Biology subjects

Hassan, B. A.

Publications and source records attributed to Hassan, B. A..

At least 19 recordsLinked to original sources

bioRxiv2026

Pten Orchestrates Neurogenic Radial Glia Lineage Progression and Tunes Neocortical Astrocyte Production

The cerebral cortex consists of immense numbers of neuronal and glial cell-types derived from radial glial progenitor (RGP) cells. How RGPs generate appropriate quantities of distinct cortical cell-types to safeguard a brain of correct size, is not well understood. However, genetic aberration in human, including mutations in PTEN, lead to cortical malformation such as macrocephaly, albeit with unknown etiology. Here we utilized Mosaic Analysis with Double Markers (MADM)-based clonal analysis and single cell phenotyping to decipher the role of Pten in neurogenic and gliogenic RGP lineage progression during cortical ontogeny. While neurogenic RGP lineage progression and projection neuron production was moderately altered in the absence of Pten, cortical astrocyte production was drastically increased. Through genetic epistasis experiments we show that the loss of Pten uncouples astrocyte generation from essential growth factor signaling hubs, funneling into MAPK. Collectively, our results suggest that Pten regulates RGP lineage progression with distinct sequential functions in cortical projection neurogenesis and astrocyte production to ensure the emergence of a correctly-sized cerebral cortex.

neuroscience↗

bioRxiv2025

Post-translational Tuning of Human Cortical Progenitor Neuronal Output

The striking expansion of the human neocortex is driven by the ability of radial glial cells (RGCs) to prolong neurogenesis without exhausting their progenitor pool, thereby producing more neurons, particularly in the upper layers. The mechanisms underlying enhanced human neurogenic output remain unresolved. We hypothesized that post-translational modification of conserved proteins could provide a flexible mechanism supporting species-specific patterns of neurogenesis. Here, we identify phosphorylation of the proneural factor NEUROGENIN2 as a regulator of human RGC neuronal output by modulating its pioneering-like activity, at the expense of its transactivation function. This, in turn, remodels the RGC chromatin landscape, resulting in a forward temporal shift of RGC gene regulatory networks and leading to increased neuronal production biased towards upper-layer neurons. Our work proposes a conceptual framework for how subtle modifications of conserved processes, and not just new genes, encode tunable mechanisms for species-specific trajectories of brain development.

developmental biology↗

bioRxiv2024

Structural Adaptations of HepI Enzymes in Proteobacteria: Insights into Evolutionary Resilience and Functional Dynamics

Since lipopolysaccharide molecules are highly variable in their structure while also being essential for bacterial cell survival, we hypothesized that enzymes involved in its biosynthesis may exhibit differences that could be used to predict the likelihood of survival in different ecological niches. We examined the sequence variability of orthologues of Heptosyltransferase I (HepI), which is found in all lipopolysaccharide (LPS) containing Gram-negative bacteria. We identified two different sequence motifs within the N-terminal domain of HepI, which correlated with differences in the Lipid A portion of the LPS. Further, we compared the protein structure of HepI from Escherichia coli with a structural model we generated that incorporated the alternate sequence motif. Molecular dynamics simulations of these two proteins, recapitulated our findings, that proteins with the E. coli-like sequence motif maintained a larger enzyme active site, while the mutated structural model undergoes rearrangements that lead to a smaller N-terminal active site pocket. This work revealed sequence-structure-function relationships that can be used to determine if a species incorporates a Heptose residue onto Lipid A chains containing one or two 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo) residues. Since research suggests that the number of Kdo residues in LPS impacts the overall immune response to the LPS endotoxin, this work could aid in our understanding of the pathogenic effects of human-bacterial interactions. Understanding the sequence-structural adaptations of HepI enzymes across proteobacteria sheds light on their evolutionary resilience and functional versatility. HighlightsO_LILipopolysaccharide (LPS) structure varies between organisms C_LIO_LIHeptosytransferase I sequence motifs allow prediction of LPS structural features C_LIO_LIAdaptations of HepI enzymes reveal evolutionary resilience and functional versatility C_LI

evolutionary biology↗

bioRxiv2023

Respective contribution of Appl to mushroom body axon growth and long-term memory in Drosophila

The Amyloid Precursor Protein (APP) is associated with Alzheimers disease. Appl is the single Drosophila APP ortholog and is expressed in all neurons throughout development. Appl was previously shown to cell-autonomously modulate axon outgrowth in the mushroom bodies (MBs), the fly olfactory memory center. However, we found that Appld, the only reported null allele, affects the normal function of vnd, the gene just proximal to Appl. To decipher developmental defects specifically due to a loss of only Appl function, we generated a precise Appl null allele (ApplC2.1) by CRISPR/Cas9 genomic engineering. With ApplC2.1, we confirmed the partial requirement for Appl in MB axon outgrowth. We also produced new CRISPR vnd alleles removing either vnd-B or vnd-A function. We report here that vnd is also required for MB {beta}-branch axon outgrowth and to a much greater extent than Appl itself. Moreover, vnd is expressed in neurons close to, but not within, the MB during development and is required non-cell-autonomously for MB axon outgrowth

neuroscience↗

bioRxiv2023

Developmental transcriptional control of mitochondrial homeostasis is required for activity-dependent synaptic connectivity

During neuronal circuit formation, local control of axonal organelles ensures proper synaptic connectivity. Whether this process is genetically encoded is unclear and if so, its developmental regulatory mechanisms remain to be identified. We hypothesized that developmental transcription factors regulate critical parameters of organelle homeostasis that contribute to circuit wiring. We combined cell type-specific transcriptomics with a genetic screen to discover such factors. We identified Telomeric Zinc finger-Associated Protein (TZAP) as a temporal developmental regulator of neuronal mitochondrial homeostasis genes, including Pink1. In Drosophila, loss of dTzap function during visual circuit development leads to loss of activity-dependent synaptic connectivity, that can be rescued by Pink1 expression. At the cellular level, loss of dTzap/TZAP leads to defects in mitochondrial morphology, attenuated calcium uptake and reduced synaptic vesicle release in fly and mammalian neurons. Our findings highlight developmental transcriptional regulation of mitochondrial homeostasis as a key factor in activity-dependent synaptic connectivity.

neuroscience↗

bioRxiv2022

Probabilistic axon targeting dynamics lead to individualized brain wiring

Developmental variation in brain-wiring contributes to behavioural individuality1,2. However, how and when individualized wiring diagrams emerge and become stable during development remains largely unknown. Here, we explored axon targeting dynamics in individual brains using live-imaging of a developing Drosophila visual circuit and discovered that targeting choice is an algorithmic multi-step growth process with variable outcomes. Using optogenetics, we found that temporally restricted Notch lateral-inhibition defines a subset of neurons with a probabilistic potential to innervate distal targets. Next, axons from NotchOFF neurons amplify into long actin-rich multi-fibre structures necessary for distal growth. A subset of these NotchOFF neurons create distal targeting axons by stabilizing microtubule growth in one of their actin fibres. Amplified axons without tubulin-stabilized fibres retract, resulting in the stochastic selection of a different number of distal targeting axons in each brain. Pharmacological microtubule destabilization suffices to inhibit this targeting. We observed a similar axonal amplification-stabilization process in the developing chick spinal cord, suggesting a conserved mechanism. Finally, early microtubule patterns predict the adult brain-wiring of an individual in a target-independent manner prior to synapse formation3,4. Thus, we show that a temporal succession of genetically encoded stochastic processes explains the emergence of individual wiring variation. One-Sentence SummaryThe temporal succession of stochastic developmental processes explains the emergence of individual wiring variation.

neuroscience↗

bioRxiv2022

Kinetic Characterization and Computational Modeling of the Escherichia coli Heptosyltransferase II: Exploring the Role of Protein Dynamics in Catalysis for a GT-B Glycosyltransferase

Glycosyltransferases (GTs) are enzymes that are uniquely adapted to promote the formation of a glycosidic bond between a sugar molecule and a wide variety of substrates. Heptosyltransferase II (HepII) is a GT involved in the lipopolysaccharide (LPS) biosynthetic pathway that transfers the seven-carbon sugar (L-glycero-D-manno-heptose; Hep) onto a lipid anchored glycopolymer (heptosylated Kdo2-Lipid A, Hep-Kdo2-Lipid A or HLA). LPS plays a key role in Gram-negative bacterial sepsis as a stimulator of the human immune response and has been used as an adjuvant in vaccines. As such, ongoing efforts towards inhibition of LPS biosynthetic enzymes to aid development of novel antimicrobial therapeutics has driven significant effort towards the characterization of these enzymes. Three heptosyltransferases are involved in the inner-core biosynthesis, with E. coli HepII being the last to be quantitatively characterized in vivo, as described herein. HepII shares modest sequence similarity with heptosyltransferase I (HepI) while maintaining a high degree of structural homology. Here we report the first kinetic and biophysical characterization of HepII and demonstrate the properties of HepII that are shared by HepI to include sugar donor promiscuity, and sugar acceptor induced secondary structural changes which results in significant thermal stabilization. HepII also has an increased catalytic efficiency and a significantly tighter binding affinity for both of its substrates, with an insensitivity to the number of acyl chains on the sugar acceptor. Additionally, a structural model of the HepII ternary complex, refined by molecular dynamics simulations, was developed to probe potentially important substrate-protein contacts and revealed the potential of Tryptophan (Trp) residues responsible for reporting on ligand binding. As was previously described for HepI, Tryptophan fluorescence in HepII allowed observation of substrate induced changes in Trp fluorescence intensity which enabled determination of substrate dissociation constants. Combined, these efforts meaningfully enhance our understanding of the Heptosyltransferase family of enzymes and will aid in future efforts to design novel, potent and specific inhibitors for this family of enzymes.

biochemistry↗

bioRxiv2022

A critical developmental interval of coupling axon branching to synaptic degradation during neural circuit formation

The emergence of neuronal wiring specificity requires stabilization of dynamic axonal branches at sites of selective synapse formation. Models that explain how axonal branching is coupled to synaptogenesis postulate molecular regulators acting in a spatiotemporally restricted fashion. We report that Epidermal Growth Factor Receptor (EGFR) activity is required in presynaptic axonal branches during two distinct temporal intervals to regulate circuit wiring in the developing Drosophila visual system. EGFR is required early to regulate primary axonal branching and independently again later to prevent autophagic degradation of the synaptic active zone protein Bruchpilot (Brp). The protection of synaptic material during this later interval of wiring ensures the stabilization of terminal branches, circuit connectivity and appropriate visual behavior. Phenotypes of EGFR inactivation were rescued by increasing Brp levels or downregulating autophagic genes. We identify a temporally restricted molecular mechanism required for coupling axonal branching and synaptic stabilization that contributes to the emergence of neuronal wiring specificity.

neuroscience↗

bioRxiv2022

Numerical discrimination in Drosophila melanogaster

Sensitivity to numbers is a crucial and evolutionarily conserved cognitive ability. The lack of experimental models amenable to systematic genetic and neural manipulation has precluded discovering circuits required for numerical cognition. Here, we demonstrate that in a two-choice task Drosophila fruit flies spontaneously prefer sets containing more objects. This preference is determined by the ratio between the two numerical quantities tested, a characteristic signature of numerical cognition across species. Individual flies maintained their numerical choice over consecutive days. Using a numerical visual conditioning paradigm, we found that flies are capable of associating sucrose with numerical quantities and can be trained to reverse their spontaneous preference for large quantities. Finally, we show that silencing LC11 neurons reduces the preference for more objects, thus identifying a neuronal substrate for numerical cognition in invertebrates. This discovery paves the way for the systematic analysis of the behavioral and neural mechanisms underlying sensitivity to numerosity.

neuroscience↗

bioRxiv2022

Transient regulation of focal adhesion via Tensin3 is required for nascent oligodendrocyte differentiation

The differentiation of oligodendroglia from oligodendrocyte precursor cells (OPCs) to complex and extensive myelinating oligodendrocytes (OLs) is a multistep process that involves largescale morphological changes with significant strain on the cytoskeleton. While key chromatin and transcriptional regulators of differentiation have been identified, their target genes responsible for the morphological changes occurring during OL myelination are still largely unknown. Here, we show that the regulator of focal adhesion, Tensin3 (Tns3), is a direct target gene of Olig2, Chd7, and Chd8, transcriptional regulators of OL differentiation. Tns3 is transiently upregulated and localized to cell processes of immature OLs, together with integrin-{beta}1, a key mediator of survival at this transient stage. Constitutive Tns3 loss-of-function leads to reduced viability in mouse and humans, with surviving knockout mice still expressing Tns3 in oligodendroglia. Acute deletion of Tns3 in vivo, either in postnatal neural stem cells (NSCs) or in OPCs, leads to a two-fold reduction in OL numbers. We find that the transient upregulation of Tns3 is required to protect differentiating OPCs and immature OLs from cell death by preventing the upregulation of p53, a key regulator of apoptosis. Altogether, our findings reveal a specific time window during which transcriptional upregulation of Tns3 in immature OLs is required for OL differentiation likely by mediating integrin-{beta}1 survival signaling to the actin cytoskeleton as OL undergo the large morphological changes required for their terminal differentiation.

neuroscience↗

bioRxiv2021

Discovery of Aminoglycosides as first in class, nanomolar inhibitors of Heptosyltransferase I

A clinically relevant inhibitor for Heptosyltransferase I (HepI) has been sought after for many years and while many have designed novel small-molecule inhibitors, these compounds lack the bioavailability and potency necessary for therapeutic use. Extensive characterization of the HepI protein has provided valuable insight into the dynamic motions necessary for catalysis that could be targeted for inhibition. With the help of molecular dynamic simulations, aminoglycoside antibiotics were shown to be putative inhibitors for HepI and in this study, they were experimentally determined to be the first in-class nanomolar inhibitors of HepI with the best inhibitor demonstrating a Ki of 600 +/- 90 nM. Detailed kinetic analyses were performed to determine the mechanism of inhibition while circular dichroism spectroscopy, intrinsic tryptophan fluorescence, docking, and MD simulations were used to corroborate kinetic experimental findings. Kinetic analysis methods include Lineweaver-Burk, Dixon, Cornish-Bowden and Mixed-Model of Inhibition which allowed for unambiguous assignment of inhibition mechanism for each inhibitor. In this study, we show that neomycin and kanamycin b are competitive inhibitors against the sugar acceptor substrate while tobramycin exhibits a mixed inhibitory effect and streptomycin is non-competitive. MD simulations also allowed us to suggest that the inhibitors bind tightly and inhibit catalytic dynamics due to a major desolvation penalty of the enzyme active site. While aminoglycosides have long been known as a class of potent antibiotics, they also have been scientifically shown to impact cell membrane stability, and we propose that inhibition of HepI contributes to this effect by disrupting lipopolysaccharide biosynthesis.

biochemistry↗

bioRxiv2021

Ligand Induced Conformational and Dynamical Changes in a GT-B Glycosyltransferase:Molecular Dynamic Simulations of Heptosyltransferase I Apo, Binary and Ternary Complexes

Understanding the dynamical motions and ligand recognition motifs of specific glycosyltransferase enzymes, like Heptosyltransferase I (HepI), is critical to discerning the behavior of other carbohydrate binding enzymes. Prior studies in our lab demonstrated that glycosyltransferases in the GT-B structural class, which are characterized by their connection of two Rossman-like domains by a linker region, have conservation of both structure and dynamical motions, despite low sequence conservation, therefore making discoveries found in HepI transferable to other GT-B enzymes. Through a series of 100 nanosecond Molecular Dynamics simulations of HepI in apo enzyme state, and also in the binary and ternary complexes with the native substrates/products. Ligand free energy analysis allowed determination of an anticipated enzymatic path for ligand binding and release. Principle component, dynamic cross correlation and network analyses of the simulation trajectories revealed that there are not only correlated motions between the N- and C-termini, but also that residues within the N-terminal domain communicate via a path that includes substrate proximal residues of the C-terminal domain. Analysis of structural changes, energetics of substrate/products binding and changes in pKa have elucidated a variety of inter- and intradomain interactions that are critical for catalysis. These data corroborate and allow visualization of previous experimental observations of protein conformational changes of HepI. This study has provided valuable insights into the regions involved in HepI conformational rearrangement upon ligand binding, and are likely to enhance efforts to develop new dynamics disrupting enzyme inhibitors for GT-B structural enzymes in the future.

biochemistry↗

bioRxiv2021

Variable brain wiring through scalable and relative synapse formation in Drosophila

Variability of synapse numbers and partners despite identical genes reveals limits of genetic determinism. Non-genetic perturbation of brain wiring can therefore reveal to what extent synapse formation is precise and absolute, or promiscuous and relative. Here, we show the role of relative partner availability for synapse formation in the fly brain through perturbation of developmental temperature. Unexpectedly, slower development at lower temperatures substantially increases axo-dendritic branching, synapse numbers and non-canonical synaptic partnerships of various neurons, while maintaining robust ratios of canonical synapses. Using R7 photoreceptors as a model, we further show that scalability of synapse numbers and ratios is preserved when relative availability of synaptic partners is changed in a DIP{gamma} mutant that ablates R7s preferred synaptic partner. Behaviorally, movement activity scales inversely with synapse numbers, while movement precision and relative connectivity are congruently robust. Hence, the fly genome encodes scalable relative connectivity to develop functional, but not identical, brains. One-Sentence SummaryNon-identical connectivity and behavior result from temperature-dependent synaptic partner availability in Drosophila.

neuroscience↗

bioRxiv2021

The Amyloid Precursor Protein regulates human cortical neurogenesis

The approximately 16 billion neurons of the human neocortex are derived from a relatively limited number of developmental neural stem cells (NSCs). During embryogenesis, human cortical NSCs initially generate neurons at a particularly slow rate while preserving their progenitor state for a relatively long time. How this balance between the progenitor state and neurogenic state is regulated, and whether it contributes to species-specific brain patterning, is poorly understood. Here we show that the characteristic potential of human NSCs to remain in a progenitor state as they generate neurons for a prolonged amount of time requires the Amyloid Precursor Protein (APP). In contrast, APP is dispensable in mouse NSCs, which undergo neurogenesis at a much faster rate. Mechanistically, loss of APP cell-autonomously accelerates neurogenesis through activation of the AP1 transcription factor and repression of WNT signaling. We propose that the fine balance between self-renewal and differentiation is homeostatically regulated by APP, which may contribute to human-specific temporal patterns of neurogenesis.

developmental biology↗

bioRxiv2021

The Amyloid Precursor Protein is a conserved Wnt receptor

The Amyloid Precursor Protein (APP) and its homologues are transmembrane proteins required for various aspects of neuronal development and activity, whose molecular function is unknown. Specifically, it is unclear whether APP acts as a receptor, and if so what its ligand(s) may be. We show that APP binds the Wnt ligands Wnt3a and Wnt5a and that this binding regulates APP protein levels. Wnt3a binding promotes full length APP (flAPP) recycling and stability. In contrast, Wnt5a promotes APP targeting to lysosomal compartments and reduces flAPP levels. A conserved Cysteine Rich Domain (CRD) in the extracellular portion of APP is required for Wnt binding, and deletion of the CRD abrogates the effects of Wnts on flAPP levels and trafficking. Finally, loss of APP results in increased axonal and reduced dendritic growth of mouse embryonic primary cortical neurons. This phenotype can be cell-autonomously rescued by full length, but not CRD-deleted, APP.

cell biology↗

bioRxiv2020

Generation of neuronal diversity from common progenitors via Notch signaling in the cerebellum

Brain neurons arise from relatively few progenitors capable of giving rise to an enormous diversity of neuronal types. Nonetheless, a cardinal feature of mammalian brain neurogenesis in both the cortex and the cerebellum is that excitatory neurons and inhibitory neurons derive from separate, spatially segregated, progenitors. Whether bi-potential progenitors with an intrinsic capacity to generate both excitatory and inhibitory lineages exist and how such a fate decision may be regulated is unknown. Using cerebellar development as a model, we discover that individual embryonic cerebellar progenitors give rise to both inhibitory and excitatory lineages. We find that gradations of Notch activity levels determine the fates of the progenitors and their daughters. Daughters with the highest levels of Notch activity retain the progenitor fate. Daughters with intermediate levels of Notch activity become fate restricted to generate inhibitory neurons, while daughters with very low levels of Notch signaling adopt the excitatory fate. Therefore, Notch mediated binary cell fate choice is a mechanism for regulating the ratio of excitatory to inhibitory neurons from common progenitors. Graphical summary O_FIG O_LINKSMALLFIG WIDTH=144 HEIGHT=200 SRC="FIGDIR/small/997205v2_ufig1.gif" ALT="Figure 1"> View larger version (25K): org.highwire.dtl.DTLVardef@c477f8org.highwire.dtl.DTLVardef@a079bforg.highwire.dtl.DTLVardef@1992877org.highwire.dtl.DTLVardef@2fd5de_HPS_FORMAT_FIGEXP M_FIG C_FIG

developmental biology↗

bioRxiv2020

The Drosophila Amyloid Precursor Protein homologue mediates neuronal survival and neuro-glial interactions

The amyloid precursor protein (APP) is a structurally and functionally conserved transmembrane protein whose physiological role in adult brain function and health is still unclear. Because mutations in APP cause familial Alzheimers disease, most research focuses on this aspect of APP biology. We investigated the physiological function of APP in the adult brain using the fruit fly Drosophila melanogaster, which harbors a single APP homologue called APP Like (APPL). Previous studies have provided evidence for the implication of APPL in neuronal wiring and axonal growth through the Wnt signaling pathway. However, like APP, APPL continues to be expressed in all neurons of the adult brain where its functions and their molecular and cellular underpinnings are unknown. We report that APPL loss of function results in the dysregulation of endolysosomal function, in both neurons and glia, with a notable enlargement of early endosomal compartment in neurons followed by neuronal cell death, the accumulation of dead neurons in the brain during a critical period at a young age and subsequent reduction in lifespan. These defects can be rescued by reduction in the levels of the early endosomal regulator Rab5, indicating a causal role of endosomal function for cell death. Finally, we show that the secreted extracellular domain of APPL is taken up by glia, regulates their endosomal morphology and this is necessary and sufficient for the clearance of neuronal debris in an axotomy model. We propose that the APP proteins represent a novel family of neuro-glial signaling proteins required for adult brain homeostasis.

neuroscience↗

bioRxiv2019

Single-cell resolution view of the transcriptional landscape of developing Drosophila eye.

Faithful and reliable quantification of gene expression at a single-cell level is an outstanding challenge in developmental biology. Most existing approaches face a trade-off between the signal to noise ratio, resolution, and sensitivity. Here, we present a novel approach for in situ quantification of gene expression in a developing tissue. Our pipeline combines computational prediction of transcription factor targets, gene tagging, fluorescent reporter imaging, state-of-the-art image analysis, and automated cell-type identification. By applying this approach to identify the sequence of quantitative changes in gene expression which govern the development of the Drosophila neural retina, we demonstrate the feasibility of our method. We analyze the targets of Atonal (Ato), a transcription factor that controls the transition from eye disc progenitor cell to photoreceptor neurons. We utilized recombineering and genomic engineering to tag all predicted Ato targets with novel transcriptional reporters. These reporters enable following the expression of both regulator and regulated genes to accurately quantify their expression levels in individual cells. Our complete computational pipeline identifies nuclei in the eye discs and detects different states of cells as they progress through differentiation. Based on detailed gene expression analysis, our technique revealed genes likely to be direct Ato targets and provided insight into how gene expression changes drive the specification of photoreceptors.

developmental biology↗