A suite of enhancer AAVs and transgenic mouse lines for genetic access to cortical cell types.

The mammalian cortex is comprised of cells with different morphological, physiological, and molecular properties that can be classified according to shared properties into cell types. Defining the contribution of each cell type to the computational and cognitive processes that are guided by the cortex is essential for understanding its function in health and disease. We use transcriptomic and epigenomic cortical cell type taxonomies from mice and humans to define marker genes and enhancers, and to build genetic tools for cortical cell types. Here, we present a large toolkit for selective targeting of cortical populations, including mouse transgenic lines and recombinant adeno-associated virus (AAV) vectors containing genomic enhancers. We report evaluation of fifteen new transgenic driver lines and over 680 different enhancer AAVs covering all major subclasses of cortical cells, with many achieving a high degree of specificity, comparable with existing transgenic lines. We find that the transgenic lines based on marker genes can provide exceptional specificity and completeness of cell type labeling, but frequently require generation of a triple-transgenic cross for best usability/specificity. On the other hand, enhancer AAVs are easy to screen and use, and can be easily modified to express diverse cargo, such as recombinases. However, their use depends on many factors, such as viral titer and route of administration. The tools reported here as well as the scaled process of tool creation provide an unprecedented resource that should enable diverse experimental strategies towards understanding mammalian cortex and brain function.

Situación epidemiológica de VIH a nivel global y nacional: Puesta al día / Epidemiological situation of HIV at global and national level: update

INTRODUCCIÓN: En las cuatro décadas de la epidemia por VIH, se han observado avances notables que han contribuido a una disminución progresiva en la incidencia de nuevas infecciones y en la mortalidad a nivel mundial. Sin embargo, no ha ocurrido lo mismo en Latinoamérica y en Chile. OBJETIVO: Esta revisión tiene como objetivo conocer la epidemiología actual a nivel global, latinoamericano y chileno. METODOLOGÍA: Se analizaron los informes epidemiológicos oficiales de infección por VIH emitidos por organismos nacionales e internacionales, más los estudios epidemiológicos nacionales. RESULTADOS: Se estima que 39 millones de personas viven con VIH en el mundo; no obstante, la mortalidad y la incidencia de nuevos casos han disminuido de forma notoria durante las últimas dos décadas, asociado a una expansión en al acceso a terapia antirretroviral en forma global. A diferencia del resto del mundo, América Latina presenta una tendencia de alza en las nuevas infecciones y Chile registra un aumento de 35% en nuevos casos durante los últimos 10 años, coincidiendo con un aumento en los flujos de migrantes que ha afectado a la región. Algunas estrategias preventivas como la profilaxis pre exposición se han implementa-do a un ritmo lento, tanto a nivel mundial, como latinoamericano. CONCLUSIONES: La epidemiología del VIH presenta características propias regionales y nacionales. En particular en Chile, diversos factores incluyendo déficit en políticas públicas de prevención y los recientes flujos migratorios han modelado nuestra actual epidemia. El desafío presente debe contemplar los esfuerzos multisectoriales para lograr los objetivos de ONUSIDA en esta década.

BACKGROUND: Over the course of the last four decades of global HIV epidemic, significant improvements have contributed to gradually reduce the frequency of new infections and global mortality rates. However, in Latin America particularly in Chile, new infections continue increasing. AIM: This review aims to comprehend the_epidemiology today on a worldwide, Latin American, and Chilean scale. METHODS: National epidemiology studies and official HIV reports from international and national organizations were reviewed. RESULTS: It is estimated that 39 million people live with HIV worldwide; however, mortality and the incidence of new cases have decreased markedly over the last two decades, associated with an expansion in access to antiretroviral therapy globally. In contrast to the rest of the world, Latin America shows an upward trend in new infections, with Chile registering a 35% increase in new cases over the last 10 years, coinciding with an increase in migratory flows that has occurred throughout the region. Some preventive strategies, such as pre-exposure prophylaxis, have been implemented at a slow pace, both globally and in Latin America. CONCLUSIONS: The epidemiology of HIV has regional and national characteristics. Specifically in Chile, several factors, including deficits in public prevention policies and recent migratory flows, have shaped our current epidemic. The present challenge must contemplate multisectoral efforts to achieve the UNAIDS objectives during this decade.

Enhancer-AAVs allow genetic access to oligodendrocytes and diverse populations of astrocytes across species.

Proper brain function requires the assembly and function of diverse populations of neurons and glia. Single cell gene expression studies have mostly focused on characterization of neuronal cell diversity; however, recent studies have revealed substantial diversity of glial cells, particularly astrocytes. To better understand glial cell types and their roles in neurobiology, we built a new suite of adeno-associated viral (AAV)-based genetic tools to enable genetic access to astrocytes and oligodendrocytes. These oligodendrocyte and astrocyte enhancer-AAVs are highly specific (usually > 95% cell type specificity) with variable expression levels, and our astrocyte enhancer-AAVs show multiple distinct expression patterns reflecting the spatial distribution of astrocyte cell types. To provide the best glial-specific functional tools, several enhancer-AAVs were: optimized for higher expression levels, shown to be functional and specific in rat and macaque, shown to maintain specific activity in epilepsy where traditional promoters changed activity, and used to drive functional transgenes in astrocytes including Cre recombinase and acetylcholine-responsive sensor iAChSnFR. The astrocyte-specific iAChSnFR revealed a clear reward-dependent acetylcholine response in astrocytes of the nucleus accumbens during reinforcement learning. Together, this collection of glial enhancer-AAVs will enable characterization of astrocyte and oligodendrocyte populations and their roles across species, disease states, and behavioral epochs.

Signature morphoelectric properties of diverse GABAergic interneurons in the human neocortex.

Human cortex transcriptomic studies have revealed a hierarchical organization of γ-aminobutyric acid-producing (GABAergic) neurons from subclasses to a high diversity of more granular types. Rapid GABAergic neuron viral genetic labeling plus Patch-seq (patch-clamp electrophysiology plus single-cell RNA sequencing) sampling in human brain slices was used to reliably target and analyze GABAergic neuron subclasses and individual transcriptomic types. This characterization elucidated transitions between PVALB and SST subclasses, revealed morphological heterogeneity within an abundant transcriptomic type, identified multiple spatially distinct types of the primate-specialized double bouquet cells (DBCs), and shed light on cellular differences between homologous mouse and human neocortical GABAergic neuron types. These results highlight the importance of multimodal phenotypic characterization for refinement of emerging transcriptomic cell type taxonomies and for understanding conserved and specialized cellular properties of human brain cell types.

[SARS-CoV-2 infection in people living with HIV. Topics on the global panorama and in Chile]. / Infección por SARS-CoV-2 en personas viviendo con VIH. Tópicos del panorama mundial y en Chile.

The COVID-19 disease is caused by the SARS-CoV-2 virus and was declared a pandemic by the WHO on March 11, 2020. To date, more than 500 million people have been infected and it has caused over 6 million deaths worldwide. People that belong to the most vulnerable risk groups, such as those at the extremes of life, patients with chronic comorbidities and those with severe immunosuppression, are especially susceptible to developing a severe form of COVID-19 infection and death. Subjects living with HIV, especially those in precarious immunological conditions or those in whom antiretroviral therapy is yet to be started, may be at risk of developing complications related to COVID-19, as observed with other infectious diseases. This review aims to determine the magnitude of the impact of the SARS-CoV-2 virus on people living with HIV in Chile.

Infección por SARS-CoV-2 en personas viviendo con VIH: tópicos del panorama mundial y en Chile / SARS-CoV-2 infection in people living with HIV: topics on the global panorama and in Chile

Resumen La enfermedad COVID-19 es una patología de origen infeccioso causada por el virus SARS-CoV-2 y fue declarada pandemia por la OMS el 11 de marzo 2020. A la fecha se registran más de 500 millones de personas infectadas y más de 6 millones de fallecidos en todo el mundo. Las personas que pertenecen a grupos de riesgo más vulnerables, particularmente aquellos que se encuentran en los extremos de la vida, con comorbilidades crónicas o inmunocomprometidas, son especialmente susceptibles a presentar un eventual mayor riesgo de infección, de gravedad y de mortalidad por esta enfermedad. Las personas que viven con VIH, especialmente aquellos en precarias condiciones inmunológicas o aquellas que aún no inician terapia antiretroviral, pueden ser un grupo especialmente susceptible de presentar complicaciones relacionadas a esta y otras enfermedades de origen infeccioso. Con esta revisión se pretende determinar la magnitud del impacto del virus SARS-CoV-2 en personas viviendo con VIH en Chile.

Abstract The COVID-19 disease is caused by the SARS-CoV-2 virus and was declared a pandemic by the WHO on March 11, 2020. To date, more than 500 million people have been infected and it has caused over 6 million deaths worldwide. People that belong to the most vulnerable risk groups, such as those at the extremes of life, patients with chronic comorbidities and those with severe immunosuppression, are especially susceptible to developing a severe form of COVID-19 infection and death. Subjects living with HIV, especially those in precarious immunological conditions or those in whom antiretroviral therapy is yet to be started, may be at risk of developing complications related to COVID-19, as observed with other infectious diseases. This review aims to determine the magnitude of the impact of the SARS-CoV-2 virus on people living with HIV in Chile.

Functional enhancer elements drive subclass-selective expression from mouse to primate neocortex.

Viral genetic tools that target specific brain cell types could transform basic neuroscience and targeted gene therapy. Here, we use comparative open chromatin analysis to identify thousands of human-neocortical-subclass-specific putative enhancers from across the genome to control gene expression in adeno-associated virus (AAV) vectors. The cellular specificity of reporter expression from enhancer-AAVs is established by molecular profiling after systemic AAV delivery in mouse. Over 30% of enhancer-AAVs produce specific expression in the targeted subclass, including both excitatory and inhibitory subclasses. We present a collection of Parvalbumin (PVALB) enhancer-AAVs that show highly enriched expression not only in cortical PVALB cells but also in some subcortical PVALB populations. Five vectors maintain PVALB-enriched expression in primate neocortex. These results demonstrate how genome-wide open chromatin data mining and cross-species AAV validation can be used to create the next generation of non-species-restricted viral genetic tools.

Ca(2+) entry into neurons is facilitated by cooperative gating of clustered CaV1.3 channels.

CaV1.3 channels regulate excitability in many neurons. As is the case for all voltage-gated channels, it is widely assumed that individual CaV1.3 channels behave independently with respect to voltage-activation, open probability, and facilitation. Here, we report the results of super-resolution imaging, optogenetic, and electrophysiological measurements that refute this long-held view. We found that the short channel isoform (CaV1.3S), but not the long (CaV1.3L), associates in functional clusters of two or more channels that open cooperatively, facilitating Ca(2+) influx. CaV1.3S channels are coupled via a C-terminus-to-C-terminus interaction that requires binding of the incoming Ca(2+) to calmodulin (CaM) and subsequent binding of CaM to the pre-IQ domain of the channels. Physically-coupled channels facilitate Ca(2+) currents as a consequence of their higher open probabilities, leading to increased firing rates in rat hippocampal neurons. We propose that cooperative gating of CaV1.3S channels represents a mechanism for the regulation of Ca(2+) signaling and electrical activity.

Graded Ca²âº/calmodulin-dependent coupling of voltage-gated CaV1.2 channels.

In the heart, reliable activation of Ca(2+) release from the sarcoplasmic reticulum during the plateau of the ventricular action potential requires synchronous opening of multiple CaV1.2 channels. Yet the mechanisms that coordinate this simultaneous opening during every heartbeat are unclear. Here, we demonstrate that CaV1.2 channels form clusters that undergo dynamic, reciprocal, allosteric interactions. This 'functional coupling' facilitates Ca(2+) influx by increasing activation of adjoined channels and occurs through C-terminal-to-C-terminal interactions. These interactions are initiated by binding of incoming Ca(2+) to calmodulin (CaM) and proceed through Ca(2+)/CaM binding to the CaV1.2 pre-IQ domain. Coupling fades as [Ca(2+)]i decreases, but persists longer than the current that evoked it, providing evidence for 'molecular memory'. Our findings suggest a model for CaV1.2 channel gating and Ca(2+)-influx amplification that unifies diverse observations about Ca(2+) signaling in the heart, and challenges the long-held view that voltage-gated channels open and close independently.

Molecular determinants controlling NMDA receptor synaptic incorporation.

Synaptic incorporation of NMDA receptors (NMDARs) is regulated by GluN2 subunits with different rules controlling GluN2A- and GluN2B-containing receptors; whereas GluN2B-containing receptors are constitutively incorporated into synapses, GluN2A incorporation is activity-dependent. We expressed electrophysiologically tagged NMDARs in rat hippocampal slices to identify the molecular determinants controlling the mode of synaptic incorporation of NMDARs. Expressing chimeric GluN2 subunits, we identified a putative N-glycosylation site present in GluN2B, but not in GluN2A, as necessary and sufficient to drive NMDARs into synapses in an activity-independent manner. This suggests a novel mechanism for regulating activity-driven changes and trafficking of NMDARs to the synapse.

Organotypic hippocampal slice cultures.

The hippocampus, a component of the limbic system, plays important roles in long-term memory and spatial navigation. Hippocampal neurons can modify the strength of their connections after brief periods of strong activation. This phenomenon, known as long-term potentiation (LTP) can last for hours or days and has become the best candidate mechanism for learning and memory. In addition, the well defined anatomy and connectivity of the hippocampus has made it a classical model system to study synaptic transmission and synaptic plasticity. As our understanding of the physiology of hippocampal synapses grew and molecular players became identified, a need to manipulate synaptic proteins became imperative. Organotypic hippocampal cultures offer the possibility for easy gene manipulation and precise pharmacological intervention but maintain synaptic organization that is critical to understanding synapse function in a more naturalistic context than routine culture dissociated neurons methods. Here we present a method to prepare and culture hippocampal slices that can be easily adapted to other brain regions. This method allows easy access to the slices for genetic manipulation using different approaches like viral infection or biolistics. In addition, slices can be easily recovered for biochemical assays, or transferred to microscopes for imaging or electrophysiological experiments.

Requirement for caspase-2 in stress-induced apoptosis before mitochondrial permeabilization.

A current view is that cytotoxic stress, such as DNA damage, induces apoptosis by regulating the permeability of mitochondria. Mitochondria sequester several proteins that, if released, kill by activating caspases, the proteases that disassemble the cell. Cytokines activate caspases in a different way, by assembling receptor complexes that activate caspases directly; in this case, the subsequent mitochondrial permeabilization accelerates cell disassembly by amplifying caspase activity. We found that cytotoxic stress causes activation of caspase-2, and that this caspase is required for the permeabilization of mitochondria. Therefore, we argue that cytokine-induced and stress-induced apoptosis act through conceptually similar pathways in which mitochondria are amplifiers of caspase activity rather than initiators of caspase activation.