarkably, aspirin strongly reduced the pro-inflammatory IL-1ß and TNF-α production, while it increased the anti-inflammatory IL-10 level in LPS-challenged cells. Moreover, aspirin differentially regulated the expression of a number of inflammation-related genes as it downregulated such pro-inflammatory genes as Nos2, Kng1, IL1ß, Ptgs2 or Ccr1, while it upregulated some anti-inflammatory genes such as IL10, Csf2, Cxcl1, Ccl5 or Tgfb1. Thus, the use of aspirin could be beneficial for the prophylaxis of certain neurodegenerative disorders as it effectively ameliorates inflammation in the brain.

The small molecule AUTEN-99 (autophagy enhancer-99) prevents the progression of neurodegenerative symptoms.

Autophagy functions as a main route for the degradation of superfluous and damaged constituents of the cytoplasm. Defects in autophagy are implicated in the development of various age-dependent degenerative disorders such as cancer, neurodegeneration and tissue atrophy, and in accelerated aging. To promote basal levels of the process in pathological settings, we previously screened a small molecule library for novel autophagy-enhancing factors that inhibit the myotubularin-related phosphatase MTMR14/Jumpy, a negative regulator of autophagic membrane formation. Here we identify AUTEN-99 (autophagy enhancer-99), which activates autophagy in cell cultures and animal models. AUTEN-99 appears to effectively penetrate through the blood-brain barrier, and impedes the progression of neurodegenerative symptoms in Drosophila models of Parkinson's and Huntington's diseases. Furthermore, the molecule increases the survival of isolated neurons under normal and oxidative stress-induced conditions. Thus, AUTEN-99 serves as a potent neuroprotective drug candidate for preventing and treating diverse neurodegenerative pathologies, and may promote healthy aging.

AUTEN-67 (Autophagy Enhancer-67) Hampers the Progression of Neurodegenerative Symptoms in a Drosophila model of Huntington's Disease.

BACKGROUND: Autophagy, a lysosome-mediated self-degradation process of eukaryotic cells, serves as a main route for the elimination of cellular damage [1-3]. Such damages include aggregated, oxidized or misfolded proteins whose accumulation can cause various neurodegenerative pathologies, including Huntington's disease (HD). OBJECTIVE: Here we examined whether enhanced autophagic activity can alleviate neurophatological features in a Drosophila model of HD (the transgenic animals express a human mutant Huntingtin protein with a long polyglutamine repeat, 128Q). METHODS: We have recently identified an autophagy-enhancing small molecule, AUTEN-67 (autophagy enhancer 67), with potent neuroprotective effects [4]. AUTEN-67 was applied to induce autophagic activity in the HD model used in this study. RESULTS: We showed that AUTEN-67 treatment interferes with the progressive accumulation of ubiquitinated proteins in the brain of Drosophila transgenic for the pathological 128Q form of human Huntingtin protein. The compound significantly improved the climbing ability and moderately extended the mean life span of these flies. Furthermore, brain tissue samples from human patients diagnosed for HD displayed increased levels of the autophagy substrate SQSTM1/p62 protein, as compared with controls. CONCLUSIONS: These results imply that AUTEN-67 impedes the progression of neurodegenerative symptoms characterizing HD, and that autophagy is a promising therapeutic target for treating this pathology. In humans, AUTEN-67 may have the potential to delay the onset and decrease the severity of HD.

Calmodulin inhibition regulates morphological and functional changes related to the actin cytoskeleton in pure microglial cells.

The roles of calmodulin (CaM), a multifunctional intracellular calcium receptor protein, as concerns selected morphological and functional characteristics of pure microglial cells derived from mixed primary cultures from embryonal forebrains of rats, were investigated through use of the CaM antagonists calmidazolium (CALMID) and trifluoperazine (TFP). The intracellular localization of the CaM protein relative to phalloidin, a bicyclic heptapeptide that binds only to filamentous actin, and the ionized calcium-binding adaptor molecule 1 (Iba1), a microglia-specific actin-binding protein, was determined by immunocytochemistry, with quantitative analysis by immunoblotting. In unchallenged and untreated (control) microglia, high concentrations of CaM protein were found mainly perinuclearly in ameboid microglia, while the cell cortex had a smaller CaM content that diminished progressively deeper into the branches in the ramified microglia. The amounts and intracellular distributions of both Iba1 and CaM proteins were altered after lipopolysaccharide (LPS) challenge in activated microglia. CALMID and TFP exerted different, sometimes opposing, effects on many morphological, cytoskeletal and functional characteristics of the microglial cells. They affected the CaM and Iba1 protein expressions and their intracellular localizations differently, inhibited cell proliferation, viability and fluid-phase phagocytosis to different degrees both in unchallenged and in LPS-treated (immunologically challenged) cells, and differentially affected the reorganization of the actin cytoskeleton in the microglial cell cortex, influencing lamellipodia, filopodia and podosome formation. In summary, these CaM antagonists altered different aspects of filamentous actin-based cell morphology and related functions with variable efficacy, which could be important in deciphering the roles of CaM in regulating microglial functions in health and disease.

Decrease of mGluR5 receptor density goes parallel with changes in enkephalin and substance P immunoreactivity in Huntington's disease: a preliminary investigation in the postmortem human brain.

Group 1 metabotropic glutamate subtype 5 receptors (mGluR5) contribute to the control of motor behavior by regulating the balance between excitation and inhibition of outputs in the basal ganglia. The density of these receptors is increased in patients with Parkinson's disease and motor complications. We hypothesized that similar changes may occur in Huntington's disease (HD) and aimed at testing this hypothesis in a preliminary experimental series in postmortem human brain material obtained from HD patients. Using autoradiography, we analyzed mGluR5 density in the putamen, caudate nucleus and cerebellum (control region) in postmortem tissue samples from three patients with HD and three controls with two mGluR5-specific radioligands ([(3)H]ABP688 and [(11)C]ABP688). The density of enkephalin (Enk)- or substance P (SP)-containing neurons was assessed using immunohistochemical and cell-counting methods. [(3)H]ABP688 binding in HD was reduced in the caudate (-70.4 %, P < 0.001), in the putamen (-33.3 %, P = 0.053), and in the cerebellum (-8.79 %, P = 0.930) vs controls. Results with [(11)C]ABP688 were similar; there was good correlation between [(11)C]ABP688 and [(3)H]ABP688 binding ratios. Total cell density was similar in all three brain regions in HD patients and controls. Neuronal density was 69 % lower in the caudate (P = 0.002) and 64 % lower in the putamen (P < 0.001) of HD patients vs controls. Both direct and indirect pathways were affected, with ≥ 90 % decrease in the density of Enk- and SP-containing neurons in the caudate and putamen of HD patients vs controls (P < 0.001). In contrast to earlier observations in PD, in HD, compared to controls, the mGluR5 density was significantly lower in the caudate nucleus. The decrease in neuronal density suggests that neuronal loss was largely responsible for the observed decrease in mGluR5.

Long-term effects of selective immunolesions of cholinergic neurons of the nucleus basalis magnocellularis on the ascending cholinergic pathways in the rat: a model for Alzheimer's disease.

Alzheimer's disease is associated with a significant decrease in the cholinergic input to the neocortex. In a rat model of this depletion, we analyzed the subsequent long-term changes in cholinergic fiber density in two well-defined areas of the frontal and parietal cortices: Fr1, the primary motor cortex, and HL, the hindlimb area of the somatosensory (parietal) cortex, two cortical cholinergic fields that receive inputs from the nucleus basalis magnocellularis (nBM). A specific cholinergic lesion was induced by the intraparenchymal injection of 192 IgG-saporin into the nBM. Choline acetyltransferase (ChAT) immunohistochemistry was applied to identify the loss of cholinergic neurons in the nBM, while acetylcholinesterase (AChE) enzyme histochemistry was used to analyze the decreases in the number of cholinoceptive neurons in the nBM and the cholinergic fiber density in the Fr1 and HL cortical areas in response to the nBM lesion. The immunotoxin differentially affected the number of ChAT- and AChE-positive neurons in the nBM. 192 IgG-saporin induced a massive, irreversible depletion of the ChAT-positive (cholinergic) neurons (to 11.7% of the control level), accompanied by a less dramatic, but similarly persistent loss of the AChE-positive (cholinoceptive) neurons (to 59.2% of the control value) in the nBM within 2 weeks after the lesion. The difference seen in the depletion of ChAT- and AChE-positive neurons is due to the specificity of the immunotoxin to cholinergic neurons. The cholinergic fiber densities in cortical areas Fr1 and HL remained similarly decreased (to 62% and 68% of the control values, respectively) up to 20 weeks. No significant rebound in AChE activity occurred either in the nBM or in the cortices during the period investigated. This study therefore demonstrated that, similarly to the very extensive reduction in the number of ChAT-positive neurons in the nBM, cortical areas Fr1 and HL underwent long-lasting reductions in the number of AChE-positive fibers in response to specific cholinergic lesioning of the nBM.

Distribution and binding of 18F-labeled and 125I-labeled analogues of ACI-80, a prospective molecular imaging biomarker of disease: a whole hemisphere post mortem autoradiography study in human brains obtained from Alzheimer's disease patients.

One of the major pathological landmarks of Alzheimer's disease and other neurodegenerative diseases is the presence of amyloid deposits in the brain. The early non-invasive visualization of amyloid is a major objective of recent diagnostic neuroimaging approaches, including positron emission tomography (PET), with an eye on follow-up of disease progression and/or therapy efficacy. The development of molecular imaging biomarkers with binding affinity to amyloid in the brain is therefore in the forefront of imaging biomarker and radiochemistry research. Recently, a dodecamer peptide (amino acid sequence=QSHYRHISPAQV; denominated D1 or ACI-80) was identified as a prospective ligand candidate, binding with high ex vivo affinity to L-Aß-amyloid (K(d): 0.4 µM). In order to assess the ligand's capacity to visualize amyloid in Alzheimer's disease (AD), two (125)I labeled and three (18)F labeled analogues of the peptide were synthesized and tested in post mortem human autoradiography experiments using whole hemisphere brain slices obtained from deceased AD patients and age matched control subjects. The (18)F-labeled radioligands showed more promising visualization capacity of amyloid that the (125)I-labeled radioligands. In the case of each (18)F radioligands the grey matter uptake in the AD brains was significantly higher than that in control brains. Furthermore, the grey matter: white matter uptake ratio was over ~2, the difference being significant for each (18)F-radioligands. The regional distribution of the uptake of the various radioligands systematically shows a congruent pattern between the high uptake regions and spots in the autoradiographic images and the disease specific signals obtained in adjacent or identical brain slices labeled with histological, immunohistochemical or autoradiographic stains for amyloid deposits or activated astrocytes. The present data, using post mortem human brain autoradiography in whole hemisphere human brains obtained from deceased AD patients and age matched control subjects, support the visualization capacity of the radiolabeled ACI-80 analogues of amyloid deposits in the human brain. Further studies are warranted to explore the usefulness of the (18)F-labeled analogues as in vivo molecular imaging biomarkers in diagnostic PET studies.

Protective effects of a phosphatidylcholine-enriched diet in lipopolysaccharide-induced experimental neuroinflammation in the rat.

Our goal was to characterize the neuroprotective properties of orally administered phosphatidylcholine (PC) in a rodent model of systemic inflammation. Sprague-Dawley rats were killed at 3 h, 1 day, 3 days, or 7 days after i.p. administration of lipopolysaccharide (LPS) to determine the plasma levels of tumor necrosis factor α (TNF-α) and interleukin 6 cytokines. The control group and one group of LPS-treated animals were nourished with standard laboratory chow, whereas another LPS-treated group received a special diet enriched with 1% PC for 5 days before the administration of LPS and thereafter during the 7-day observation period. Immunohistochemistry was performed to visualize the bromodeoxyuridine and doublecortin-positive neuroprogenitor cells and Iba1-positive microglia in the hippocampus, whereas the degree of mucosal damage was evaluated on ileal and colon biopsy samples after hematoxylin-eosin staining. The activities of proinflammatory myeloperoxidase and xanthine-oxidoreductase and the tissue nitrite/nitrate (NOx) level were additionally determined, and the cognitive functions were monitored via Morris water maze testing. The inflammatory challenge transiently increased the hippocampal NOx level and led to microglia accumulation and decreased neurogenesis. The intestinal damage, mucosal myeloperoxidase, xanthine-oxidoreductase, and NOx changes were less pronounced, and long-lasting behavioral alterations were not observed. Phosphatidylcholine pretreatment reduced the plasma TNF-α and hippocampal NOx changes and prevented the decreased neurogenesis. These data demonstrated the relative susceptibility of the brain to the consequences of transient peripheral inflammatory stimuli. Phosphatidylcholine supplementation did not reduce the overall extent of peripheral inflammatory activation, but efficiently counteracted the disturbed hippocampal neurogenesis by lowering circulating TNF-α concentrations.

Direct projection from the visual associative cortex to the caudate nucleus in the feline brain.

Recent morphological and physiological studies support the assumption that the extrageniculate ascending tectofugal pathways send visual projection to the caudate nucleus (CN) in amniotes. In the present study we investigate the anatomical connection between the visual associative cortex along the anterior ectosylvian sulcus (AES) and the CN in adult domestic cats. An anterograde tracer - fluoro-dextrane-amine - was injected into the AES cortex. The distribution of labeled axons was not uniform in the CN. The majority of labeled axons and terminal like puncta was found only in a limited area in the dorsal part of the CN between the coordinates anterior 12-15. Furthermore, a retrograde tracer - choleratoxin-B - was injected into the dorsal part of the CN between anterior 12 and 13. We detected a large number of labeled neurons in the fundus and the dorsal part of the AES between the coordinates anterior 12-14. Based upon our recent results we argue that there is a direct monosynaptic connection between the visual associative cortex along the AES and the CN. Beside the posterior thalamus, the AES cortex should also participate in the transmission of the tectal visual information to the CN. This pathway is likely to convey complex information containing both sensory and motor components toward the basal ganglia, which supports their integrative function in visuomotor actions such as motion and novelty detection and saccade generation.

A comprehensive study on the putative δ-opioid receptor (sub)types using the highly selective δ-antagonist, Tyr-Tic-(2S,3R)-ß-MePhe-Phe-OH.

The goal of our work was a throughout characterization of the pharmacology of the TIPP-analog, Tyr-Tic-(2S,3R)-ß-MePhe-Phe-OH and see if putative δ-opioid receptor subtypes can be distinguished. Analgesic latencies were assessed in mouse tail-flick assays after intrathecal administration. In vitro receptor autoradiography, binding and ligand-stimulated [(35)S]GTPγS functional assays were performed in the presence of putative δ(1)-(DPDPE: agonist, BNTX: antagonist), δ(2)-(agonist: deltorphin II, Ile(5,6)-deltorphin II, antagonist: naltriben) and µ-(DAMGO: agonist) opioid ligands. The examined antagonist inhibited the effect of DPDPE by 60%, but did not antagonize δ(2)- and µ-agonist induced analgesia. The radiolabeled form identified binding sites with K(D)=0.18 nM and receptor densities of 102.7 fmol/mg protein in mouse brain membranes. The binding site distribution of the [(3)H]Tyr-Tic-(2S,3R)-ß-MePhe-Phe-OH agreed well with that of [(3)H]Ile(5,6)-deltorphin II as revealed by receptor autoradiography. Tyr-Tic-(2S,3R)-ß-MePhe-Phe-OH displayed 2.49±0.06 and 0.30±0.01 nM potency against DPDPE and deltorphin II in the [(35)S]GTPγS functional assay, respectively. The rank order of potency of putative δ(1)- and δ(2)-antagonists against DPDPE and deltorphin was similar in brain and CHO cells expressing human δ-opioid receptors. Deletion of the DOR-1 gene resulted in no residual binding of the radioligand and no significant DPDPE effect on G-protein activation. Tyr-Tic-(2S,3R)-ß-MePhe-Phe-OH is a highly potent and δ-opioid specific antagonist both in vivo and in vitro. However, the putative δ(1)- and δ(2)-opioid receptors could not be unequivocally distinguished in vitro.