Tau therapeutic candidates have been chiefly screened in vivo in transgenic tau mouse models, but this is expensive and laborious, and requires large numbers of aged mice in order to assess effects on tau aggregation and burden. Although there is usually affordable consensus that protein aggregation is usually tightly associated with neurodegeneration, there is limited understanding regarding (1) how protein aggregation impacts neurodegeneration, (2) what events trigger protein aggregation in the absence of mutations or overexpression, and (3) whether therapeutically targeting this aggregation leads to disease modification. Decades of research into neurodegenerative proteinopathies using in vivo and in vitro models have linked mutations and overexpression of these aggregation-prone proteins to the development of inclusions (Forman et al., 2004; Rademakers et al., 2004; Golde et al., 2013a; Goedert et al., 2017). Despite this intensive body of work in the field, mechanistic insights and therapeutic development have been limited by a lack of facile in vitro models that fully recapitulate proteinopathies found in humans. Exciting observations and preclinical development have mostly been conducted in vivo in mammalian models. Specifically, in the case of tau pathology, such as that observed in Alzheimers disease (AD), strong neurofibrillary tangle (NFT) development and pathology are only observed in transgenic rodent models (Lewis et al., 2000; Allen et al., 2002; Bue et al., 2010; Noble et al., 2010). These models restrict throughput and are expensive to maintain and age. Phenotypic variability in transgenic tau mice has been reported (Woerman et al., 2017), with gender differences and other confounding variables often cited (Noble et al., 2010; Jankowsky and Zheng, 2017), thereby hindering both preclinical therapeutic studies and studies probing mechanisms regulating tau pathology and tau-induced neurodegeneration. Nonmammalian models have been useful in enabling behavioral screening and the study of Avatrombopag tau phosphorylation, but no evidence of true tau inclusion pathology has been observed (Jackson et al., 2002; Kraemer et al., 2006; Brandt et al., 2009). Primary neuronal cultures or neuronally differentiated human induced pluripotent stem cell cultures have been used in efforts to create a reliable culture system to recapitulate inclusion pathology reflective of that observed in AD or Parkinsons disease (PD; Choi et al., 2014; Sposito et al., 2015). However, none have reproducibly and robustly shown mature neurofibrillary pathologies resembling those in human tauopathies or Lewy body (LB) pathology reminiscent of those found in PD. Further, these systems are not comprised of all the central nervous system (CNS) cell types, which may play a role in disease (Choi et al., 2014; Sposito et al., 2015). Indeed, in AD, where a genetic role of microglia has emerged recently (Guerreiro et al., 2013; Tejera and Heneka, 2016; Sims et al., 2017), an accessible system that enables the study of all the neuronal and nonneuronal cell types and their interactions in an environment where anatomical planes of connectivity are maintained would be highly useful. On this basis, we explored the feasibility of combining over a decade of experience in our laboratories optimizing CNS delivery of recombinant adeno-associated viruses (rAAVs) with a three-dimensional intact brain slice culture (BSC) system to see if we could develop more robust ex vivo models of AD and PD inclusion pathologies. These three-dimensional BSCs are functionally and physiologically relevant (Beach et al., 1982; Bahr, 1995; De Simoni et al., 2003), can be derived from brain areas involved in the human disease, and are comprised of neuronal and nonneuronal cell types. In addition, BSCs can often predict in vivo findings such as acute treatment of BSCs with small molecule compounds, recapitulating data obtained in in vivo studies (Croft et al., 2017a); other similarities and differences between in vivo models and BSCs are reviewed extensively by others (Sundstrom et al., 2005; Humpel, 2015). We find that rAAVs can be used to efficiently target neurons, astrocytes, microglia, and oligodendrocytes in this system and that expression is usually sustained long-term in culture. In.The rAAV-transduced BSCs will be an excellent tool to evaluate aggregate formation of additional tau variants in relevant CNS cell types as well as effects on cellular function. from neurons, microglia, astrocytes, and oligodendrocytes, alone or in combination, with transgene expression lasting for many months. These rAAV-based BSC models provide a cost-effective and facile alternative to in vivo studies, and in the future can become a widely adopted methodology to explore physiological and pathological mechanisms related to brain function and dysfunction. Introduction Genetic, pathological, and experimental modeling data all provide strong evidence that numerous neurodegenerative diseases are proteinopathies brought on by the accumulation of proteins within the brain (Forman et al., 2004; Golde et al., 2013a). Although there is usually affordable consensus that protein aggregation is tightly associated with neurodegeneration, there is limited understanding regarding (1) how protein aggregation impacts neurodegeneration, (2) what events trigger protein aggregation in the absence of mutations or overexpression, and (3) whether therapeutically targeting this aggregation leads to disease modification. Decades of research into neurodegenerative proteinopathies using in vivo and in vitro models have linked mutations and overexpression of these aggregation-prone proteins to the development of inclusions (Forman et al., 2004; Rademakers et al., 2004; Golde et al., 2013a; Goedert et al., 2017). Despite this intensive body of work in the field, mechanistic insights and therapeutic development have been limited by a lack of facile in vitro models that fully recapitulate proteinopathies found in humans. Exciting observations and preclinical development have mostly been conducted in vivo in mammalian models. Specifically, in the case of tau pathology, such as that observed in Alzheimers disease (AD), strong neurofibrillary tangle (NFT) development and pathology are Avatrombopag only observed in transgenic rodent models (Lewis et al., 2000; Allen et al., 2002; Bue et al., 2010; Noble et al., 2010). These models restrict throughput and are expensive to maintain and age. Phenotypic variability in transgenic tau mice has been reported (Woerman et al., 2017), with gender differences and other confounding variables often cited (Noble et al., 2010; Jankowsky and Zheng, 2017), thereby hindering both preclinical therapeutic studies and studies probing mechanisms regulating tau pathology and tau-induced neurodegeneration. Nonmammalian models have been useful in BMP10 enabling behavioral screening and the study of tau phosphorylation, but no evidence of true tau inclusion pathology has been observed (Jackson et al., 2002; Kraemer et al., 2006; Brandt et al., 2009). Primary neuronal cultures or neuronally differentiated human induced pluripotent stem cell cultures have been used in efforts to create a reliable culture system to recapitulate inclusion pathology reflective of that observed in AD or Parkinsons disease (PD; Choi et al., 2014; Sposito et al., 2015). However, none have reproducibly and robustly shown mature neurofibrillary pathologies resembling those in human tauopathies or Lewy body (LB) pathology reminiscent of those found in PD. Further, these systems are not comprised of all the central nervous system (CNS) cell types, which may play a role in disease (Choi et al., 2014; Sposito et al., 2015). Indeed, in AD, where a genetic role of microglia has emerged recently (Guerreiro et al., 2013; Tejera and Heneka, 2016; Sims et al., 2017), an accessible system that enables the study of all the neuronal and nonneuronal cell types and their interactions in an environment where anatomical planes of connectivity are maintained would be highly useful. On this basis, we explored the feasibility of combining over a decade of experience in our laboratories optimizing CNS delivery of recombinant adeno-associated viruses (rAAVs) with a three-dimensional intact brain slice culture (BSC) system to see if we could develop Avatrombopag more robust ex vivo models of AD and PD inclusion pathologies. These three-dimensional BSCs are functionally and physiologically relevant (Beach et al., 1982; Bahr, 1995; De Simoni et al., 2003),.