CRISPRi-based screens in iAssembloids to elucidate neuron-glia interactions.

The sheer complexity of the brain has complicated our ability to understand its cellular mechanisms in health and disease. Genome-wide association studies have uncovered genetic variants associated with specific neurological phenotypes and diseases. In addition, single-cell transcriptomics have provided molecular descriptions of specific brain cell types and the changes they undergo during disease. Although these approaches provide a giant leap forward towards understanding how genetic variation can lead to functional changes in the brain, they do not establish molecular mechanisms. To address this need, we developed a 3D co-culture system termed iAssembloids (induced multi-lineage assembloids) that enables the rapid generation of homogenous neuron-glia spheroids. We characterize these iAssembloids with immunohistochemistry and single-cell transcriptomics and combine them with large-scale CRISPRi-based screens. In our first application, we ask how glial and neuronal cells interact to control neuronal death and survival. Our CRISPRi-based screens identified that GSK3ß inhibits the protective NRF2-mediated oxidative stress response in the presence of reactive oxygen species elicited by high neuronal activity, which was not previously found in 2D monoculture neuron screens. We also apply the platform to investigate the role of APOE-ε4, a risk variant for Alzheimer's Disease, in its effect on neuronal survival. This platform expands the toolbox for the unbiased identification of mechanisms of cell-cell interactions in brain health and disease.

Tuberous sclerosis complex is associated with a novel human tauopathy.

Tuberous sclerosis complex (TSC) is a neurogenetic disorder leading to epilepsy, developmental delay, and neurobehavioral dysfunction. The syndrome is caused by pathogenic variants in TSC1 (coding for hamartin) or TSC2 (coding for tuberin). Recently, we reported a progressive frontotemporal dementia-like clinical syndrome in a patient with a mutation in TSC1, but the neuropathological changes seen in adults with TSC with or without dementia have yet to be systematically explored. Here, we examined neuropathological findings in adults with TSC (n = 11) aged 30-58 years and compared them to age-matched patients with epilepsy unrelated to TSC (n = 9) and non-neurological controls (n = 10). In 3 of 11 subjects with TSC, we observed a neurofibrillary tangle-predominant "TSC tauopathy" not seen in epilepsy or non-neurological controls. This tauopathy was observed in the absence of pathological amyloid beta, TDP-43, or alpha-synuclein deposition. The neurofibrillary tangles in TSC tauopathy showed a unique pattern of post-translational modifications, with apparent differences between TSC1 and TSC2 mutation carriers. Tau acetylation (K274, K343) was prominent in both TSC1 and TSC2, whereas tau phosphorylation at a common phospho-epitope (S202) was observed only in TSC2. TSC tauopathy was observed in selected neocortical, limbic, subcortical, and brainstem sites and showed a 3-repeat greater than 4-repeat tau isoform pattern in both TSC1 and TSC2 mutation carriers, but no tangles were immunolabeled with MC1 or p62 antibodies. The findings suggest that individuals with TSC are at risk for a unique tauopathy in mid-life and that tauopathy pathogenesis may involve TSC1, TSC2, and related molecular pathways.

Endocytic trafficking determines cellular tolerance of presynaptic opioid signaling.

Opioid tolerance is well-described physiologically but its mechanistic basis remains incompletely understood. An important site of opioid action in vivo is the presynaptic terminal, where opioids inhibit transmitter release. This response characteristically resists desensitization over minutes yet becomes gradually tolerant over hours, and how this is possible remains unknown. Here, we delineate a cellular mechanism underlying this longer-term form of opioid tolerance in cultured rat medium spiny neurons. Our results support a model in which presynaptic tolerance is mediated by a gradual depletion of cognate receptors from the axon surface through iterative rounds of receptor endocytosis and recycling. For the µ-opioid receptor (MOR), we show that the agonist-induced endocytic process which initiates iterative receptor cycling requires GRK2/3-mediated phosphorylation of the receptor's cytoplasmic tail, and that partial or biased agonist drugs with reduced ability to drive phosphorylation-dependent endocytosis in terminals produce correspondingly less presynaptic tolerance. We then show that the δ-opioid receptor (DOR) conforms to the same general paradigm except that presynaptic endocytosis of DOR, in contrast to MOR, does not require phosphorylation of the receptor's cytoplasmic tail. Further, we show that DOR recycles less efficiently than MOR in axons and, consistent with this, that DOR tolerance develops more strongly. Together, these results delineate a cellular basis for the development of presynaptic tolerance to opioids and describe a methodology useful for investigating presynaptic neuromodulation more broadly.

Cross-species screening platforms identify EPS-8 as a critical link for mitochondrial stress and actin stabilization.

The dysfunction of mitochondria is associated with the physiological consequences of aging and many age-related diseases. Therefore, critical quality control mechanisms exist to protect mitochondrial functions, including the unfolded protein response of the mitochondria (UPRMT). However, it is still unclear how UPRMT is regulated in mammals with mechanistic discrepancies between previous studies. Here, we reasoned that a study of conserved mechanisms could provide a uniquely powerful way to reveal previously uncharacterized components of the mammalian UPRMT. We performed cross-species comparison of genetic requirements for survival under­and in response to­mitochondrial stress between karyotypically normal human stem cells and the nematode Caenorhabditis elegans. We identified a role for EPS-8/EPS8 (epidermal growth factor receptor pathway substrate 8), a signaling protein adaptor, in general mitochondrial homeostasis and UPRMT regulation through integrin-mediated remodeling of the actin cytoskeleton. This study also highlights the use of cross-species comparisons in genetic screens to interrogate cellular pathways.

UPRER promotes lipophagy independent of chaperones to extend life span.

Longevity is dictated by a combination of environmental and genetic factors. One of the key mechanisms to regulate life-span extension is the induction of protein chaperones for protein homeostasis. Ectopic activation of the unfolded protein response of the endoplasmic reticulum (UPRER) specifically in neurons is sufficient to enhance organismal stress resistance and extend life span. Here, we find that this activation not only promotes chaperones but also facilitates ER restructuring and ER function. This restructuring is concomitant with lipid depletion through lipophagy. Activation of lipophagy is distinct from chaperone induction and is required for the life-span extension found in this paradigm. Last, we find that overexpression of the lipophagy component, ehbp-1, is sufficient to deplete lipids, remodel ER, and promote life span. Therefore, UPR induction in neurons triggers two distinct programs in the periphery: the proteostasis arm through protein chaperones and metabolic changes through lipid depletion mediated by EH domain binding protein 1 (EHBP-1).

Spatial regulation of the actin cytoskeleton by HSF-1 during aging.

There are many studies suggesting an age-associated decline in the actin cytoskeleton, and this has been adopted as common knowledge in the field of aging biology. However, a direct identification of this phenomenon in aging multicellular organisms has not been performed. Here, we express LifeAct::mRuby in a tissue-specific manner to interrogate cytoskeletal organization as a function of age. We show for the first time in Caenorhabditis elegans that the organization and morphology of the actin cytoskeleton deteriorate at advanced age in the muscles, intestine, and hypodermis. Moreover, hsf-1 is essential for regulating cytoskeletal integrity during aging, so that knockdown of hsf-1 results in premature aging of actin and its overexpression protects actin cytoskeletal integrity in the muscles, the intestine, and the hypodermis. Finally, hsf-1 overexpression in neurons alone is sufficient to protect cytoskeletal integrity in nonneuronal cells.

Acupuntura: percepción de usuarios en terapia por dolor / Acupuntura: percepción de usuarios en terapia por dolor

El presente estudio es una investigación que responde al paradigma interpretativo, de tipo exploratorio y metodología cualitativa, desde el enfoque fenomenológico. Esta investigación fue realizada en Santiago de Chile el año 2013 en un centro de acupuntura ubicado en la comuna de Independencia. Se realizaron siete entrevistas semi estructuradas a personas en terapia de acupuntura, obteniéndose las siguientes conclusiones: La acupuntura posee buena aceptación por parte de los usuarios, generando una opinión positiva hacia el uso de esta de terapia, debido a que se obtiene en la mayoría de los casos una resolución al problema de salud por el cual consultaron. Siendo considerada como un complemento o una alternativa, confiable y resolutiva de las patologías que presentan los entrevistados, a la medicina convencional.