Dr. Eugene Roberts.

This is an obituary for Eugene Roberts (1920-2016), an outstanding neurobiochemist who was the first to report on the discovery of gamma-aminobutyric acid (GABA) in the brain, and whose research focused on the important role of GABA in human health and disease.

Deconvolution of the confounding variations for reverse transcription quantitative real-time polymerase chain reaction by separate analysis of biological replicate data.

Reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) uses threshold cycles (Ct values) for measuring relative gene expression. Ct values are signal-to-noise data composed of target gene expression and multiple sources of confounding variations. Data analysis is to minimize technical noises, evaluate biological variances, and estimate treatment-attributable expression changes of particular genes. However, this function is not sufficiently fulfilled in current analytic methods. An important but unrecognizable problem is that Ct values from all biological replicates and technical repeats are pooled across genes and treatment types. This violates the sample-specific association between target and reference genes, leading to inefficient removal of technical noises. To resolve this problem, here we propose to separate Ct values into replicate-specific data subsets and iteratively analyze expression ratios for individual data subsets. The individual expression ratios, rather than the raw Ct values, are pooled to determine the final expression change. The variances of all biological replicates and technical repeats across all target and reference genes are summed up. Our results from example data demonstrate that this separated method can substantially minimize RT-qPCR variance compared with the traditional methods using pooled Ct profiles. This analytic strategy is more effective in control of technical noises and improves the fidelity of RT-qPCR quantification.

Brain aging and Aß1₋42 neurotoxicity converge via deterioration in autophagy-lysosomal system: a conditional Drosophila model linking Alzheimer's neurodegeneration with aging.

Aging is known to be the most prominent risk factor for Alzheimer's disease (AD); however, the underlying mechanism linking brain aging with AD pathogenesis remains unknown. The expression of human amyloid beta 42 peptide (Aß1₋42), but not Aß1₋40 in Drosophila brain induces an early onset and progressive autophagy-lysosomal neuropathology. Here we show that the natural process of brain aging also accompanies a chronic and late-onset deterioration of neuronal autophagy-lysosomal system. This process is characterized by accumulation of dysfunctional autophagy-lysosomal vesicles, a compromise of these vesicles leading to damage of intracellular membranes and organelles, necrotic-like intraneuronal destruction and neurodegeneration. In addition, conditional activation of neuronal autophagy in young animals is protective while late activation is deleterious for survival. Intriguingly, conditional Aß1₋42 expression limited to young animals exacerbates the aging process to a greater extent than Aß1₋42 expression in old animals. These data suggest that the neuronal autophagy-lysosomal system may shift from a functional and protective state to a pathological and deleterious state either during brain aging or via Aß1₋42 neurotoxicity. A chronic deterioration of the neuronal autophagy-lysosomal system is likely to be a key event in transitioning from normal brain aging to pathological aging leading to Alzheimer's neurodegeneration.

Functional and intracellular localization properties of U6 promoter-expressed siRNAs, shRNAs, and chimeric VA1 shRNAs in mammalian cells.

RNA polymerase III (Pol III) expression systems for short hairpin RNAs (U6 shRNAs or chimeric VA1 shRNAs) or individually expressed sense/antisense small interfering RNA (siRNA) strands have been used to trigger RNA interference (RNAi) in mammalian cells. Here we show that individually expressed siRNA expression constructs produce 21-nucleotide siRNAs that strongly accumulate as duplex siRNAs in the nucleus of human cells, exerting sequence-specific silencing activity similar to cytoplasmic siRNAs derived from U6 or VA1-expressed hairpin precursors. In contrast, 29-mer siRNAs separately expressed as sense/antisense strands fail to elicit RNAi activity, despite accumulation of these RNAs in the nucleus. Our findings delineate different intracellular accumulation patterns for the three expression strategies and suggest the possibility of a nuclear RNAi pathway that requires 21-mer duplexes.

Developmental changes in beta-subunit composition of Na,K-ATPase in the Drosophila eye.

The Drosophila genome contains at least three loci for the Na,K-ATPase beta-subunit; however, only the protein products of nrv1 and nrv2 have been characterized hitherto. Here, we provide evidence that nrv3 also encodes for a functional Na,K-ATPase beta-subunit, as its protein product co-precipitates with the Na,K-ATPase alpha-subunit. Nrv3 expression in adult flies is restricted to the nervous system in which Nrv3 is enriched in selective types of sensory cells. Because Nrv3 expression is especially prominent in the compound eye, we have analyzed the subcellular and developmental distribution of Nrv3 within the visual cells and related this distribution to those of the alpha-subunit and of the beta-subunits Nrv1 and Nrv2. Prospective visual cells express Nrv2 in the third larval instar stage and during the first half of pupal development. During the last third of pupal life, Nrv3 gradually replaces Nrv2 as the Na,K-ATPase beta-subunit in the photoreceptor cells. Adult photoreceptors express Nrv3 as their major beta-subunit; the visual cells R1-R6 co-express Nrv2 at a low level, whereas R7 and R8 co-express Nrv1. Notably, beta-subunits do not co-distribute exactly with the alpha-subunit at some developmental stages, supporting the concept that the alpha-subunit and beta-subunit can exist in the plasma membrane without being engaged in alpha/beta heterodimers. The non-visual cells within the compound eye express almost exclusively Nrv2, which segregates together with the alpha-subunit to septate junctions throughout development.

Focused Ultrasound Stimulates ER Localized Mechanosensitive PANNEXIN-1 to Mediate Intracellular Calcium Release in Invasive Cancer Cells.

Focused ultrasound (FUS) is a rapidly developing stimulus technology with the potential to uncover novel mechanosensory dependent cellular processes. Since it is non-invasive, it holds great promise for future therapeutic applications in patients used either alone or as a complement to boost existing treatments. For example, FUS stimulation causes invasive but not non-invasive cancer cell lines to exhibit marked activation of calcium signaling pathways. Here, we identify the membrane channel PANNEXIN1 (PANX1) as a mediator for activation of calcium signaling in invasive cancer cells. Knockdown of PANX1 decreases calcium signaling in invasive cells, while PANX1 overexpression enhances calcium elevations in non-invasive cancer cells. We demonstrate that FUS may directly stimulate mechanosensory PANX1 localized in endoplasmic reticulum to evoke calcium release from internal stores. This process does not depend on mechanosensory stimulus transduction through an intact cytoskeleton and does not depend on plasma membrane localized PANX1. Plasma membrane localized PANX1, however, plays a different role in mediating the spread of intercellular calcium waves via ATP release. Additionally, we show that FUS stimulation evokes cytokine/chemokine release from invasive cancer cells, suggesting that FUS could be an important new adjuvant treatment to improve cancer immunotherapy.

A Human Embryonic Stem Cell Model of Aß-Dependent Chronic Progressive Neurodegeneration.

We describe the construction and phenotypic analysis of a human embryonic stem cell model of progressive Aß-dependent neurodegeneration (ND) with potential relevance to Alzheimer's disease (AD). We modified one allele of the normal APP locus to directly express a secretory form of Aß40 or Aß42, enabling expression from this edited allele to bypass the normal amyloidogenic APP processing pathway. Following neuronal differentiation, edited cell lines specifically accumulate intracellular aggregated/oligomeric Aß, exhibit a synaptic deficit, and have an abnormal accumulation of endolysosomal vesicles. Edited cultures progress to a stage of overt ND. All phenotypes appear at earlier culture times for Aß42 relative to Aß40. Whole transcriptome RNA-Seq analysis identified 23 up and 70 down regulated genes (differentially expressed genes) with similar directional fold change but larger absolute values in the Aß42 samples suggesting common underlying pathogenic mechanisms. Pathway/annotation analysis suggested that down regulation of extracellular matrix and cilia functions is significantly overrepresented. This cellular model could be useful for uncovering mechanisms directly linking Aß to neuronal death and as a tool to screen for new therapeutic agents that slow or prevent human ND.

miR-302b maintains "stemness" of human embryonal carcinoma cells by post-transcriptional regulation of Cyclin D2 expression.

Embryonic stem cells (ESCs) and embryonal carcinoma cells (ECCs) possess the remarkable property of self-renewal and differentiation potency. They are model preparations for investigating the underlying mechanisms of "stemness". microRNAs are recently discovered small noncoding RNAs with a broad spectrum of functions, especially in control of development. Here, we show that miR-302b indirectly regulates expression of the pluripotent stem cell marker Oct4, and it directly regulates expression of Cyclin D2 protein, a developmental regulator during gastrulation. Using loss-of function and gain-of function approaches, we demonstrate that functional miR-302b is necessary to maintain stem cell self-renewal and inhibit neuronal differentiation of human ECCs. During retinoic acid-induced neuronal differentiation, Cyclin D2 protein but not mRNA expression is strongly increased, concurrent with the down-regulation of miR-302b and Oct4. Our results suggest that miR-302b plays an important role in maintaining the pluripotency of ECCs and probably ESCs, by post-transcriptional regulation of Cyclin D2 expression.

Abnormal chemosensory jump 6 is a positive transcriptional regulator of the cholinergic gene locus in Drosophila olfactory neurons.

Cholinergic neurons acquire their neurotransmitter phenotype, in part, by expressing the cholinergic gene locus. Previous studies have indicated that the 5' flanking DNA of the locus contains both positive and negative regulatory elements important for expression in different subsets of cholinergic neurons in Drosophila and other animals. Approximately 300 bases of proximal 5' flanking DNA control expression in Drosophila CNS neurons essential for viability, whereas more distal regulatory elements are important for expression in PNS sensory neurons. In this study we identify the POU domain transcription factor abnormal chemosensory jump 6 (Acj6) as a necessary positive transcriptional regulator for cholinergic locus expression in primary olfactory neurons. Choline acetyltransferase enzyme activity, protein levels, mRNA, and a fluorescent cholinergic reporter gene are all decreased in olfactory neurons of acj6 mutants. Decreased cholinergic expression was observed in both adults and larvae. The presence of a specific Acj6 binding site has been identified in the cholinergic locus 5' flanking DNA, suggesting that Acj6 may play a direct role in specifying the cholinergic neurotransmitter phenotype of most olfactory neurons. Transgenic expression of two different isoforms of Acj6 restricted to olfactory neurons indicates that additional trans factors may be required for cholinergic locus expression. Transgenic expression in all cholinergic neurons, however, results in lethality when a POU IV box element is absent but is essentially benign when present, indicating the importance of this motif in specifying different functional roles for Acj6.

Regulation of synaptic connectivity: levels of Fasciclin II influence synaptic growth in the Drosophila CNS.

Much of our understanding of synaptogenesis comes from studies that deal with the development of the neuromuscular junction (NMJ). Although well studied, it is not clear how far the NMJ represents an adequate model for the formation of synapses within the CNS. Here we investigate the role of Fasciclin II (Fas II) in the development of synapses between identified motor neurons and cholinergic interneurons in the CNS of Drosophila. Fas II is a neural cell adhesion molecule homolog that is involved in both target selection and synaptic plasticity at the NMJ in Drosophila. In this study, we show that levels of Fas II are critical determinants of synapse formation and growth in the CNS. The initial establishment of synaptic contacts between these identified neurons is seemingly independent of Fas II. The subsequent proliferation of these synaptic connections that occurs postembryonically is, in contrast, significantly retarded by the absence of Fas II. Although the initial formation of synaptic connectivity between these neurons is seemingly independent of Fas II, we show that their formation is, nevertheless, significantly affected by manipulations that alter the relative balance of Fas II in the presynaptic and postsynaptic neurons. Increasing expression of Fas II in either the presynaptic or postsynaptic neurons, during embryogenesis, is sufficient to disrupt the normal level of synaptic connectivity that occurs between these neurons. This effect of Fas II is isoform specific and, moreover, phenocopies the disruption to synaptic connectivity observed previously after tetanus toxin light chain-dependent blockade of evoked synaptic vesicle release in these neurons.

Non-coding RNAs derived from an alternatively spliced REST transcript (REST-003) regulate breast cancer invasiveness.

RE1-Silencing Transcription factor (REST) has a well-established role in regulating transcription of genes important for neuronal development. Its role in cancer, though significant, is less well understood. We show that REST downregulation in weakly invasive MCF-7 breast cancer cells converts them to a more invasive phenotype, while REST overexpression in highly invasive MDA-MB-231 cells suppresses invasiveness. Surprisingly, the mechanism responsible for these phenotypic changes does not depend directly on the transcriptional function of REST protein. Instead, it is driven by previously unstudied mid-size (30-200 nt) non-coding RNAs (ncRNAs) derived from the first exon of an alternatively spliced REST transcript: REST-003. We show that processing of REST-003 into ncRNAs is controlled by an uncharacterized serine/arginine repeat-related protein, SRRM3. SRRM3 expression may be under REST-mediated transcriptional control, as it increases following REST downregulation. The SRRM3-dependent regulation of REST-003 processing into ncRNAs has many similarities to recently described promoter-associated small RNA-like processes. Targeting ncRNAs that control invasiveness could lead to new therapeutic approaches to limit breast cancer metastasis.

Early development of the Drosophila brain: IV. Larval neuropile compartments defined by glial septa.

In this study, we have analyzed the architecture of the brain neuropile of the Drosophila larva, which is formed by two main structural elements: long axon tracts and terminal axonal/dendritic arborizations carrying synapses. By using several molecular markers expressed in neurons and glial cells, we show that the early larval neuropile is subdivided by glial sheaths into numerous compartments. The three-dimensional layout of these compartments and their relationship to the pattern of long axon tracts described in the accompanying article (Nassif et al. [2003] J. Comp. Neurol 417-434) was modeled by using a three-dimensional illustration computer software. On the basis of their location relative to each other and to long axon tracts, larval brain compartments can be identified with compartments defined by structural and functional criteria for the adult fly brain. We find that small precursors of most of the compartments of the adult central brain can be identified in the early larva. Changes in brain compartmental organization occurring during larval growth are described. Neuropile compartments, representing easily identifiable landmark structures, will assist in future analyses of Drosophila brain development in which the exact location of neurons and their axonal trajectories is of importance.

Accumulation of amyloid-like Aß1-42 in AEL (autophagy-endosomal-lysosomal) vesicles: potential implications for plaque biogenesis.

Abnormal accumulation of Aß (amyloid ß) within AEL (autophagy-endosomal-lysosomal) vesicles is a prominent neuropathological feature of AD (Alzheimer's disease), but the mechanism of accumulation within vesicles is not clear. We express secretory forms of human Aß1-40 or Aß1-42 in Drosophila neurons and observe preferential localization of Aß1-42 within AEL vesicles. In young animals, Aß1-42 appears to associate with plasma membrane, whereas Aß1-40 does not, suggesting that recycling endocytosis may underlie its routing to AEL vesicles. Aß1-40, in contrast, appears to partially localize in extracellular spaces in whole brain and is preferentially secreted by cultured neurons. As animals become older, AEL vesicles become dysfunctional, enlarge and their turnover appears delayed. Genetic inhibition of AEL function results in decreased Aß1-42 accumulation. In samples from older animals, Aß1-42 is broadly distributed within neurons, but only the Aß1-42 within dysfunctional AEL vesicles appears to be in an amyloid-like state. Moreover, the Aß1-42-containing AEL vesicles share properties with AD-like extracellular plaques. They appear to be able to relocate to extracellular spaces either as a consequence of age-dependent neurodegeneration or a non-neurodegenerative separation from host neurons by plasma membrane infolding. We propose that dysfunctional AEL vesicles may thus be the source of amyloid-like plaque accumulation in Aß1-42-expressing Drosophila with potential relevance for AD.

A novel dual-color reporter for identifying insulin-producing beta-cells and classifying heterogeneity of insulinoma cell lines.

Many research studies use immortalized cell lines as surrogates for primary beta- cells. We describe the production and use of a novel "indirect" dual-fluorescent reporter system that leads to mutually exclusive expression of EGFP in insulin-producing (INS(+)) beta-cells or mCherry in non-beta-cells. Our system uses the human insulin promoter to initiate a Cre-mediated shift in reporter color within a single transgene construct and is useful for FACS selection of cells from single cultures for further analysis. Application of our reporter to presumably clonal HIT-T15 insulinoma cells, as well as other presumably clonal lines, indicates that these cultures are in fact heterogeneous with respect to INS(+) phenotype. Our strategy could be easily applied to other cell- or tissue-specific promoters. We anticipate its utility for FACS purification of INS(+) and glucose-responsive beta-like-cells from primary human islet cell isolates or in vitro differentiated pluripotent stem cells.

Robust RT-qPCR data normalization: validation and selection of internal reference genes during post-experimental data analysis.

Reverse transcription and real-time PCR (RT-qPCR) has been widely used for rapid quantification of relative gene expression. To offset technical confounding variations, stably-expressed internal reference genes are measured simultaneously along with target genes for data normalization. Statistic methods have been developed for reference validation; however normalization of RT-qPCR data still remains arbitrary due to pre-experimental determination of particular reference genes. To establish a method for determination of the most stable normalizing factor (NF) across samples for robust data normalization, we measured the expression of 20 candidate reference genes and 7 target genes in 15 Drosophila head cDNA samples using RT-qPCR. The 20 reference genes exhibit sample-specific variation in their expression stability. Unexpectedly the NF variation across samples does not exhibit a continuous decrease with pairwise inclusion of more reference genes, suggesting that either too few or too many reference genes may detriment the robustness of data normalization. The optimal number of reference genes predicted by the minimal and most stable NF variation differs greatly from 1 to more than 10 based on particular sample sets. We also found that GstD1, InR and Hsp70 expression exhibits an age-dependent increase in fly heads; however their relative expression levels are significantly affected by NF using different numbers of reference genes. Due to highly dependent on actual data, RT-qPCR reference genes thus have to be validated and selected at post-experimental data analysis stage rather than by pre-experimental determination.

A central role for autophagy in Alzheimer-type neurodegeneration.

The macroautophagy (autophagy) pathway is thought to be involved in a variety of neurodegenerative diseases, including Alzheimer disease (AD). It is not clear however, if autophagy plays a causative role, a protective role or is a consequence of the disease process itself. Using a Drosophila model of neuron-limited expression of AD-associated amyloid beta (Abeta) peptides, we have demonstrated an autophagy-mediated neurodegenerative cascade that is initiated by Abeta(1-42) and enhanced by aging. Our results suggest a central role for the autophagy pathway in AD type neurodegeneration and a new framework to understand seemingly unrelated AD phenotypes.

Abeta42-induced neurodegeneration via an age-dependent autophagic-lysosomal injury in Drosophila.

The mechanism of widespread neuronal death occurring in Alzheimer's disease (AD) remains enigmatic even after extensive investigation during the last two decades. Amyloid beta 42 peptide (Abeta(1-42)) is believed to play a causative role in the development of AD. Here we expressed human Abeta(1-42) and amyloid beta 40 (Abeta(1-40)) in Drosophila neurons. Abeta(1-42) but not Abeta(1-40) causes an extensive accumulation of autophagic vesicles that become increasingly dysfunctional with age. Abeta(1-42)-induced impairment of the degradative function, as well as the structural integrity, of post-lysosomal autophagic vesicles triggers a neurodegenerative cascade that can be enhanced by autophagy activation or partially rescued by autophagy inhibition. Compromise and leakage from post-lysosomal vesicles result in cytosolic acidification, additional damage to membranes and organelles, and erosive destruction of cytoplasm leading to eventual neuron death. Neuronal autophagy initially appears to play a pro-survival role that changes in an age-dependent way to a pro-death role in the context of Abeta(1-42) expression. Our in vivo observations provide a mechanistic understanding for the differential neurotoxicity of Abeta(1-42) and Abeta(1-40), and reveal an Abeta(1-42)-induced death execution pathway mediated by an age-dependent autophagic-lysosomal injury.

Cyclical expression of Na+/K+-ATPase in the visual system of Drosophila melanogaster.

In the first (lamina) and second (medulla) optic neuropils of Drosophila melanogaster, sodium pump subunit expression changes during the day and night, controlled by a circadian clock. We examined alpha-subunit expression from the intensity of immunolabeling. For the beta-subunit, encoded by Nervana 2 (Nrv2), we used Nrv2-GAL4 to drive expression of GFP, and measured the resultant fluorescence in whole heads and specific optic lobe cells. All optic neuropils express the alpha-subunit, highest at the beginning of night in both lamina and medulla in day/night condition and the oscillation was maintained in constant darkness. This rhythm was lacking in the clock arrhythmic per(0) mutant. GFP driven by Nrv2 was mostly detected in glial cells, mainly in the medulla. There, GFP expression occurs in medulla neuropil glia (MNGl), which express the clock gene per, and which closely contact the terminals of clock neurons immunoreactive to pigment dispersing factor. GFP fluorescence exhibited circadian oscillation in whole heads from Nrv2-GAL4+UAS-S65T-GFP flies, although significant GFP oscillations were lacking in MNGl, as they were for both subunit mRNAs in whole-head homogenates. In the dissected brain tissues, however, the mRNA of the alpha-subunit showed a robust daily rhythm in concentration changes while changes in the beta-subunit mRNA were weaker and not statistically significant. Thus in the brain, the genes for the sodium pump subunits, at least the one encoding the alpha-subunit, seem to be clock-controlled and the abundance of their corresponding proteins mirrors daily changes in mRNA, showing cyclical accumulation in cells.

Novel method for visualizing and modeling the spatial distribution of neural stem cells within intracranial glioma.

Neural stem cells (NSCs) hold great promise for glioma therapy due to their inherent tumor-tropic properties, enabling them to deliver therapeutic agents directly to invasive tumor sites. In the present study, we visualized and quantitatively analyzed the spatial distribution of tumor-tropic NSCs in a mouse model of orthotopic glioma in order to predict the therapeutic efficacy of a representative NSC-based glioma therapy. U251.eGFP human glioma was established in the brain of athymic mice, followed by stereotactic injection of CM-DiI-labeled human NSCs posterior-lateral to the tumor site. Confocal microscopy, three-dimensional modeling and mathematical algorithms were used to visualize and characterize the spatial distribution of NSCs throughout the tumor. The pattern of NSC distribution showed a gradient with higher densities toward the centroid of the tumor mass. We estimate that NSC-mediated therapy would eradicate 70-90% of the primary tumor mass and the majority of invasive tumor foci. Our method may serve as a model for optimizing the efficacy of NSC-based glioma therapy.

Cytoplasmic and nuclear retained DMPK mRNAs are targets for RNA interference in myotonic dystrophy cells.

Small interfering RNA (siRNA) duplexes induce the specific cleavage of target RNAs in mammalian cells. Their involvement in down-regulation of gene expression is termed RNA interference (RNAi). It is widely believed that RNAi predominates in the cytoplasm. We report here the co-existence of cytoplasmic and nuclear RNAi phenomena in primary human myotonic dystrophy type 1 (DM1) cells by targeting myotonic dystrophy protein kinase (DMPK) mRNAs. Heterozygote DM1 myoblasts from a human DM1 fetus produce a nuclear retained mutant DMPK transcript with large CUG repeats ( approximately 3,200) from one allele of the DMPK gene and a wild type transcript with 18 CUG repeats, thus providing for both a nuclear and cytoplasmic expression profile to be evaluated. We demonstrate here for the first time down-regulation of the endogenous nuclear retained mutant DMPK mRNAs targeted with lentivirus-delivered short hairpin RNAs (shRNAs). This nuclear RNAi(-like) phenomenon was not observed when synthetic siRNAs were delivered by cationic lipids, suggesting either a link between processing of the shRNA and nuclear import or a separate pathway for processing shRNAs in the nuclei. Our observation of simultaneous RNAi on both cytoplasmic and nuclear retained DMPK has important implications for post-transcriptional gene regulation in both compartments of mammalian cells.