Apolipoprotein E intersects with amyloid-ß within neurons.

Apolipoprotein E4 (ApoE4) is the most important genetic risk factor for Alzheimer's disease (AD). Among the earliest changes in AD is endosomal enlargement in neurons, which was reported as enhanced in ApoE4 carriers. ApoE is thought to be internalized into endosomes of neurons, whereas ß-amyloid (Aß) accumulates within neuronal endosomes early in AD. However, it remains unknown whether ApoE and Aß intersect intracellularly. We show that internalized astrocytic ApoE localizes mostly to lysosomes in neuroblastoma cells and astrocytes, whereas in neurons, it preferentially localizes to endosomes-autophagosomes of neurites. In AD transgenic neurons, astrocyte-derived ApoE intersects intracellularly with amyloid precursor protein/Aß. Moreover, ApoE4 increases the levels of endogenous and internalized Aß42 in neurons. Taken together, we demonstrate differential localization of ApoE in neurons, astrocytes, and neuron-like cells, and show that internalized ApoE intersects with amyloid precursor protein/Aß in neurons, which may be of considerable relevance to AD.

Brain region-specific microglial and astrocytic activation in response to systemic lipopolysaccharides exposure.

Microglia cells are the macrophage population within the central nervous system, which acts as the first line of the immune defense. These cells present a high level of heterogeneity among different brain regions regarding morphology, cell density, transcriptomes, and expression of different inflammatory mediators. This region-specific heterogeneity may lead to different neuroinflammatory responses, influencing the regional involvement in several neurodegenerative diseases. In this study, we aimed to evaluate microglial response in 16 brain regions. We compared different aspects of the microglial response, such as the extension of their morphological changes, sensitivity, and ability to convert an acute inflammatory response to a chronic one. Then, we investigated the synaptic alterations followed by acute and chronic inflammation in substantia nigra. Moreover, we estimated the effect of partial ablation of fractalkine CX3C receptor 1 (CX3CR1) on microglial response. In the end, we briefly investigated astrocytic heterogeneity and activation. To evaluate microglial response in different brain regions and under the same stimulus, we induced a systemic inflammatory reaction through a single intraperitoneal (i.p.) injection of lipopolysaccharides (LPS). We performed our study using C57BL6 and CX3CR1+/GFP mice to investigate microglial response in different regions and the impact of CX3CR1 partial ablation. We conducted a topographic study quantifying microglia alterations in 16 brain regions through immunohistochemical examination and computational image analysis. Assessing Iba1-immunopositive profiles and the density of the microglia cells, we have observed significant differences in region-specific responses of microglia populations in all parameters considered. Our results underline the peculiar microglial inflammation in the substantia nigra pars reticulata (SNpr). Here and in concomitance with the acute inflammatory response, we observed a transient decrease of dopaminergic dendrites and an alteration of the striato-nigral projections. Additionally, we found a significant decrease in microglia response and the absence of chronic inflammation in CX3CR1+/GFP mice compared to the wild-type ones, suggesting the CX3C axis as a possible pharmacological target against neuroinflammation induced by an increase of systemic tumor necrosis factor-alpha (TNFα) or/and LPS. Finally, we investigated astrocytic heterogeneity in this model. We observed different distribution and morphology of GFAP-positive astrocytes, a heterogeneous response under inflammatory conditions, and a decrease in their activation in CX3CR1 partially ablated mice compared with C57BL6 mice. Altogether, our data confirm that microglia and astrocytes heterogeneity lead to a region-specific inflammatory response in presence of a systemic TNFα or/and LPS treatment.

CXCR4 and CD74 together enhance cell survival in response to macrophage migration-inhibitory factor in chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL) is characterized by the accumulation of small, mature CD5+ B lymphocytes in the blood, marrow, and lymphoid organs. Cell survival depends on interaction with the leukemic microenvironment. However, the mechanisms controlling CLL cell survival are still incompletely understood. Macrophage migration-inhibitory factor (MIF), a pro-inflammatory and immunoregulatory chemokine-like cytokine, interacts with CXCR4, a major chemokine receptor, as well as with CD74/invariant chain, a single-pass type II receptor. In this study, we analyzed the roles of CXCR4, CD74, and MIF in CLL. Mononuclear cells from patients with hematological malignancies were analyzed for coexpression of CXCR4 and CD74 by flow cytometry. Strong co- and overexpression of CXCR4 and CD74 were observed on B cells of CLL patients (n = 10). Survival and chemotaxis assays indicated that CXCR4 and CD74 work together to enhance the survival and migration of malignant cells in CLL. Blockade of the receptors, either individually or in combination, promoted cell death and led to an abrogation of MIF-driven migration responses in murine and human CLL cells, suggesting that joint activation of both receptors is crucial for CLL cell survival and mobility. These findings indicate that the MIF/CXCR4/CD74 axis represents a novel therapeutic target in CLL.

Aß/Amyloid Precursor Protein-Induced Hyperexcitability and Dysregulation of Homeostatic Synaptic Plasticity in Neuron Models of Alzheimer's Disease.

Alzheimer's disease (AD) is increasingly seen as a disease of synapses and diverse evidence has implicated the amyloid-ß peptide (Aß) in synapse damage. The molecular and cellular mechanism(s) by which Aß and/or its precursor protein, the amyloid precursor protein (APP) can affect synapses remains unclear. Interestingly, early hyperexcitability has been described in human AD and mouse models of AD, which precedes later hypoactivity. Here we show that neurons in culture with either elevated levels of Aß or with human APP mutated to prevent Aß generation can both induce hyperactivity as detected by elevated calcium transient frequency and amplitude. Since homeostatic synaptic plasticity (HSP) mechanisms normally maintain a setpoint of activity, we examined whether HSP was altered in AD transgenic neurons. Using methods known to induce HSP, we demonstrate that APP protein levels are regulated by chronic modulation of activity and that AD transgenic neurons have an impaired adaptation of calcium transients to global changes in activity. Further, AD transgenic compared to WT neurons failed to adjust the length of their axon initial segments (AIS), an adaptation known to alter excitability. Thus, we show that both APP and Aß influence neuronal activity and that mechanisms of HSP are disrupted in primary neuron models of AD.

Monitoring the interactions between alpha-synuclein and Tau in vitro and in vivo using bimolecular fluorescence complementation.

Parkinson's disease (PD) and Alzheimer's disease (AD) are characterized by pathological accumulation and aggregation of different amyloidogenic proteins, α-synuclein (aSyn) in PD, and amyloid-ß (Aß) and Tau in AD. Strikingly, few PD and AD patients' brains exhibit pure pathology with most cases presenting mixed types of protein deposits in the brain. Bimolecular fluorescence complementation (BiFC) is a technique based on the complementation of two halves of a fluorescent protein, which allows direct visualization of protein-protein interactions. In the present study, we assessed the ability of aSyn and Tau to interact with each other. For in vitro evaluation, HEK293 and human neuroblastoma cells were used, while in vivo studies were performed by AAV6 injection in the substantia nigra pars compacta (SNpc) of mice and rats. We observed that the co-expression of aSyn and Tau led to the emergence of fluorescence, reflecting the interaction of the proteins in cell lines, as well as in mouse and rat SNpc. Thus, our data indicates that aSyn and Tau are able to interact with each other in a biologically relevant context, and that the BiFC assay is an effective tool for studying aSyn-Tau interactions in vitro and in different rodent models in vivo.

RGS14414-Mediated Activation of the 14-3-3ζ in Rodent Perirhinal Cortex Induces Dendritic Arborization, an Increase in Spine Number, Long-Lasting Memory Enhancement, and the Prevention of Memory Deficits.

The remedy of memory deficits has been inadequate, as all potential candidates studied thus far have shown limited to no effects and a search for an effective strategy is ongoing. Here, we show that an expression of RGS14414 in rat perirhinal cortex (PRh) produced long-lasting object recognition memory (ORM) enhancement and that this effect was mediated through the upregulation of 14-3-3ζ, which caused a boost in BDNF protein levels and increase in pyramidal neuron dendritic arborization and dendritic spine number. A knockdown of the 14-3-3ζ gene in rat or the deletion of the BDNF gene in mice caused complete loss in ORM enhancement and increase in BDNF protein levels and neuronal plasticity, indicating that 14-3-3ζ-BDNF pathway-mediated structural plasticity is an essential step in RGS14414-induced memory enhancement. We further observed that RGS14414 treatment was able to prevent deficits in recognition, spatial, and temporal memory, which are types of memory that are particularly affected in patients with memory dysfunctions, in rodent models of aging and Alzheimer's disease. These results suggest that 14-3-3ζ-BDNF pathway might play an important role in the maintenance of the synaptic structures in PRh that support memory functions and that RGS14414-mediated activation of this pathway could serve as a remedy to treat memory deficits.

Astrocytic and Neuronal Apolipoprotein E Isoforms Differentially Affect Neuronal Excitability.

Synaptic changes and neuronal network dysfunction are among the earliest changes in Alzheimer's disease (AD). Apolipoprotein E4 (ApoE4), the major genetic risk factor in AD, has been shown to be present at synapses and to induce hyperexcitability in mouse knock-in brain regions vulnerable to AD. ApoE in the brain is mainly generated by astrocytes, however, neurons can also produce ApoE under stress conditions such as aging. The potential synaptic function(s) of ApoE and whether the cellular source of ApoE might affect neuronal excitability remain poorly understood. Therefore, the aim of this study was to elucidate the synaptic localization and effects on neuronal activity of the two main human ApoE isoforms from different cellular sources in control and AD-like in vitro cultured neuron models. In this study ApoE is seen to localize at or near to synaptic terminals. Additionally, we detected a cellular source-specific effect of ApoE isoforms on neuronal activity measured by live cell Ca2+ imaging. Neuronal activity increases after acute but not long-term administration of ApoE4 astrocyte medium. In contrast, ApoE expressed by neurons appears to induce the highest neuronal firing rate in the presence of ApoE3, rather than ApoE4. Moreover, increased neuronal activity in APP/PS1 AD transgenic compared to wild-type neurons is seen in the absence of astrocytic ApoE and the presence of astrocytic ApoE4, but not ApoE3. In summary, ApoE can target synapses and differentially induce changes in neuronal activity depending on whether ApoE is produced by astrocytes or neurons. Astrocytic ApoE induces the strongest neuronal firing with ApoE4, while the most active and efficient neuronal activity induced by neuronal ApoE is caused by ApoE3. ApoE isoforms also differentially affect neuronal activity in AD transgenic compared to wild-type neurons.

FRET-Based Screening Identifies p38 MAPK and PKC Inhibition as Targets for Prevention of Seeded α-Synuclein Aggregation.

Aggregation of α-synuclein is associated with neurodegeneration and a hallmark pathology in synucleinopathies. These aggregates are thought to function as prion-like particles where the conformation of misfolded α-synuclein determines the traits of the induced pathology, similar to prion diseases. Still, little is known about the molecular targets facilitating the conformation-specific biological effects, but their identification could form the basis for new therapeutic interventions. High-throughput screening of annotated compound libraries could facilitate mechanistic investigation by identifying targets with impact on α-synuclein aggregation. To this end, we developed a FRET-based cellular reporter in HEK293T cells, with sensitivity down to 6.5 nM α-synuclein seeds. Using this model system, we identified GF109203X, SB202190, and SB203580 as inhibitors capable of preventing induction of α-synuclein aggregation via inhibition of p38 MAPK and PKC, respectively. We further investigated the mechanisms underlying the protective effects and found alterations in the endo-lysosomal system to be likely candidates of the protection. We found the changes did not stem from a reduction in uptake but rather alteration of lysosomal abundance and degradative capacity. Our findings highlight the value high-throughput screening brings to the mechanistic investigation of α-synuclein aggregation while simultaneously identifying novel therapeutic compounds.

Amyloid Structural Changes Studied by Infrared Microspectroscopy in Bigenic Cellular Models of Alzheimer's Disease.

Alzheimer's disease affects millions of lives worldwide. This terminal disease is characterized by the formation of amyloid aggregates, so-called amyloid oligomers. These oligomers are composed of ß-sheet structures, which are believed to be neurotoxic. However, the actual secondary structure that contributes most to neurotoxicity remains unknown. This lack of knowledge is due to the challenging nature of characterizing the secondary structure of amyloids in cells. To overcome this and investigate the molecular changes in proteins directly in cells, we used synchrotron-based infrared microspectroscopy, a label-free and non-destructive technique available for in situ molecular imaging, to detect structural changes in proteins and lipids. Specifically, we evaluated the formation of ß-sheet structures in different monogenic and bigenic cellular models of Alzheimer's disease that we generated for this study. We report on the possibility to discern different amyloid signatures directly in cells using infrared microspectroscopy and demonstrate that bigenic (amyloid-ß, α-synuclein) and (amyloid-ß, Tau) neuron-like cells display changes in ß-sheet load. Altogether, our findings support the notion that different molecular mechanisms of amyloid aggregation, as opposed to a common mechanism, are triggered by the specific cellular environment and, therefore, that various mechanisms lead to the development of Alzheimer's disease.