RESUMEN

The aggregation of the microtubule-associated protein tau is a defining feature of Alzheimer's disease and other tauopathies. Tau pathology is believed to be driven by free tau aggregates and tau carried within exosome-like extracellular vesicles, both of which propagate trans-synaptically and induce tau pathology in recipient neurons by a corrupting process of seeding. Here, we performed a genome-wide CRISPRi screen in tau biosensor cells and identified cellular regulators shared by both mechanisms of tau seeding. We identified ANKLE2, BANF1, NUSAP1, EIF1AD, and VPS18 as the top validated regulators that restrict tau aggregation initiated by both exosomal and vesicle-free tau seeds. None of our validated hits affected the uptake of either form of tau seeds, supporting the notion that they operate through a cell-autonomous mechanism downstream of the seed uptake. Lastly, validation studies with human brain tissue also revealed that several of the identified protein hits are down-regulated in the brains of Alzheimer's patients, suggesting that their decreased activity may be required for the emergence or progression of tau pathology in the human brain.

Asunto(s)

Enfermedad de Alzheimer , Exosomas , Tauopatías , Humanos , Proteínas tau/genética , Proteínas tau/metabolismo , Tauopatías/genética , Tauopatías/metabolismo , Tauopatías/patología , Enfermedad de Alzheimer/metabolismo , Encéfalo/metabolismo , Neuronas/metabolismo , Exosomas/genética

RESUMEN

The microtubule-associated protein tau has a critical role in Alzheimer's disease and other tauopathies. A proposed pathomechanism in the progression of tauopathies is the trans-synaptic spreading of tau seeds, with a role for exosomes which are secretory nanovesicles generated by late endosomes. Our previous work demonstrated that brain-derived exosomes isolated from tau transgenic rTg4510 mice encapsulate tau seeds with the ability to induce tau aggregation in recipient cells. We had also shown that exosomes can hijack the endosomal pathway to spread through interconnected neurons. Here, we reveal how tau seeds contained within internalized exosomes exploit mechanisms of lysosomal degradation to escape the endosome and induce tau aggregation in the cytosol of HEK293T-derived 'tau biosensor cells'. We found that the majority of the exosome-containing endosomes fused with lysosomes to form endolysosomes. Exosomes induced their permeabilization, irrespective of the presence of tau seeds, or whether the exosomal preparations originated from mouse brains or HEK293T cells. We also found that permeabilization is a conserved mechanism, operating in both non-neuronal tau biosensor cells and primary neurons. However, permeabilization of endolysosomes only occurred in a small fraction of cells, which supports the notion that permeabilization occurs by a thresholded mechanism. Interestingly, tau aggregation was only induced in cells that exhibited permeabilization, presenting this as an escape route of exosomal tau seeds into the cytosol. Overexpression of RAB7, which is required for the formation of endolysosomes, strongly increased tau aggregation. Conversely, inhibition of lysosomal function with alkalinizing agents, or by knocking-down RAB7, decreased tau aggregation. Together, we conclude that the enzymatic activities of lysosomes permeabilize exosomal and endosomal membranes, thereby facilitating access of exosomal tau seeds to cytosolic tau to induce its aggregation. Our data underscore the importance of endosomal membrane integrity in mechanisms of cellular invasion by misfolded proteins that are resistant to lysosomal degradation.

Asunto(s)

Citosol/metabolismo , Exosomas/fisiología , Lisosomas/fisiología , Proteínas tau/metabolismo , Animales , Autofagia , Endosomas/metabolismo , Células HEK293 , Humanos , Lentivirus/crecimiento & desarrollo , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Permeabilidad , Deficiencias en la Proteostasis , Tauopatías , Proteínas de Unión al GTP rab/biosíntesis , Proteínas de Unión al GTP rab/genética , Proteínas de Unión a GTP rab7

RESUMEN

Environmental enrichment can dramatically affect both the development and function of neural circuits. This is accomplished, at least in part, by the regulation of inhibitory cellular networks and related extracellular matrix glycoprotein structures known as perineuronal nets. The degree to which enhanced housing can influence brain areas involved in the planning and execution of actions is not well known. We examined the effect of enriching mice from birth on parvalbumin expression and perineuronal net formation in developing and adult striatum. This input nucleus of the basal ganglia consists of topographically discernible regions that serve different functions, providing a means of simultaneously examining the influence of environmental factors on discrete, but related networks. Greater densities of striatal parvalbumin positive cells and wisteria floribunda agglutinin labelled perineuronal nets were present in enriched pups during the second postnatal week, primarily within the lateral portion of the nucleus. Housing conditions continued to have an impact into adulthood, with enriched mice exhibiting higher parvalbumin positive cell densities in both medial and lateral striatum. Curiously, no differences due to housing conditions were detected in striatal perineuronal net densities of mature animals. The degree of overlap between striatal parvalbumin expression and perineuronal net formation was also increased, suggesting that heightened neural activity associated with enrichment may have contributed to greater engagement of networks affiliated with cells that express the calcium binding protein. Brain derived neurotrophic factor, an important regulator of inhibitory network maturation, is also subtly, but significantly affected within the striatum of enriched cohorts. Together, these findings suggest that environmental enrichment can exert cell specific effects within different divisions of an area vital for the regulation of action.