Molecular Biology seminars

Corticospinal neurons (CSN) are the cortical projection neurons that innervate the spinal cord and some brainstem targets with segmental precision to control voluntary movement of specific functional motor groups, limb sections, or individual digits, yet molecular regulation over CSN segmental target specificity is essentially unknown. CSN subpopulations exhibit striking axon targeting specificity from development into maturity: Evolutionarily newer rostrolateral CSN exclusively innervate bulbar-cervical targets (CSNBC-lat), while evolutionarily older caudomedial CSN (CSNmed) are more heterogeneous, with distinct subpopulations extending axons to either bulbar-cervical or thoraco-lumbar segments. The cervical cord, with its evolutionarily enhanced precision of forelimb movement, is innervated by multiple CSN subpopulations, suggesting inter-neuronal interactions in establishing corticospinal connectivity. I identify that Lumican, previously unrecognized in axon development, controls the specificity of cervical spinal cord innervation by CSN. Remarkably, Lumican, an extracellular matrix protein expressed by CSNBC-lat, non-cell-autonomously suppresses axon collateralization in the cervical cord by CSNmed. Intersectional viral labeling and mouse genetics further identify that Lumican controls axon collateralization by multiple subpopulations in caudomedial sensorimotor cortex. These results identify inter-axonal molecular crosstalk between CSN subpopulations as a novel mechanism controlling corticospinal connectivity and competitive specificity. Further, this mechanism has potential implications for evolutionary diversification of corticospinal circuitry with finer scale precision. "" Complementing this work, to comprehensively elucidate related axon projection mechanisms functioning at tips of growing CSN axons in vivo, I am currently applying experimental and analytic approaches recently developed in my postdoc lab (Poulopoulos*, Murphy*, Nature, 2019) to quantitatively and subcellularly “map” RNA and protein molecular machinery of subtype-specific growth cones, in parallel to their parent somata, isolated directly in vivo from developing subcerebral projection neurons (SCPN; the broader cortical output neuron population targeting both brainstem and spinal cord; includes CSN). I am investigating both normal development and GC-soma dysregulation with mutation of central CSN-SCPN transcriptional regulator Ctip2/Bcl11b.

Transposable elements, known colloquially as ‘jumping genes’, constitute approximately 45% of the human genome. Cells utilize epigenetic defenses to limit transposable element jumping, including formation of silencing heterochromatin and generation of piwi-interacting RNAs (piRNAs), small RNAs that facilitate clearance of transposable element transcripts. We have utilized fruit flies, mice and postmortem human brain samples to identify transposable element dysregulation as a key mediator of neuronal death in tauopathies, a group of neurodegenerative disorders that are pathologically characterized by deposits of tau protein in the brain. Mechanistically, we find that heterochromatin decondensation and reduction of piwi and piRNAs drive transposable element dysregulation in tauopathy. We further report a significant increase in transcripts of the endogenous retrovirus class of transposable elements in human Alzheimer’s disease and progressive supranuclear palsy, suggesting that transposable element dysregulation is conserved in human tauopathy. Taken together, our data identify heterochromatin decondensation, piwi and piRNA depletion and consequent transposable element dysregulation as a pharmacologically targetable, mechanistic driver of neurodegeneration in tauopathy.

Esta conferencia pretende ofrecer una visión panorámica de los progresos en el conocimiento del cerebro, desde la fundación por Cajal de la moderna neurociencia hasta los muy recientes hallazgos aportados por la genética, la biología molecular, la microscopia y la electrofisiología al conocimiento de la estructura, conectividad y función de las células nerviosas, asi como sobre el funcionamiento integrado del cerebro humano aportado por las nuevas técnicas de imagen y el registro y estimulación selectivos de las distintas áreas cerebrales y su análisis con técnicas de computación. Finalmente se discutirán las repercusiones médicas y sociales que implica un mejor conocimiento del cerebro, sus limitaciones en el momento actual y los riesgo que conlleva el mal uso de los avances científicos de la neurociencia.

The occurrence of seizures at specific times of the day has been consistently observed for centuries in individuals with epilepsy. Electrophysiological recordings provide evidence that seizures have a higher probability of occurring at a given time during the night and day cycle in individuals with epilepsy – the seizure rush hour. Which mechanisms underly such circadian rhythmicity of seizures? Why don’t they occur every day at the same time? Which mechanisms may underly their occurrence outside the rush hour? I shall present a hypothesis: MORE - Molecular Oscillations and Rhythmicity of Epilepsy, a conceptual framework to study and understand the mechanisms underlying the circadian rhythmicity of seizures and their probabilistic nature. The core of the hypothesis is the existence of circa 24h oscillations of gene and protein expression throughout the body in different cells and organs. The orchestrated molecular oscillations control the rhythmicity of numerous body events, such as feeding and sleep. The concept developed here is that molecular oscillations may favor seizure genesis at preferred times, generating the condition for a seizure rush hour. However, the condition is not sufficient, as other factors are necessary for a seizure to occur. Studying these molecular oscillations may help us understand seizure genesis mechanisms and find new therapeutic targets and predictive biomarkers. The MORE hypothesis can be generalized to comorbidities and the slower multidien (week/month period) rhythmicity of seizures.

Human genes associated with brain-related diseases are being discovered at an accelerating pace. A major challenge is an identification of the mechanisms through which these genes act, and of potential therapeutic strategies. To elucidate such mechanisms in human cells, we established a CRISPR-based platform for genetic screening in human iPSC-derived neurons, astrocytes and microglia. Our approach relies on CRISPR interference (CRISPRi) and CRISPR activation (CRISPRa), in which a catalytically dead version of the bacterial Cas9 protein recruits transcriptional repressors or activators, respectively, to endogenous genes to control their expression, as directed by a small guide RNA (sgRNA). Complex libraries of sgRNAs enable us to conduct genome-wide or focused loss-of-function and gain-of-function screens. Such screens uncover molecular players for phenotypes based on survival, stress resistance, fluorescent phenotypes, high-content imaging and single-cell RNA-Seq. To uncover disease mechanisms and therapeutic targets, we are conducting genetic modifier screens for disease-relevant cellular phenotypes in patient-derived neurons and glia with familial mutations and isogenic controls. In a genome-wide screen, we have uncovered genes that modulate the formation of disease-associated aggregates of tau in neurons with a tauopathy-linked mutation (MAPT V337M). CRISPRi/a can also be used to model and functionally evaluate disease-associated changes in gene expression, such as those caused by eQTLs, haploinsufficiency, or disease states of brain cells. We will discuss an application to Alzheimer’s Disease-associated genes in microglia.

The distribution of pathological tau in the brain of patients with AD is highly predicable, and as disease worsens, it spreads transynaptically from initial regions of vulnerability. The reason why only some neurons are vulnerable to the accumulation and propagation of pathological forms of tau, and the mechanisms by which tauopathy spreads through the brain are not well understood. Using a combination of immunohistochemistry and computational analysis we have examined pathway differences between vulnerable and resistant neurons. How tau spreads across a synapse has been examined in vitro using different model systems. Our data show that dysregulation of tau homeostasis determines the cellular and regional vulnerability of specific neurons to tau pathology (H. Fu et al. 2019. Nat. Neuro. 22 (1):47-56) and that deficits in tau homeostasis can exacerbate tau accumulation and propagation. Aging appears to impact similar neuronal populations. Mechanisms and consequences of abnormal tau accumulation within neurons, its transfer between cells, pathology propagation and therapeutic opportunities will be discussed.

The vagus nerve contains a diversity of sensory neurons that detect peripheral stimuli such as blood pressure changes at the aortic arch, lung expansion during breathing, meal-induced stomach distension, and chemotherapeutics that induce nausea. Underlying vagal sensory mechanisms are largely unresolved at a molecular level, presenting tremendously important problems in sensory biology. We charted vagal sensory neurons by single cell RNA sequencing, identifying novel cell surface receptors and classifying a staggering diversity of sensory neuron types. We then generated a collection of ires-Cre knock-in mice to target each neuron type, and adapted genetic tools for Cre-based anatomical mapping, in vivo imaging, targeted ablation, and optogenetic control of vagal neuron activity. We found different sensory neuron types that innervate the lung and exert powerful effects on breathing, others that monitor and control the digestive system, and yet others that innervate that innervate the larynx and protect the airways. Together with Ardem Patapoutian, we also identified a critical role for Piezo mechanoreceptors in the sensation of airway stretch, which underlies a classical respiratory reflex termed the Hering-Breuer inspiratory reflex, as well as in the neuronal sensation of blood pressure and the baroreceptor reflex.

This talk aims to show how new physical methods can advance biological and biomedical research. A major advance in physical chemistry in the last two decades has been the development of quantitative methods to directly observe individual molecules in solution, attached to surfaces, in the membrane of live cells or more recently inside live cells. These single-molecule fluorescence studies have now reached a stage where they can provide new insights into important biological problems. After presenting the principles of these methods, I will give some examples from our current research to probe the molecular basis of neurodegeneration. Here we have used single-molecule fluorescence to detect and analyse the low concentrations of soluble protein aggregates thought to be responsible for Alzheimer’s disease and determine the mechanisms by which they damage neurons. Lastly, I will describe how fundamental science aimed at watching single molecules incorporating nucleotides into DNA gave rise to a new rapid method to sequence DNA that is now widely used.

Fred Ramsdell traces the discovery of FOXP3 from the striking immune phenotype of the scurfy mouse to the mapping and cloning of the responsible gene. He explains how linking a disease phenotype to its genetic cause opened a route to understanding the regulation of immune responses and the biology of regulatory T cells. The lecture considers FOXP3 as a regulator of immune function and the implications of this history for therapeutic intervention. It argues for the continuing value of phenotype-first research and naturally occurring disease mutations in discovering biological mechanisms and identifying potential treatment targets, even as genome sequencing becomes increasingly routine.