Specific Akt Family Members Impair Stress-Mediated Transactivation of Viral Promoters and Enhance Neuronal Differentiation: Important Functions for Maintaining Latency.

Neurotropic Alphaherpesvirinae subfamily members such as bovine herpesvirus 1 (BoHV-1) and herpes simplex virus 1 (HSV-1) establish and maintain lifelong latent infections in neurons. Following infection of ocular, oral, or nasal cavities, sensory neurons within trigeminal ganglia (TG) are an important site for latency. Certain external stressors can trigger reactivation from latency, in part because activation of the glucocorticoid receptor (GR) stimulates productive infection and promoters that drive expression of key viral transcriptional regulators. The Akt serine/threonine protein kinase family is linked to maintaining latency. For example, Akt3 is detected in more TG neurons during BoHV-1 latency than in reactivation and uninfected calves. Furthermore, Akt signaling correlates with maintaining HSV-1 latency in certain neuronal models of latency. Finally, an active Akt protein kinase is crucial for the ability of the HSV-1 latency-associated transcript (LAT) to inhibit apoptosis in neuronal cell lines. Consequently, we hypothesized that viral and/or cellular factors impair stress-induced transcription and reduce the incidence of reactivation triggered by low levels of stress. New studies demonstrate that Akt1 and Akt2, but not Akt3, significantly reduced GR-mediated transactivation of the BoHV-1 immediate early transcription unit 1 (IEtu1) promoter, the HSV-1 infected cell protein 0 (ICP0) promoter, and the mouse mammary tumor virus long terminal repeat (MMTV-LTR). Akt3, but not Akt1 or Akt2, significantly enhanced neurite formation in mouse neuroblastoma cells, which correlates with repairing damaged neurons. These studies suggest that unique biological properties of the three Akt family members promote the maintenance of latency in differentiated neurons.IMPORTANCE External stressful stimuli are known to increase the incidence of reactivation of Alphaherpesvirinae subfamily members. Activation of the glucocorticoid receptor (GR) by the synthetic corticosteroid dexamethasone (DEX) stimulates bovine herpesvirus 1 (BoHV-1) and herpes simplex virus 1 (HSV-1) reactivation. Furthermore, GR and dexamethasone stimulate productive infection and promoters that drive expression of viral transcriptional regulators. These observations lead us to predict that stress-induced transcription is impaired by factors abundantly expressed during latency. Interestingly, activation of the Akt family of serine/threonine protein kinases is linked to maintenance of latency. New studies reveal that Akt1 and Ak2, but not Akt3, impaired GR- and dexamethasone-mediated transactivation of the BoHV-1 immediate early transcription unit 1 and HSV-1 ICP0 promoters. Strikingly, Akt3, but not Akt1 or Akt2, stimulated neurite formation in mouse neuroblastoma cells, a requirement for neurogenesis. These studies provide insight into how Akt family members may promote the maintenance of lifelong latency.

Impact of TREM2 risk variants on brain region-specific immune activation and plaque microenvironment in Alzheimer's disease patient brain samples.

Identification of multiple immune-related genetic risk factors for sporadic AD (sAD) have put the immune system center stage in mechanisms underlying this disorder. Comprehensive analysis of microglia in different stages of AD in human brains revealed microglia activation to follow the progression of AD neuropathological changes and requiring the co-occurrence of beta-Amyloid (Aß) and tau pathology. Carriers of AD-associated risk variants in TREM2 (Triggering receptor expressed on myeloid cells 2) showed a reduction of plaque-associated microglia and a substantial increase in dystrophic neurites and overall pathological tau compared with age and disease stage matched AD patients without TREM2 risk variants. These findings were substantiated by digital spatial profiling of the plaque microenvironment and targeted gene expression profiling on the NanoString nCounter system, which revealed striking brain region dependent differences in immune response patterns within individual cases. The demonstration of profound brain region and risk-variant specific differences in immune activation in human AD brains impacts the applicability of immune-therapeutic approaches for sAD and related neurodegenerative diseases.

SARM1, Not MyD88, Mediates TLR7/TLR9-Induced Apoptosis in Neurons.

Neuronal apoptosis is a key aspect of many different neurologic diseases, but the mechanisms remain unresolved. Recent studies have suggested a mechanism of innate immune-induced neuronal apoptosis through the stimulation of endosomal TLRs in neurons. TLRs are stimulated both by pathogen-associated molecular patterns as well as by damage-associated molecular patterns, including microRNAs released by damaged neurons. In the present study, we identified the mechanism responsible for TLR7/TLR9-mediated neuronal apoptosis. TLR-induced apoptosis required endosomal localization of TLRs but was independent of MyD88 signaling. Instead, apoptosis required the TLR adaptor molecule SARM1, which localized to the mitochondria following TLR activation and was associated with mitochondrial accumulation in neurites. Deficiency in SARM1 inhibited both mitochondrial accumulation in neurites and TLR-induced apoptosis. These studies identify a non-MyD88 pathway of TLR7/ TLR9 signaling in neurons and provide a mechanism for how innate immune responses in the CNS directly induce neuronal damage.

Neurons preferentially respond to self-MHC class I allele products regardless of peptide presented.

Studies of mice lacking MHC class I (MHC I)-associated proteins have demonstrated a role for MHC I in neurodevelopment. A central question arising from these observations is whether neuronal recognition of MHC I has specificity for the MHC I allele product and the peptide presented. Using a well-established embryonic retina explant system, we observed that picomolar levels of a recombinant self-MHC I molecule inhibited neurite outgrowth. We then assessed the neurobiological activity of a panel of recombinant soluble MHC Is, consisting of different MHC I heavy chains with a defined self- or nonself-peptide presented, on cultured embryonic retinas from mice with different MHC I haplotypes. We observed that self-MHC I allele products had greater inhibitory neuroactivity than nonself-MHC I molecules, regardless of the nature of the peptide presented, a pattern akin to MHC I recognition by some innate immune system receptors. However, self-MHC I molecules had no effect on retinas from MHC I-deficient mice. These observations suggest that neuronal recognition of MHC I may be coordinated with the inherited MHC I alleles, as occurs in the innate immune system. Consistent with this notion, we show that MHC I and MHC I receptors are coexpressed by precursor cells at the earliest stages of retina development, which could enable such coordination.

Inhibition of cyclin-dependent kinase 5 but not of glycogen synthase kinase 3-ß prevents neurite retraction and tau hyperphosphorylation caused by secretable products of human T-cell leukemia virus type I-infected lymphocytes.

Human T-cell leukemia virus type I (HTLV-I)-associated myelopathy/tropical spastic paraparesis (HAM/TSP) is a neurodegenerative disease characterized by selective loss of axons and myelin in the corticospinal tracts. This central axonopathy may originate from the impairment of anterograde axoplasmic transport. Previous work showed tau hyperphosphorylation at T(181) in cerebrospinal fluid of HAM/TSP patients. Similar hyperphosphorylation occurs in SH-SY5Y cells incubated with supernatant from MT-2 cells (HTLV-I-infected lymphocytes secreting viral proteins, including Tax) that produce neurite shortening. Tau phosphorylation at T(181) is attributable to glycogen synthase kinase 3-ß (GSK3-ß) and cyclin-dependent kinase 5 (CDK5) activation. Here we investigate whether neurite retraction in the SH-SY5Y model associates with concurrent changes in other tau hyperphosphorylable residues. Threonine 181 turned out to be the only tau hyperphosphorylated residue. We also evaluate the role of GSK3-ß and CDK5 in this process by using specific kinase inhibitors (LiCl, TDZD-8, and roscovitine). Changes in both GSK3-ß active and inactive forms were followed by measuring the regulatory phosphorylable sites (S(9) and Y(216) , inactivating and activating phosphorylation, respectively) together with changes in ß-catenin protein levels. Our results showed that LiCl and TDZD-8 were unable to prevent MT-2 supernatant-mediated neurite retraction and also that neither Y(216) nor S(9) phosphorylations were changed in GSK3-ß. Thus, GSK3-ß seems not to play a role in T(181) hyperphosphorylation. On the other hand, the CDK5 involvement in tau phosphorylation was confirmed by both the increase in its enzymatic activity and the absence of MT-2 neurite retraction in the presence of roscovitine or CDK5 siRNA transfection.

Phenolic and Steroidal Metabolites from the Cultivated Edible Inonotus hispidus Mushroom and Their Bioactivities.

A chemical study on the fruiting bodies of cultivated edible mushroom Inonotus hispidus resulted in 14 metabolites including three new hispolon congeners, named inonophenols A-B and one new lanostane triterpenoid, named inonoterpene A. These structures were identified by NMR, high-resolution electrospray ionization mass spectrometry (HRESIMS), and electronic circular dichroism (ECD) data analysis. All metabolites were assessed for neurotrophic, anti-inflammatory, and antioxidative activities. Among them, inonophenols B and C were the most active in promoting PC-12 cell neurite outgrowth at a concentration of 10 µM. The phenolic derivatives reduced NO generation by lipopolysaccharide (LPS)-induced BV-2 microglial cells by suppressing the expression of toll-like receptor-4 (TLR-4) and the nuclear factor-kappa-B (NF-κB) signaling pathway as well as the inflammatory mediators including inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2). Moreover, the phenolics showed antioxidant effects in DPPH scavenging assay with the IC50 values of 9.82-21.43 µM. These findings showed that I. hispidus may be a new source of neurotrophic and protective agents against neurodegenerative disorders.

The occurrence of aging-dependent reticulon 3 immunoreactive dystrophic neurites decreases cognitive function.

Reticulon 3 (RTN3) has been shown to mark a distinct and abundant population of dystrophic neurites named RTN3 immunoreactive dystrophic neurites (RIDNs) in patients' brains of Alzheimer disease (AD). Transgenic mice expressing RTN3 (Tg-RTN3) also spontaneously develop RIDNs. To determine whether RIDNs formed in Tg-RTN3 mice would ever naturally occur in the nontransgenic mouse brain, we targeted our examination to elderly mouse brains on the basis that AD is an age-dependent neurodegenerative disease where the decline in cognitive function becomes progressively increased during the course of the disease. Here, we demonstrate that the distribution of RIDNs is abundant, rather than sporadic, in elderly but not young mouse brains. RIDNs in the elderly brain have two distinct populations: abundantly dispersed RIDNs that can only be marked by RTN3, and less abundantly clustered RIDNs that can be marked by multiple proteins including RTN3, ubiquitin, and phosphorylated neurofilament. The abundance of RIDNs in Tg-RTN3 mice at the age of 3 months resembles that of 24-month-old wild type mice, suggesting that this animal model mimics and accelerates the natural occurrence of RIDNs. Importantly, we demonstrate that preformed RIDNs appear to reduce dendritic spine density and synaptic function. Further analysis from mechanistic studies suggests that elevated levels of RTN3 lead to an imbalance in the axonal transport of RTN3, which results in the accumulation of RTN3 in swollen neurites. Collectively, these results suggest that blocking the formation of RIDNs may be a promising strategy to impede cognitive decline in the elderly and in AD patients.

Neuroprotective and axon growth-promoting effects following inflammatory stimulation on mature retinal ganglion cells in mice depend on ciliary neurotrophic factor and leukemia inhibitory factor.

After optic nerve injury retinal ganglion cells (RGCs) normally fail to regenerate axons in the optic nerve and undergo apoptosis. However, lens injury (LI) or intravitreal application of zymosan switch RGCs into an active regenerative state, enabling these neurons to survive axotomy and to regenerate axons into the injured optic nerve. Several factors have been proposed to mediate the beneficial effects of LI. Here, we investigated the contribution of glial-derived ciliary neurotrophic factor (CNTF) to LI-mediated regeneration and neuroprotection using wild-type and CNTF-deficient mice. In wild-type mice, CNTF expression was strongly upregulated in retinal astrocytes, the JAK/STAT3 pathway was activated in RGCs, and RGCs were transformed into an active regenerative state after LI. Interestingly, retinal LIF expression was correlated with CNTF expression after LI. In CNTF-deficient mice, the neuroprotective and axon growth-promoting effects of LI were significantly reduced compared with wild-type animals, despite an observed compensatory upregulation of LIF expression in CNTF-deficient mice. The positive effects of LI and also zymosan were completely abolished in CNTF/LIF double knock-out mice, whereas LI-induced glial and macrophage activation was not compromised. In culture CNTF and LIF markedly stimulated neurite outgrowth of mature RGCs. These data confirm a key role for CNTF in directly mediating the neuroprotective and axon regenerative effects of inflammatory stimulation in the eye and identify LIF as an additional contributing factor.

Accelerated Nerve Regeneration in Mice by upregulated expression of interleukin (IL) 6 and IL-6 receptor after trauma.

In this study we aimed to examine a role for interleukin 6 (IL-6) and its receptor (IL-6R) in peripheral nerve regeneration in vivo. We first observed that cultured mouse embryonic dorsal root ganglia exhibited dramatic neurite extension by simultaneous addition of IL-6 and soluble IL-6R (sIL-6R), a complex that is known to interact with and activate a signal transducing receptor component, gp130. After injury in the hypoglossal nerve in adult mice by ligation, immunoreactivity to IL-6 was upregulated in Schwann cells at the lesional site as well as in the cell bodies of hypoglossal neurons in the brain stem. In the latter, upregulation of the immunoreactivity to IL-6R was also observed. Regeneration of axotomized hypoglossal nerve in vivo was significantly retarded by the administration of anti-IL-6R antibody. Surprisingly, accelerated regeneration of the axotomized nerve was achieved in transgenic mice constitutively expressing both IL-6 and IL-6R, as compared with nontransgenic controls. These results suggest that the IL-6 signal may play an important role in nerve regeneration after trauma in vivo.

Reduction of aggregated Tau in neuronal processes but not in the cell bodies after Abeta42 immunisation in Alzheimer's disease.

Alzheimer's disease (AD) pathology is characterised by aggregation in the brain of amyloid-beta (Abeta) peptide and hyperphosphorylated tau (phospho-tau), although how these proteins interact in disease pathogenesis is unclear. Abeta immunisation results in removal of Abeta from the brain but cognitive decline continues to progress, possibly due to persistent phospho-tau. We quantified phospho-tau and Abeta42 in the brains of 10 AD patients (iAD) who were actively immunised with Abeta42 (AN1792, Elan Pharmaceuticals) compared with 28 unimmunised AD cases (cAD). The phospho-tau load was lower in the iAD than the cAD group in the cerebral cortex (cAD 1.08% vs. iAD 0.72%, P = 0.048), CA1 hippocampus (cAD 2.26% vs. iAD 1.05%; P = 0.001), subiculum (cAD 1.60% vs. iAD 0.31%; P = 0.001) and entorhinal cortex (cAD 1.10% vs. iAD 0.18%; P < 0.001). Assessment of the localisation within neurons of phospho-tau indicated that the Abeta immunotherapy-associated reduction was confined to neuronal processes, i.e. neuropil threads and dystrophic neurites. However, the phospho-tau accumulation in the neuronal cell bodies, contributing to neurofibrillary tangles, appeared not to be affected. In showing that Abeta immunisation can influence phospho-tau pathology, we confirm the position of Abeta as a target for modifying tau accumulation in AD and demonstrate a link between these proteins. However, the continuing progression of cognitive decline in AD patients after Abeta immunisation may be explained by its lack of apparent effect on tangles.

Collapsin Response Mediator Proteins: Novel Targets for Alzheimer's Disease.

Numerous experimental and postmortem studies have increasingly reported dystrophic axons and dendrites, and alterations of dendritic spine morphology and density in the hippocampus as prominent changes in the early stages of Alzheimer's disease (AD). Furthermore, these alterations tend to correlate well with the progressive cognitive decline observed in AD. For these reasons, and because these neurite structures have a capacity to re-grow, re-establish lost connections, and are critical for learning and memory, there is compelling evidence to suggest that therapeutic interventions aimed at preventing their degradation or promoting their regrowth may hold tremendous promise in preventing the progression of AD. In this regard, collapsin response mediator proteins (CRMPs), a family of phosphoproteins playing a major role in axon guidance and dendritic growth, are especially interesting. The roles these proteins play in neurons and immune cells are reviewed here.

Chicken acidic leucine-rich EGF-like domain containing brain protein (CALEB), a neural member of the EGF family of differentiation factors, is implicated in neurite formation.

Chicken acidic leucine-rich EGF-like domain containing brain protein (CALEB) was identified by combining binding assays with immunological screens in the chicken nervous system as a novel member of the EGF family of differentiation factors. cDNA cloning indicates that CALEB is a multidomain protein that consists of an NH2-terminal glycosylation region, a leucine-proline-rich segment, an acidic box, a single EGF-like domain, a transmembrane, and a short cytoplasmic stretch. In the developing nervous system, CALEB is associated with glial and neuronal surfaces. CALEB is composed of a 140/130-kD doublet, an 80-kD band, and a chondroitinsulfate-containing 200-kD component. The latter two components are expressed in the embryonic nervous system and are downregulated in the adult nervous system. CALEB binds to the extracellular matrix glycoproteins tenascin-C and -R. In vitro antibody perturbation experiments reveal a participation of CALEB in neurite formation in a permissive environment.

BDNF, NT-3 and Trk receptor agonist monoclonal antibodies promote neuron survival, neurite extension, and synapse restoration in rat cochlea ex vivo models relevant for hidden hearing loss.

Neurotrophins and their mimetics are potential treatments for hearing disorders because of their trophic effects on spiral ganglion neurons (SGNs) whose connections to hair cells may be compromised in many forms of hearing loss. Studies in noise or ototoxin-exposed animals have shown that local delivery of NT-3 or BDNF has beneficial effects on SGNs and hearing. We evaluated several TrkB or TrkC monoclonal antibody agonists and small molecules, along with BDNF and NT-3, in rat cochlea ex vivo models. The TrkB agonists BDNF and a monoclonal antibody, M3, had the greatest effects on SGN survival, neurite outgrowth and branching. In organotypic cochlear explants, BDNF and M3 enhanced synapse formation between SGNs and inner hair cells and restored these connections after excitotoxin-induced synaptopathy. Loss of these synapses has recently been implicated in hidden hearing loss, a condition characterized by difficulty hearing speech in the presence of background noise. The unique profile of M3 revealed here warrants further investigation, and the broad activity profile of BDNF observed underpins its continued development as a hearing loss therapeutic.

Anti-Abeta antibody treatment promotes the rapid recovery of amyloid-associated neuritic dystrophy in PDAPP transgenic mice.

Neuritic plaques are a defining feature of Alzheimer disease (AD) pathology. These structures are composed of extracellular accumulations of amyloid-beta peptide (Abeta) and other plaque-associated proteins, surrounded by large, swollen axons and dendrites (dystrophic neurites) and activated glia. Dystrophic neurites are thought to disrupt neuronal function, but whether this damage is static, dynamic, or reversible is unknown. To address this, we monitored neuritic plaques in the brains of living PDAPP;Thy-1:YFP transgenic mice, a model that develops AD-like pathology and also stably expresses yellow fluorescent protein (YFP) in a subset of neurons in the brain. Using multiphoton microscopy, we observed and monitored amyloid through cranial windows in PDAPP;Thy-1:YFP double-transgenic mice using the in vivo amyloid-imaging fluorophore methoxy-X04, and individual YFP-labeled dystrophic neurites by their inherent fluorescence. In vivo studies using this system suggest that amyloid-associated dystrophic neurites are relatively stable structures in PDAPP;Thy-1:YFP transgenic mice over several days. However, a significant reduction in the number and size of dystrophic neurites was seen 3 days after Abeta deposits were cleared by anti-Abeta antibody treatment. This analysis suggests that ongoing axonal and dendritic damage is secondary to Abeta and is, in part, rapidly reversible.

CD40 ligation mediates plaque-associated tau phosphorylation in beta-amyloid overproducing mice.

Neuritic dystrophy with amyloid burden and neurofibrillary tangles are pathological hallmarks of Alzheimer's disease. Genetic disruption of CD40 or CD40L alleviates amyloid burden, astrocytosis, and microgliosis in transgenic animal models of Alzheimer's disease. It has been reported that phosphorylated tau-positive dystrophic neurites are observed in transgenic mice over-expressing human mutant beta-amyloid precursor protein (Tg2576). Here, we studied the pattern of phosphorylated tau (labeled with AT8, CP13, PG5, and PHF1 antibodies) and plaques using immunohistochemical techniques. Phosphorylated tau-positive dystrophic neurites were exclusively associated with Congo red-positive plaques as previously reported. Further, we show that CD40L or CD40 deficiency reduces the mean ratio of dystrophic neurite area to congophilic plaque area and the level of expression of cdk5 and p35/p25 in mice. In addition, we show that in a human neuroblastoma cell line treated with CD40L, cdk5 and p35/p25 are increased. Together, our data suggest that CD40-CD40L interaction has an effect on tau phosphorylation independent of beta-amyloid pathology, and that this effect may occur through a decrease of cdk5 and p35/p25.

Meningeal B-cell follicles in secondary progressive multiple sclerosis associate with early onset of disease and severe cortical pathology.

Intrathecal antibody production is a hallmark of multiple sclerosis and humoral immunity is thought to play an important role in the inflammatory response and development of demyelinated lesions. The presence of lymphoid follicle-like structures in the cerebral meninges of some multiple sclerosis patients indicates that B-cell maturation can be sustained locally within the CNS and contribute to the establishment of a compartmentalized humoral immune response. In this study we examined the distribution of ectopic B-cell follicles in multiple sclerosis cases with primary and secondary progressive clinical courses to determine their association with clinical and neuropathological features. A detailed immunohistochemical and morphometric analysis was performed on post-mortem brain tissue samples from 29 secondary progressive (SP) and 7 primary progressive (PP) multiple sclerosis cases. B-cell follicles were detected in the meninges entering the cerebral sulci of 41.4% of the SPMS cases, but not in PPMS cases. The SPMS cases with follicles significantly differed from those without with respect to a younger age at multiple sclerosis onset, irreversible disability and death and more pronounced demyelination, microglia activation and loss of neurites in the cerebral cortex. Cortical demyelination in these SPMS cases was also more severe than in PPMS cases. Notably, all meningeal B-cell follicles were found adjacent to large subpial cortical lesions, suggesting that soluble factors diffusing from these structures have a pathogenic role. These data support an immunopathogenetic mechanism whereby B-cell follicles developing in the multiple sclerosis meninges exacerbate the detrimental effects of humoral immunity with a subsequent major impact on the integrity of the cortical structures.

Neural architecture in lymphoid organs: Hard-wired antigen presenting cells and neurite networks in antigen entrance areas.

INTRODUCTION: Recently, we found abundant innervation of antigen presenting cells that were reached and enclosed by single neurites. These neurally hard-wired antigen presenting cells (wAPC) could be observed in the T-cell zone of superficial cervical lymph nodes of rats and other mammalians, including humans. METHODS: As a consequence, we investigated lymph nodes at many different anatomical positions as well as all primary and secondary lymphoid organs (SLO) in rodents for a similar morphology of innervation regarding antigen presenting cells known in those tissues. RESULTS: As a result, we confirmed wAPC in lymph nodes independent from their draining areas and anatomical positions but also in all other T-cell zones of lymphoid organs, like Peyer's patches, NALT and BALT, as well as in the thymic medulla. Other cells were innervated in a similar fashion but with seemingly missing antigen presenting capacity. Both types of innervated immune cells were observed as being also present in the dermis of the skin. Only in the spleen wAPC could not be detected. Beyond this systematic finding, we also found another regular phenomenon: a dense network of neurites that stained for neurofilament always in antigen entrance areas of lymphoid organs (subsinoidal layer of lymph nodes, subepithelial dome of Peyer's patches, subsinoidal layer of the splenic white pulp, margins of NALT and BALT). Lastly, also thymic epithelial cells (TEC) restricted to the corticomedullary junction of the thymus showed similar neurofilament staining. CONCLUSIONS: Therefore, we propose much more hard-wired and probably afferent connections between lymphoid organs and the central nervous system than is hitherto known.

Galanin plays a role in the conditioning lesion effect in sensory neurons.

Sensory neurons show enhanced neurite outgrowth in vivo and in vitro following a conditioning lesion. Previous studies have shown that these effects are dependent on two members of the gp130 family of cytokines, leukemia inhibitory factor and interleukin-6. Here, we asked whether galanin, a neuropeptide induced by these cytokines, plays a role in the conditioning lesion response. Following a conditioning lesion, neurite outgrowth in culture was reduced in sensory neurons from galanin -/- mice compared with those from wild type controls. In neurons from wild type mice, the length of the longest neurite was increased 2.4-fold after a conditioning lesion, compared with 1.8-fold in neurons from knockout animals. The results indicate that the induction of galanin plays an important role in triggering the conditioning lesion response.

Amyloid-beta antibody treatment leads to rapid normalization of plaque-induced neuritic alterations.

The accumulation of amyloid-beta into insoluble plaques is a characteristic feature of Alzheimer's disease. Neuronal morphology is distorted by plaques: rather than being essentially straight, they are substantially more curved than those in control tissue, their trajectories become altered, and they are frequently distended or swollen, presumably affecting synaptic transmission. Clearance of plaques by administration of antibodies to amyloid-beta is a promising therapeutic approach to the treatment of Alzheimer's disease, leading to stabilization of dementia by an unknown cellular mechanism. The effect of plaque clearance on plaque-induced neuronal alterations has not been studied previously. Here we show that both plaques and neuritic lesions are reversible in a strikingly short period of time after administration of a single dose of amyloid-beta antibody. Amyloid clearance and recovery of normal neuronal geometries were observed as early as 4 d and lasted at least 32 d after a single treatment. These results demonstrate that, once plaques are cleared, neuronal morphology is self-correcting and that passive antibody treatment has the potential to reverse neuronal damage caused by Alzheimer's disease and, hence, directly impact cognitive decline. Moreover, the rapid normalization of neuritic dystrophy suggests an unexpected degree of plasticity in the adult nervous system.

[Confocal laser scanning microscopy to study molecular mechanism of mast cell activation].

In the immune system, mast cells are a key cell type in the pathogenesis of immunoglobulin E (IgE)-dependent hypersensitivity reactions. Engagement of the high-affinity IgE receptors by multivalent antigens initiates the downstream activation of signal-transducing enzymes and evokes degranulation and cytokine production via an increase in the intracellular Ca2+ concentration. In addition, mast cells also play a prominent role in non-IgE-mediated hypersensitivity reactions. Mast cells are closely apposed to nerves in vivo and are likely to be regulated functionally by nerves. However, the molecular mechanisms for mast cell activation in an IgE-dependent and -independent manner have not been fully clarified. Confocal laser scanning microscopy has played an essential role in cell biology by allowing visualization of specific intracellular signaling molecules with high spatiotemporal resolution in living cells. We have studied intracellular movements of Ca2+ using a specific fluorescent probe and several types of signaling molecules using derivatives of green fluorescent protein in a living single mast cell using a microscopic strategy. We here describe our imaging analysis of the calcium signals to the nucleus, the movement of secretory granules in the degranulation process, and the nucleocytoplasmic shuttling of mitogen-activated protein kinase in mast cells. Further, we demonstrate that direct communication between mast cells and nerves occurs. These findings provide useful information from a new perspective to understand the molecular mechanisms of allergic reaction and inflammation.