Morphometric imaging biomarker identifies Alzheimer's disease even among mixed dementia patients.

A definitive diagnosis of Alzheimer's disease (AD), even in the presence of co-morbid neuropathology (occurring in > 50% of AD cases), is a significant unmet medical need that has obstructed the discovery of effective AD therapeutics. An AD-biomarker, the Morphometric Imaging (MI) assay on cultured skin fibroblasts, was used in a double-blind, allcomers (ages 55-90) trial of 3 patient cohorts: AD dementia patients, N = 25, all autopsy confirmed, non-AD dementia patients, N = 21-all autopsy or genetically confirmed; and non-demented control (AHC) patients N = 27. Fibroblasts cells isolated from 3-mm skin punch biopsies were cultured on a 3-D Matrigel matrix with movement dynamics quantified by image analysis. From counts of all aggregates (N) in a pre-defined field image and measures of the average area (A) of aggregates per image, the number-to-area ratios in a natural logarithmic form Ln(A/N) were determined for all patient samples. AD cell lines formed fewer large aggregates (cells clustered together) than non-AD or AHC cell lines. The cut-off value of Ln(A/N) = 6.98 was determined from the biomarker values of non-demented apparently healthy control (AHC) cases. Unequivocal validation by autopsy, genetics, and/or dementia criteria was possible for all 73 patient samples. The samples were collected from multiple centers-four US centers and one center in Japan. The study found no effect of center-to-center variation in fibroblast isolation, cell growth, or cell aggregation values (Ln(A/N)). The autopsy-confirmed MI Biomarker distinguished AD from non-AD dementia (non-ADD) patients and correctly diagnosed AD even in the presence of other co-morbid pathologies at autopsy (True Positive = 25, False Negative = 0, False Positive = 0, True Negative = 21, and Accuracy = 100%. Sensitivity and specificity were calculated as 100% (95% CI = 84 to 100.00%). From these findings, the MI assay appears to detect AD with great accuracy-even with abundant co-morbidity.

Cytokines and Cytokine Receptors Involved in the Pathogenesis of Alzheimer's Disease.

Inflammatory mechanisms are implicated in the pathology of Alzheimer's disease (AD). However, it is unclear whether inflammatory alterations are a cause or consequence of neurodegeneration leading to dementia. Clarifying this issue would provide valuable insight into the early diagnosis and therapeutic management of AD. To address this, we compared the mRNA expression profiles of cytokines in the brains of AD patients with "non-demented individuals with AD pathology" and non-demented healthy control (ND) individuals. "Non-demented individuals with AD pathology" are referred to as high pathology control (HPC) individuals that are considered an intermediate subset between AD and ND. HPC represents a transition between normal aging and early stage of AD, and therefore, is useful for determining whether neuroinflammation is a cause or consequence of AD pathology. We observed that immunological conditions that produce cytokines in the HPC brain were more representative of ND than AD. To validate these result, we investigated the expression of inflammatory mediators at the protein level in postmortem brain tissues. We examined the protein expression of tumor necrosis factor (TNF)α and its receptors (TNFRs) in the brains of AD, HPC, and ND individuals. We found differences in soluble TNFα and TNFRs expression between AD and ND groups and between AD and HPC groups. Expression in the temporal cortex was lower in the AD brains than HPC and ND. Our findings indicate that alterations in immunological conditions involving TNFR-mediated signaling are not the primary events initiating AD pathology, such as amyloid plaques and tangle formation. These may be early events occurring along with synaptic and neuronal changes or later events caused by these changes. In this review, we emphasize that elucidating the temporal expression of TNFα signaling molecules during AD is important to understand the selective tuning of these pathways required to develop effective therapeutic strategies for AD.

Acute Motor-dominant Polyneuropathy as Guillain-Barré Syndrome and Multiple Mononeuropathies in a Patient with Sjögren's Syndrome.

A patient with xerostomia and xerophthalmia due to Sjögren's syndrome presented with acute motor-dominant polyneuropathy and multiple mononeuropathy with antiganglioside antibodies. Nerve conduction studies and a sural nerve biopsy revealed the neuropathy as a mixture of segmental demyelination and axonal degeneration. Positive results were obtained for several antiganglioside antibodies. Corticosteroid treatment proved effective. The neuropathy was considered to represent a mixture of polyneuropathy as Guillain-Barré syndrome and multiple mononeuropathy via Sjögren's syndrome. We speculate that Guillain-Barré syndrome occurred in the patient and Guillain-Barré syndrome itself activated multiple mononeuropathy via Sjögren's syndrome.

Alpha1-chimaerin, a Rac1 GTPase-activating protein, is expressed at reduced mRNA levels in the brain of Alzheimer's disease patients.

Alpha1-chimaerin is a GTPase-activating protein (GAP) for Rac1, a member of the Rho small GTPase family, whose action leads to the inactivation of Rac1. Rac1 activity is upregulated in Alzheimer's disease, but little is known about the role of α1-chimaerin. In this study, we investigated the expression and localization of α1-chimaerin mRNA in postmortem human brains from patients with Alzheimer's disease and control subjects. In situ hybridization studies demonstrated that α1-chimaerin was expressed by neurons in the neo-cortex of the temporal lobe and the hippocampus of both controls and Alzheimer's disease cases, with the signal intensity dramatically decreased in patients with Alzheimer's disease. Real-time PCR analysis confirmed a significant reduction of α1-chimaerin mRNA expression in the temporal cortex of Alzheimer's disease cases. In contrast, α2-chimaerin mRNA levels showed no significant difference between the groups. The present study showed reduced α1-chimaerin expression in the brain of Alzheimer's disease cases, suggesting a role in the upregulation of Rac1 activity during the disease process.

Deficiency of GDNF Receptor GFRα1 in Alzheimer's Neurons Results in Neuronal Death.

We have recently developed aged cortical neuron cultures from autopsied human brains with Alzheimer's disease (AD). During the culturing process, we found that glutamatergic cortical neurons from the AD brain lacked a response to glial cell line-derived neurotrophic factor (GDNF), including no axonal regrowth, and were starting to undergo apoptosis. Here we showed that, in cortical neurons from age- and gender-matched cognitively normal control (NC) subjects (NC neurons), GDNF enhanced the expression of GDNF family receptor subtype α1 (GFRα1), but not the other three subtypes (GFRα2, GFRα3, and GFRα4), whereas GDNF failed to induce GFRα1 expression in cortical neurons from the AD brain (AD neurons). The exogenous introduction of GFRα1, but not of its binding partner α1-neural cell adhesion molecule, or RET into AD neurons restored the effect of GDNF on neuronal survival. Moreover, between NC and AD neurons, the AMPA receptor blocker CNQX and the NMDA receptor blocker AP-5 had opposite effects on the GFRα1 expression induced by GDNF. In NC neurons, the presence of glutamate receptors was necessary for GDNF-linked GFRα1 expression, while in AD neurons the absence of glutamate receptors was required for GFRα1 expression by GDNF stimulation. These results suggest that, in AD neurons, specific impairments of GFRα1, which may be linked to glutamatergic neurotransmission, shed light on developing potential therapeutic strategies for AD by upregulation of GFRα1 expression.

Effluent syntaxin3 from dying cells affords protection against apoptosis in epidermal keratinocytes.

Ultra-violet B (UVB)-induced oxidative stress crucially perturbs the epidermal homeostasis, and the skin is endowed with protective mechanisms to take action against such damage. Here, we show the possible involvement of t-SNARE protein syntaxin3, a membrane fusion mediator of cytoplasmic vesicles, and which is released from dying keratinocytes, to play a role in this response. UVB irradiation, which generates reactive oxidative stress in cells, was shown to lead to the keratinocyte cell death accompanied by a release of cytoplasmic syntaxin3. We found that such extracellularly sourced syntaxin3 completely blocked the processing of a crucial effector for apoptotic cell death, caspase-3, and thus facilitated the survival of keratinocytes damaged by oxidative stress. These results demonstrate the latent prosurvival function of syntaxin3 and underline the importance of intracellular molecular elements for the maintenance of homeostasis in epidermal keratinocytes.

Reduction of ß-amyloid accumulation by reticulon 3 in transgenic mice.

Inhibition of the ß-secretase, BACE1, which cleaves amyloid precursor protein (APP) to produce ß-amyloid protein (Aß), is thought to be a feasible therapeutic strategy for Alzheimer's disease. Reticulon (RTN) proteins such as RTN3 have been identified as membrane proteins that interact with BACE1 and inhibit its Aß-generating activity. In this study, we investigated whether RTN3 can regulate Aß production in vivo, using transgenic (Tg) mice expressing APP with Swedish and London mutations (APP Tg mice) and those expressing RTN3; the latter mice showed ~1.4-fold higher expression levels of RTN3 protein in the cerebral cortex than non-Tg controls. We analyzed the brains of single APP Tg and double APP/RTN3 Tg mice at the age of approximately 15 months. The levels of secreted APP-ß, a direct BACE1 cleavage product of APP, in Tris-soluble fraction were considerably reduced in the hippocampus and cerebral cortex of APP/RTN3 Tg mice relative to those in APP Tg mice. Immunohistochemical analyses demonstrated that Aß burden and plaques were significantly (by approximately 50%) decreased in both the hippocampus and cerebral cortex of double Tg mice compared to APP Tg mice. Furthermore, the levels of guanidine-soluble Aß40 and Aß42 in these brain regions of APP/RTN3 Tg mice were relatively lower than those in APP Tg mice. These findings indicate that even a small increase in RTN3 expression exerts suppressive effects on amyloidogenic processing of APP and Aß accumulation through modulation of BACE1 activity in vivo, and suggest that induction of RTN3 might be an effective therapeutic strategy against Alzheimer's disease.

Expression and localization of mitochondrial ferritin mRNA in Alzheimer's disease cerebral cortex.

Mitochondrial ferritin (MtF) has been identified as a novel ferritin encoded by an intron-lacking gene with specific mitochondrial localization located on chromosome 5q23.1. MtF has been associated with neurodegenerative disorders such as Friedreich ataxia and restless leg syndrome. However, little information is available about MtF in Alzheimer's disease (AD). In this study, therefore, we investigated the expression and localization of MtF messenger RNA (mRNA) in the cerebral cortex of AD and control cases using real-time polymerase chain reaction (PCR) as well as in situ hybridization histochemistry. We also examined protein expression using western-blot assay. In addition, we used in vitro methods to further explore the effect of oxidative stress and ß-amyloid peptide (Aß) on MtF expression. To do this we examined MtF mRNA and protein expression changes in the human neuroblastoma cell line, IMR-32, after treatment with Aß, H2O2, or both. The neuroprotective effect of MtF on oxidative stress induced by H(2)O(2) was measured by MTT assay. The in situ hybridization studies revealed that MtF mRNA was detected mainly in neurons to a lesser degree in glial cells in the cerebral cortex. The staining intensity and the number of positive cells were increased in the cerebral cortex of AD patients. Real-time PCR and western-blot confirmed that MtF expression levels in the cerebral cortex were significantly higher in AD cases than that in control cases at both the mRNA and the protein level. Cell culture experiments demonstrated that the expression of both MtF mRNA and protein were increased by treatment with H2O2 or a combination of Aß and H2O2, but not with Aß alone. Finally, MtF expression showed a significant neuroprotective effect against H2O2-induced oxidative stress (p<0.05). The present study suggests that MtF is involved in the pathology of AD and may play a neuroprotective role against oxidative stress.

Expression profiles of cytokines in the brains of Alzheimer's disease (AD) patients compared to the brains of non-demented patients with and without increasing AD pathology.

Neuroinflammation is involved in the pathology of Alzheimer's disease (AD). Our major focus was to clarify whether neuroinflammation plays an important role in AD pathogenesis, particularly prior to the manifestation of overt dementia. We analyzed cytokine expression profiles of the brain, with focus on non-demented patients with increasing AD pathology, referred to as high pathology control (HPC) patients, who provide an intermediate subset between AD and normal control subjects, referred to as low pathology control (LPC) patients. With real-time PCR techniques, we found significant differences in interleukin (IL)-1ß, 10, 13, 18, and 33, tumor necrosis factor-α (TNFα) converting enzyme (TACE), and transforming growth factor ß1 (TGFß1) mRNA expression ratios between HPC and AD patients, while no significant differences in the expression ratios of any cytokine tested here were observed between LPC and HPC patients. The cytokine mRNA expression ratios were determined as follows: first, cytokine mRNA levels were normalized to mRNA levels of a housekeeping gene, peptidyl-prolyl isomerase A (PPIA), which showed the most stable expression among ten housekeeping genes tested here; then, the normalized data of cytokine levels in the temporal cortex were divided by those in the cerebellum, which is resistant to AD pathology. Subsequently, the expression ratios of the temporal cortex to cerebellum were compared among LPC, HPC, and AD patient groups. Our results indicate that cytokines are more mobilized and implicated in the later AD stage when a significant cognitive decline occurs and develops than in the developmental course of AD pathology prior to the manifestation of overt dementia.

Infiltration of T lymphocytes and expression of icam-1 in the hippocampus of patients with hippocampal sclerosis.

We and others have previously shown that reactive microglia express the major histocompatibility complex (MHC) class I and class II antigens in the hippocampus of patients suffering from epilepsy. Although the MHC glycoproteins serve as restriction elements for T lymphocytes, there is little information available regarding T lymphocytes in hippocampal sclerosis. In the present study, we investigated T lymphocyte infiltration in human hippocampi in four cases of epilepsy with hippocampal sclerosis, as well as in four control cases without neurological disease. No CD8- or CD4-positive T lymphocytes were seen in hippocampi from the control cases. In contrast, CD8- and CD4-positive T lymphocytes had infiltrated into the hippocampi of patients with hippocampal sclerosis. In addition, expression of intercellular adhesion molecule-1 was diffusely upregulated in the hippocampi with hippocampal sclerosis. These results indicate that T lymphocyte infiltration is involved in the pathology of hippocampal sclerosis.

Expression and localization of lactotransferrin messenger RNA in the cortex of Alzheimer's disease.

We and others have previously reported that lactotransferrin (LF), acting both as an iron-binding protein and inflammatory modulator, is greatly up-regulated in the brain of patients with Alzheimer's disease (AD). However, it remains unknown which type of cells express LF in the brain of AD. In this study, therefore, we investigated the expression and localization of LF messenger RNA (mRNA) in the cerebral cortex of AD and control cases using real-time polymerase chain reaction (PCR) and in situ hybridization histochemistry. Real-time PCR demonstrated that LF mRNA expression in the cortex of AD cases was significantly greater than that in control cases. LF mRNA-positive granules were observed in the cortex by in situ hybridization histochemistry, and the number of positive granules was increased in AD cases compared to controls. The double staining technique of LF mRNA in situ hybridisation and D-related human leukocyte antigen (HLA-DR) immunohistochemistry revealed that positive granules were localized in a subpopulation of HLA-DR-positive reactive microglia. In addition, LF mRNA-positive granules were observed in some cells that were negative for HLA-DR. These cells were also negative for CD4 and CD8 but positive for leukocyte common antigen (CD45RB), suggesting they were monocytes/macrophages. These results indicate that reactive microglia in the cerebral cortex and monocytes/macrophages infiltrating from the circulation might be responsible for synthesizing LF in AD brain.

Multiple cytokines are involved in the early events leading to the Alzheimer's disease pathology.

It is likely that neuroinflammation begins well before detectable cognitive impairment in Alzheimer's disease (AD) occurs. Clarifying the alterations occurring prior to the clinical manifestation of overt AD dementia may provide valuable insight into the early diagnosis and management of AD. Herein, to address the issue that neuroinflammation precedes development of AD pathology, we analyzed cytokine expression profiles of the brain, with focus on non-demented control patients with increasing AD pathology, referred to as high pathology control (HPC) cases, who provide an intermediate subset between AD and normal control cases referred to as low pathology control (LPC) cases. With a semi-quantitative analysis of cytokine mRNA, among 15 cytokines and their related molecules tested, we found the involvement of eight: interleukin-1(IL-1) receptor antagonist (IL-1ra), IL-1 converting enzyme (ICE), IL-2, IL-6, IL-8, tumor necrosis factor (TNF) α, macrophage-colony stimulating factor (M-CSF) and transforming growth factor (TGF) ß1 during the development from LPC to HPC, while decreases in IL-1ra, IL-8, MCP-1 and TNFα, and an increase in TACE were implicated in the later development from HPC to AD. These findings indicate that neuroinflammation precedes the clinical manifestation of overt dementia, rather than being involved at the later stages of AD.

Autopsy case of pure akinesia showing pallidonigro-luysian atrophy.

A 60-year-old man developed levodopa-resistant pure akinesia. The patient gradually became more akinetic without accompanying gaze palsies, nuchal dystonia, or other parkinsonian features such as rigidity or tremor. At the age of 71, he died of bronchopneumonia. Neuropathologically, bilateral marked neuronal loss and gliosis were restrictedly observed in the globus pallidus, substantia nigra and corpus luysii, whereas mild gliosis without neuronal loss was found in the brain stem. With Gallyas-Braak silver stain, numerous argyrophilic fibrous structures partly surrounding glial nuclei were observed in the three major affected regions. With Bodian stain, however, they were rarely recognized. The structures were partly positive for tau protein. Rare neurofibrillary tangles were found in the three areas and brain stem. They were relatively more numerous but still sparse in the hippocampus and the parahippocampus. The present case was diagnosed as having pallidonigro-luysian atrophy based on two characteristic findings: (i) the distribution of lesions showing neuronal loss with gliosis; and (ii) significant presence of tau-positive argyrophilic fibrous structures related to glia but with the absence of neurofibrillary tangles in the major affected regions and the brain stem. As our present case uniquely showed pure akinesia for the whole clinical course, it is noteworthy to report it here with a full neuropathological evaluation. In addition, a moderate number of diffuse plaques positive for beta-amyloid were distributed in the thalamus.

Immunohistochemical distribution of cation-dependent mannose 6-phosphate receptors in the mouse central nervous system: comparison with that of cation-independent mannose 6-phophate receptors.

Most mammalian cells express two types of mannose 6-phosphate (M6P) receptors (MPRs), which are involved in the sorting of lysosomal enzymes within the cells. They are referred to as cation-dependent (CD-) MPR and cation-independent (CI-) MPR/insulin-like growth factor II receptor (IGF-IIR), based on their divalent cation requirements and the ability to bind IGF-II. The complementary actions of these two related but distinct MPRs in the sorting function suggest that they have different immunohistochemical distributions. To address this issue, we investigated the cellular distribution of CD-MPR immunoreactivity in the adult mouse central nervous system (CNS), and compared it with that of CI-MPR/IGF-IIR immunoreactivity, which we had previously investigated. These two immunoreactivities were localized in neurons of the CNS, with more intense labeling in the medial septal nucleus, the nucleus of the Broca's diagonal band, layers IV-VI of the cerebral neocortex, layers II-III of the entorhinal cortex, the habenular nucleus, the median eminence, several nuclei and structures of the brainstem, the Purkinje cell layer of the cerebellum, and in the ventral horn of the spinal cord. Although intense immunoreactivities of both MPRs were observed in the same groups of neurons in the same regions, the spatial differences in immunoreactive intensity for CI-MPR/IGF-IIR were greater, particularly in the telencephalon such as the basal forebrain and cerebral cortex, than those for CD-MPR. These findings suggest that CD-MPR is ubiquitously necessary for the general function of neurons, whereas CI-MPR/IGF-IIR is selectively necessary for certain region- and neurotransmitter-specific functions of neurons.

Tumor necrosis factor death receptor signaling cascade is required for amyloid-beta protein-induced neuron death.

Tumor necrosis factor type I receptor (TNFRI), a death receptor, mediates apoptosis and plays a crucial role in the interaction between the nervous and immune systems. A direct link between death receptor activation and signal cascade-mediated neuron death in brains with neurodegenerative disorders remains inconclusive. Here, we show that amyloid-beta protein (Abeta), a major component of plaques in the Alzheimer's diseased brain, induces neuronal apoptosis through TNFRI by using primary neurons overexpressing TNFRI by viral infection or neurons from TNFRI knock-out mice. This was mediated via alteration of apoptotic protease-activating factor (Apaf-1) expression that in turn induced activation of nuclear factor kappaB (NF-kappaB). Abeta-induced neuronal apoptosis was reduced with lower Apaf-1 expression, and little NF-kappaB activation was found in the neurons with mutated Apaf-1 or a deletion of TNFRI compared with the cells from wild-type (WT) mice. Our studies suggest a novel neuronal response of Abeta, which occurs through a TNF receptor signaling cascade and a caspase-dependent death pathway.

The temporal localization of frame-shift ubiquitin-B and amyloid precursor protein, and complement proteins in the brain of non-demented control patients with increasing Alzheimer's disease pathology.

Transcriptional misreading of dinucleotide repeats that generates deletions in RNA and produces frame-shift proteins with loss of function has been reported in Alzheimer's disease (AD). Here frame-shift ubiquitin-B and amyloid precursor protein were immunochemically shown to exist in the brain of high pathology control (HPC) patients with AD pathology but without prior dementia. These proteins were absent in low pathology control patients with limited AD pathology and no dementia. Since the HPC patients can be regarded as preclinical AD patients, our results suggest the accumulation of these proteins involved in the initial steps of AD pathogenesis. By contrast, complement proteins were detected in the AD patients, whereas only trace amounts were found in the HPC patients, indicating the involvement of complement proteins in the later stage of AD dementia.

Isolation of living neurons from human elderly brains using the immunomagnetic sorting DNA-linker system.

Isolation and culture of mature neurons from affected brain regions during diseased states provide a well-suited in vitro model system to study age-related neurodegeneration under dynamic conditions at cellular levels. We have developed a novel technique to isolate living neurons from rapidly autopsied human elderly brains, and have succeeded in keeping them alive in vitro. Specifically, the parietal cortex blocks were fractionated by density gradients and further enriched for neurons by an immunomagnetic sorting DNA-linker technique. The postmortem interval averaged 2.6 hours. After isolation and purification of neurons using this technology, the cells were maintained in vitro for 2 weeks. Our evaluation revealed that 80% of the isolated cells were neurons and they exhibited neurotransmitter phenotypes (glutamate and gamma-aminobutyric acid) as well as glutamate receptors. Studies on cell viability and calcium influx suggest that these isolated living cortical neurons still retain their typical neuronal functions. Our present study demonstrates that neurons isolated from human elderly brain autopsies can survive in vitro and maintain their functional properties. Our study has opened an opportunity to apply such neurons to dynamic pharmacological studies of neurological disorders at the single-cell level.

Target depletion of distinct tumor necrosis factor receptor subtypes reveals hippocampal neuron death and survival through different signal transduction pathways.

Tumor necrosis factor receptor-I (TNFRI) and TNFRII are two TNFR subtypes in the immune system, but their roles in the brain remain unclear. Here we present a novel interaction between TNFR subtypes and TNF-alpha in the brain. Our studies on target-depleted TNFR in mice show that TNF-alpha has little effect on hippocampal neurons in which TNFRI, containing an "intracellular death domain," is absent (TNFRI -/-), whereas neurons from TNFRII knock-out mice are vulnerable to TNF-alpha even at low doses. Moreover, little nuclear factor-kappaB (NF-kappaB) translocation is induced by TNF-alpha in neurons of TNFRI -/-, whereas NF-kappaB subunit p65 is still translocated from the cytoplasm into the nucleus in neurons from wild-type and TNFRII -/- mice. Furthermore, p38 mitogen-activated protein (MAP) kinase activity is upregulated in neurons from both wild-type and TNFRI -/-, but no alteration of p38 MAP kinase was found in neurons from TNFRII. Results from overexpression of TNF receptors further support the above findings. NT2 neuronal-like cells transiently transfected with TNFRI are very sensitive to TNF-alpha, whereas TNF-alpha is not toxic and even seems to be trophic to the cells with TNFRII overexpression. Last, our radioligand-binding experiments demonstrate that TNF-alpha binds TNFRI with high affinity (K(d) of 0.6 nm), whereas TNFRII shows lower binding affinity (K(d) of 1.14 nm) to TNF-alpha in NT2 transfected cells. Together, these studies reveal novel neuronal responses of TNF-alpha in mediating consequences of TNF receptor activation differently. Subsequent neuronal death or survival may ultimately depend on a particular subtype of TNF receptor that is predominately expressed in neurons of the brain during neural development or with neurological diseases.

Suppressive effects of phosphodiesterase type IV inhibitors on rat cultured microglial cells: comparison with other types of cAMP-elevating agents.

We investigated the effects of inhibitors of cAMP-specific phosphodiesterase type IV (PDE IV) on cultured rat microglial cells. Microglial cells expressed mRNA encoding PDE IV. Rolipram and RO-20-1724, specific inhibitors of PDE IV, elevated the intracellular cAMP level much higher than the other types of PDE inhibitors. cAMP in astrocytes but not in cerebrocortical neurons was similarly increased in response to treatment with PDE IV inhibitors examined. The PDE IV inhibitors, a beta-adrenergic agonist isoproterenol and an adenylyl cyclase stimulant forskolin suppressed the proliferation of microglial cells as revealed by PCNA-immunocytochemical staining. The PDE IV inhibitors suppressed release of TNF alpha and nitric oxide (NO) from lipopolysaccharide-activated microglial cells in pure culture, while they did not affect NO release from microglial cells in neuron-microglia coculture. The PDE IV inhibitors also suppressed superoxide anion production by phorbol ester-treated microglial cells. Isoproterenol and forskolin similarly suppressed the macrophage-like functions of activated microglial cells. However, the PDE IV inhibitors displayed novel effects distinct from those of isoproterenol, forskolin and 8Br-cAMP, regarding expression of mRNAs encoding PDE IV, metallothionein-1 and hemeoxigenase-1. The present data showed that the PDE IV inhibitors can be available to control microglial function and that their effects on glial cells should be taken into account when PDE IV inhibitors are used for treatment of brain diseases, such as multiple sclerosis.