Neuronally Derived Soluble Abeta Evokes Cell-Wide Astrocytic Calcium Dysregulation in Absence of Amyloid Plaques in Vivo.

The key pathologic entities driving the destruction of synaptic function and integrity during the evolution of Alzheimer's disease (AD) remain elusive. Astrocytes are structurally and functionally integrated within synaptic and vascular circuitry and use calcium-based physiology to modulate basal synaptic transmission, vascular dynamics, and neurovascular coupling, which are central to AD pathogenesis. We used high-resolution multiphoton imaging to quantify all endogenous calcium signaling arising spontaneously throughout astrocytic somata, primary processes, fine processes, and capillary endfeet in the brain of awake APP/PS1 transgenic mice (11 male and 6 female mice). Endogenous calcium signaling within capillary endfeet, while surprisingly as active as astrocytic fine processes, was reduced ∼50% in the brain of awake APP/PS1 mice. Cortical astrocytes, in the presence of amyloid plaques in awake APP/PS1 mice, had a cell-wide increase in intracellular calcium associated with an increased frequency, amplitude, and duration of spontaneous calcium signaling. The cell-wide astrocytic calcium dysregulation was not directly related to distance to amyloid plaques. We could re-create the cell-wide intracellular calcium dysregulation in the absence of amyloid plaques following acute exposure to neuronally derived soluble Abeta from Tg2576 transgenic mice, in the living brain of male C57/Bl6 mice. Our findings highlight a role for astrocytic calcium pathophysiology in soluble-Abeta mediated neurodegenerative processes in AD. Additionally, therapeutic strategies aiming to protect astrocytic calcium physiology from soluble Abeta-mediated toxicity may need to pharmacologically enhance calcium signaling within the hypoactive capillary endfeet while reducing the hyperactivity of spontaneous calcium signaling throughout the rest of the astrocyte.SIGNIFICANCE STATEMENT Astrocytic calcium signaling is functionally involved in central pathologic processes of Alzheimer's disease. We quantified endogenous calcium signaling arising spontaneously in the brain of awake APP/PS1 mice, as general anesthesia suppressed astrocytic calcium signaling. Cell-wide astrocytic calcium dysregulation was not related to distance to amyloid plaques but mediated in part by neuronally derived soluble Abeta, supporting a role for astrocytes in soluble-Abeta mediated neurodegeneration. Spontaneous calcium signaling is largely compartmentalized and capillary endfeet were as active as fine processes but hypoactive in the presence of amyloid plaques, while the rest of the astrocyte became hyperactive. The cell-wide calcium pathophysiology in astrocytes may require a combination therapeutic strategy for hypoactive endfeet and astrocytic hyperactivity.

The lipolysis pathway sustains normal and transformed stem cells in adult Drosophila.

Cancer stem cells (CSCs) may be responsible for tumour dormancy, relapse and the eventual death of most cancer patients. In addition, these cells are usually resistant to cytotoxic conditions. However, very little is known about the biology behind this resistance to therapeutics. Here we investigated stem-cell death in the digestive system of adult Drosophila melanogaster. We found that knockdown of the coat protein complex I (COPI)-Arf79F (also known as Arf1) complex selectively killed normal and transformed stem cells through necrosis, by attenuating the lipolysis pathway, but spared differentiated cells. The dying stem cells were engulfed by neighbouring differentiated cells through a draper-myoblast city-Rac1-basket (also known as JNK)-dependent autophagy pathway. Furthermore, Arf1 inhibitors reduced CSCs in human cancer cell lines. Thus, normal or cancer stem cells may rely primarily on lipid reserves for energy, in such a way that blocking lipolysis starves them to death. This finding may lead to new therapies that could help to eliminate CSCs in human cancers.

The PDZ-GEF Gef26 regulates synapse development and function via FasII and Rap1 at the Drosophila neuromuscular junction.

Guanine nucleotide exchange factors (GEFs) are essential for small G proteins to activate their downstream signaling pathways, which are involved in morphogenesis, cell adhesion, and migration. Mutants of Gef26, a PDZ-GEF (PDZ domain-containing guanine nucleotide exchange factor) in Drosophila, exhibit strong defects in wings, eyes, and the reproductive and nervous systems. However, the precise roles of Gef26 in development remain unclear. In the present study, we analyzed the role of Gef26 in synaptic development and function. We identified significant decreases in bouton number and branch length at larval neuromuscular junctions (NMJs) in Gef26 mutants, and these defects were fully rescued by restoring Gef26 expression, indicating that Gef26 plays an important role in NMJ morphogenesis. In addition to the observed defects in NMJ morphology, electrophysiological analyses revealed functional defects at NMJs, and locomotor deficiency appeared in Gef26 mutant larvae. Furthermore, Gef26 regulated NMJ morphogenesis by regulating the level of synaptic Fasciclin II (FasII), a well-studied cell adhesion molecule that functions in NMJ development and remodeling. Finally, our data demonstrate that Gef26-specific small G protein Rap1 worked downstream of Gef26 to regulate the level of FasII at NMJs, possibly through a ßPS integrin-mediated signaling pathway. Taken together, our findings define a novel role of Gef26 in regulating NMJ development and function.

A Novel NIR-FRET Biosensor for Reporting PS/γ-Secretase Activity in Live Cells.

Presenilin (PS)/γ-secretase plays a pivotal role in essential cellular events via proteolytic processing of transmembrane proteins that include APP and Notch receptors. However, how PS/γ-secretase activity is spatiotemporally regulated by other molecular and cellular factors and how the changes in PS/γ-secretase activity influence signaling pathways in live cells are poorly understood. These questions could be addressed by engineering a new tool that enables multiplexed imaging of PS/γ-secretase activity and additional cellular events in real-time. Here, we report the development of a near-infrared (NIR) FRET-based PS/γ-secretase biosensor, C99 720-670 probe, which incorporates an immediate PS/γ-secretase substrate APP C99 with miRFP670 and miRFP720 as the donor and acceptor fluorescent proteins, respectively. Extensive validation demonstrates that the C99 720-670 biosensor enables quantitative monitoring of endogenous PS/γ-secretase activity on a cell-by-cell basis in live cells (720/670 ratio: 2.47 ± 0.66 (vehicle) vs. 3.02 ± 1.17 (DAPT), ** p < 0.01). Importantly, the C99 720-670 and the previously developed APP C99 YPet-Turquoise-GL (C99 Y-T) biosensors simultaneously report PS/γ-secretase activity. This evidences the compatibility of the C99 720-670 biosensor with cyan (CFP)-yellow fluorescent protein (YFP)-based FRET biosensors for reporting other essential cellular events. Multiplexed imaging using the novel NIR biosensor C99 720-670 would open a new avenue to better understand the regulation and consequences of changes in PS/γ-secretase activity.

Disruption of MAGI2-RapGEF2-Rap1 signaling contributes to podocyte dysfunction in congenital nephrotic syndrome caused by mutations in MAGI2.

The essential role of membrane associated guanylate kinase 2 (MAGI2) in podocytes is indicated by the phenotypes of severe glomerulosclerosis of both MAGI2 knockout mice and in patients with congenital nephrotic syndrome (CNS) caused by mutations in MAGI2. Here, we show that MAGI2 forms a complex with the Rap1 guanine nucleotide exchange factor, RapGEF2, and that this complex is lost when expressing MAGI2 CNS variants. Co-expression of RapGEF2 with wild-type MAGI2, but not MAGI2 CNS variants, enhanced activation of the small GTPase Rap1, a central signaling node in podocytes. In mice, podocyte-specific RapGEF2 deletion resulted in spontaneous glomerulosclerosis, with qualitative glomerular features comparable to MAGI2 knockout mice. Knockdown of RapGEF2 or MAGI2 in human podocytes caused similar reductions in levels of Rap1 activation and Rap1-mediated downstream signaling. Furthermore, human podocytes expressing MAGI2 CNS variants show severe abnormalities of cellular morphology and dramatic loss of actin cytoskeletal organization, features completely rescued by pharmacological activation of Rap1 via a non-MAGI2 dependent upstream pathway. Finally, immunostaining of kidney sections from patients with congenital nephrotic syndrome and MAGI2 mutations showed reduced podocyte Rap1-mediated signaling. Thus, MAGI2-RapGEF2-Rap1 signaling is essential for normal podocyte function. Hence, disruption of this pathway is an important cause of the renal phenotype induced by MAGI2 CNS mutations.

Cancer Stem Cells and Stem Cell Tumors in Drosophila.

Accumulative studies suggest that a fraction of cells within a tumor, known as cancer stem cells (CSCs) that initiate tumors, show resistance to most of the therapies, and causes tumor recurrence and metastasis. CSCs could be either transformed normal stem cells or reprogrammed differentiated cells. The eventual goal of CSC research is to identify pathways that selectively regulate CSCs and then target these pathways to eradicate CSCs. CSCs and normal stem cells share some common features, such as self-renewal, the production of differentiated progeny, and the expression of stem-cell markers, however, CSCs vary from normal stem cells in forming tumors. Specifically, CSCs are normally resistant to standard therapies. In addition, CSCs and non-CSCs can be mutually convertible in response to different signals or microenvironments. Even though CSCs are involved in human cancers, the biology of CSCs, is still not well understood, there are urgent needs to study CSCs in model organisms. In the last several years, discoveries in Drosophila have greatly contributed to our understanding of human cancer. Stem-cell tumors in Drosophila share various properties with human CSCs and maybe used to understand the biology of CSCs. In this chapter, we first briefly review CSCs in mammalian systems, then discuss stem-cell tumors in the Drosophila posterior midgut and Malpighian tubules (kidney) and their unique properties as revealed by studying oncogenic Ras protein (RasV12)-transformed stem-cell tumors in the Drosophila kidney and dominant-negative Notch (NDN)-transformed stem-cell tumors in the Drosophila intestine. At the end, we will discuss potential approaches to eliminate CSCs and achieve tumor regression. In future, by screening adult Drosophila neoplastic stem-cell tumor models, we hope to identify novel and efficacious compounds for the treatment of human cancers.

Enteroendocrine cells are generated from stem cells through a distinct progenitor in the adult Drosophila posterior midgut.

Functional mature cells are continually replenished by stem cells to maintain tissue homoeostasis. In the adult Drosophila posterior midgut, both terminally differentiated enterocyte (EC) and enteroendocrine (EE) cells are generated from an intestinal stem cell (ISC). However, it is not clear how the two differentiated cells are generated from the ISC. In this study, we found that only ECs are generated through the Su(H)GBE(+) immature progenitor enteroblasts (EBs), whereas EEs are generated from ISCs through a distinct progenitor pre-EE by a novel lineage-tracing system. EEs can be generated from ISCs in three ways: an ISC becoming an EE, an ISC becoming a new ISC and an EE through asymmetric division, or an ISC becoming two EEs through symmetric division. We further identified that the transcriptional factor Prospero (Pros) regulates ISC commitment to EEs. Our data provide direct evidence that different differentiated cells are generated by different modes of stem cell lineage specification within the same tissues.

Tomographic phosphorescence lifetime multiplexing.

We present a tomographic reconstruction algorithm for recovering distributions of multiple phosphorescent dyes within turbid media from time-resolved measurements, using either point or spatially patterned sources and detectors. The algorithm employs a multi-exponential analysis of time-resolved data, followed by tomographic inversion of the decay amplitudes to recover independent yield distributions for each lifetime present in the medium. Using Monte Carlo simulations, we computationally demonstrate that this two-step inversion approach provides several-fold improvement in quantitative and localization accuracy compared to a direct inversion of the time domain phosphorescence. We also demonstrate the tomographic reconstruction of up to three phosphorescent lifetimes embedded in thick tissue. The proposed algorithm can allow quantitative multiplexed tomography of luminescent and phosphorescent dyes for a wide range of in vivo applications.

The Nuclear Matrix Protein Megator Regulates Stem Cell Asymmetric Division through the Mitotic Checkpoint Complex in Drosophila Testes.

In adult Drosophila testis, asymmetric division of germline stem cells (GSCs) is specified by an oriented spindle and cortically localized adenomatous coli tumor suppressor homolog 2 (Apc2). However, the molecular mechanism underlying these events remains unclear. Here we identified Megator (Mtor), a nuclear matrix protein, which regulates GSC maintenance and asymmetric division through the spindle assembly checkpoint (SAC) complex. Loss of Mtor function results in Apc2 mis-localization, incorrect centrosome orientation, defective mitotic spindle formation, and abnormal chromosome segregation that lead to the eventual GSC loss. Expression of mitotic arrest-deficient-2 (Mad2) and monopolar spindle 1 (Mps1) of the SAC complex effectively rescued the GSC loss phenotype associated with loss of Mtor function. Collectively our results define a new role of the nuclear matrix-SAC axis in regulating stem cell maintenance and asymmetric division.

The Osa-containing SWI/SNF chromatin-remodeling complex regulates stem cell commitment in the adult Drosophila intestine.

The proportion of stem cells versus differentiated progeny is well balanced to maintain tissue homeostasis, which in turn depends on the balance of the different signaling pathways involved in stem cell self-renewal versus lineage-specific differentiation. In a screen for genes that regulate cell lineage determination in the posterior midgut, we identified that the Osa-containing SWI/SNF (Brahma) chromatin-remodeling complex regulates Drosophila midgut homeostasis. Mutations in subunits of the Osa-containing complex result in intestinal stem cell (ISC) expansion as well as enteroendocrine (EE) cell reduction. We further demonstrated that Osa regulates ISC self-renewal and differentiation into enterocytes by elaborating Notch signaling, and ISC commitment to differentiation into EE cells by regulating the expression of Asense, an EE cell fate determinant. Our data uncover a unique mechanism whereby the commitment of stem cells to discrete lineages is coordinately regulated by chromatin-remodeling factors.

Optimal estimator for tomographic fluorescence lifetime multiplexing.

We use the model resolution matrix to analytically derive an optimal Bayesian estimator for multiparameter inverse problems that simultaneously minimizes inter-parameter cross talk and the total reconstruction error. Application of this estimator to time-domain diffuse fluorescence imaging shows that the optimal estimator for lifetime multiplexing is identical to a previously developed asymptotic time-domain (ATD) approach, except for the inclusion of a diagonal regularization term containing decay amplitude uncertainties. We show that, while the optimal estimator and ATD provide zero cross talk, the optimal estimator provides lower reconstruction error, while ATD results in superior relative quantitation. The framework presented here is generally applicable to other multiplexing problems where the simultaneous and accurate relative quantitation of multiple parameters is of interest.

Comparison of tomographic fluorescence spectral and lifetime multiplexing.

Multispectral and lifetime imaging in turbid media can be mathematically described in two steps, involving spectral or temporal mixing of the fluorophores and the diffuse light transport in the turbid medium. We show that the order of fluorophore mixing and diffuse propagation is reversed in spectral and lifetime multiplexing, resulting in a fundamental difference in their multiplexing capabilities, regardless of the measurement conditions. Using the resolution matrix to define a quantitative measure for inter-fluorophore cross-talk, we show that lifetime multiplexing, using the asymptotic time domain approach, provides zero cross-talk, while spectral multiplexing can achieve zero cross-talk under special conditions. We also compare the performance of spectral and lifetime multiplexing for tomographic inversion of two overlapping fluorophores in a heterogeneous digital mouse atlas.

Rapid cell death is preceded by amyloid plaque-mediated oxidative stress.

Neuronal loss is the ultimate outcome in a variety of neurodegenerative diseases and central nerve system disorders. Understanding the sequelae of events that leads to cell death would provide insight into neuroprotective approaches. We imaged neurons in the living brain of a mouse model of Alzheimer's disease that overexpresses mutant human amyloid precursor protein and presenilin 1 and followed the death of individual neurons in real time. This mouse model exhibited limited neurodegeneration and atrophy, but we were able to identify a small fraction of vulnerable cells that would not have been detectable by using standard approaches. By exploiting a genetically encoded reporter of oxidative stress, we identified susceptible neurons by their increased redox potential. The oxidative stress was most dramatic in neurites near plaques, propagated to cell bodies, and preceded activation of caspases that led to cell death within 24 h. Thus, local oxidative stress surrounding plaques contributes to long-range toxicity and selective neuronal death in Alzheimer's disease.

Broad relays hormone signals to regulate stem cell differentiation in Drosophila midgut during metamorphosis.

Like the mammalian intestine, the Drosophila adult midgut is constantly replenished by multipotent intestinal stem cells (ISCs). Although it is well known that adult ISCs arise from adult midgut progenitors (AMPs), relatively little is known about the mechanisms that regulate AMP specification. Here, we demonstrate that Broad (Br)-mediated hormone signaling regulates AMP specification. Br is highly expressed in AMPs temporally during the larva-pupa transition stage, and br loss of function blocks AMP differentiation. Furthermore, Br is required for AMPs to develop into functional ISCs. Conversely, br overexpression drives AMPs toward premature differentiation. In addition, we found that Br and Notch (N) signaling function in parallel pathways to regulate AMP differentiation. Our results reveal a molecular mechanism whereby Br-mediated hormone signaling directly regulates stem cells to generate adult cells during metamorphosis.

Tomographic lifetime imaging using combined early- and late-arriving photons.

We present a novel, hybrid approach for time domain fluorescence tomography that efficiently combines lifetime multiplexing using late-arriving or asymptotic photons, with the high spatial resolution capability of early photon tomography. We also show that a decay amplitude-based asymptotic approach is superior to direct inversion of late-arriving photons for tomographic lifetime imaging within turbid media. The hybrid reconstruction approach is experimentally shown to recover fluorescent inclusions separated as close as 1.4 mm, with improved resolution and reduced cross talk compared to just using early photons or the asymptotic approach alone.

Long noncoding RNAs heat shock RNA omega nucleates TBPH and promotes intestinal stem cell differentiation upon heat shock.

In Drosophila, long noncoding RNA Hsrω rapidly assembles membraneless organelle omega speckles under heat shock with unknown biological function. Here, we identified the distribution of omega speckles in multiple tissues of adult Drosophila melanogaster and found that they were selectively distributed in differentiated enterocytes but not in the intestinal stem cells of the midgut. We mimicked the high expression level of Hsrω via overexpression or intense heat shock and demonstrated that the assembly of omega speckles nucleates TBPH for the induction of ISC differentiation. Additionally, we found that heat shock stress promoted cell differentiation, which is conserved in mammalian cells through paraspeckles, resulting in large puncta of TDP-43 (a homolog of TBPH) with less mobility and the differentiation of human induced pluripotent stem cells. Overall, our findings confirm the role of Hsrω and omega speckles in the development of intestinal cells and provide new prospects for the establishment of stem cell differentiation strategies.

Recording γ-secretase activity in living mouse brains.

γ-Secretase plays a pivotal role in the central nervous system. Our recent development of genetically encoded Förster resonance energy transfer (FRET)-based biosensors has enabled the spatiotemporal recording of γ-secretase activity on a cell-by-cell basis in live neurons in culture . Nevertheless, how γ-secretase activity is regulated in vivo remains unclear. Here we employ the near-infrared (NIR) C99 720-670 biosensor and NIR confocal microscopy to quantitatively record γ-secretase activity in individual neurons in living mouse brains. Intriguingly, we uncovered that γ-secretase activity may influence the activity of γ-secretase in neighboring neurons, suggesting a potential "cell non-autonomous" regulation of γ-secretase in mouse brains. Given that γ-secretase plays critical roles in important biological events and various diseases, our new assay in vivo would become a new platform that enables dissecting the essential roles of γ-secretase in normal health and diseases.

Real-time imaging of mitochondrial redox reveals increased mitochondrial oxidative stress associated with amyloid ß aggregates in vivo in a mouse model of Alzheimer's disease.

BACKGROUND: Reactive oxidative stress is a critical player in the amyloid beta (Aß) toxicity that contributes to neurodegeneration in Alzheimer's disease (AD). Damaged mitochondria are one of the main sources of reactive oxygen species and accumulate in Aß plaque-associated dystrophic neurites in the AD brain. Although Aß causes neuronal mitochondria reactive oxidative stress in vitro, this has never been directly observed in vivo in the living mouse brain. Here, we tested for the first time whether Aß plaques and soluble Aß oligomers induce mitochondrial oxidative stress in surrounding neurons in vivo, and whether this neurotoxic effect can be abrogated using mitochondrial-targeted antioxidants. METHODS: We expressed a genetically encoded fluorescent ratiometric mitochondria-targeted reporter of oxidative stress in mouse models of the disease and performed intravital multiphoton microscopy of neuronal mitochondria and Aß plaques. RESULTS: For the first time, we demonstrated by direct observation in the living mouse brain exacerbated mitochondrial oxidative stress in neurons after both Aß plaque deposition and direct application of soluble oligomeric Aß onto the brain, and determined the most likely pathological sequence of events leading to oxidative stress in vivo. Oxidative stress could be inhibited by both blocking calcium influx into mitochondria and treating with the mitochondria-targeted antioxidant SS31. Remarkably, the latter ameliorated plaque-associated dystrophic neurites without impacting Aß plaque burden. CONCLUSIONS: Considering these results, combination of mitochondria-targeted compounds with other anti-amyloid beta or anti-tau therapies hold promise as neuroprotective drugs for the prevention and/or treatment of AD.

Interstitial fluid drainage is impaired in ischemic stroke and Alzheimer's disease mouse models.

The interstitial fluid (ISF) drainage pathway has been hypothesized to underlie the clearance of solutes and metabolites from the brain. Previous work has implicated the perivascular spaces along arteries as the likely route for ISF clearance; however, it has never been demonstrated directly. The accumulation of amyloid ß (Aß) peptides in brain parenchyma is one of the pathological hallmarks of Alzheimer disease (AD), and it is likely related to an imbalance between production and clearance of the peptide. Aß drainage along perivascular spaces has been postulated to be one of the mechanisms that mediate the peptide clearance from the brain. We therefore devised a novel method to visualize solute clearance in real time in the living mouse brain using laser guided bolus dye injections and multiphoton imaging. This methodology allows high spatial and temporal resolution and revealed the kinetics of ISF clearance. We found that the ISF drains along perivascular spaces of arteries and capillaries but not veins, and its clearance exhibits a bi-exponential profile. ISF drainage requires a functional vasculature, as solute clearance decreased when perfusion was impaired. In addition, reduced solute clearance was observed in transgenic mice with significant vascular amyloid deposition; we suggest the existence of a feed-forward mechanism, by which amyloid deposition promotes further amyloid deposition. This important finding provides a mechanistic link between cerebrovascular disease and Alzheimer disease and suggests that facilitation of Aß clearance along the perivascular pathway should be considered as a new target for therapeutic approaches to Alzheimer disease and cerebral amyloid angiopathy.