Behavioral and neuropathological characterization over the adult lifespan of the human tau knock-in mouse

Tau is a microtubule-associated protein with a diverse functional repertoire linked to neurodegenerative disease. Recently, a human tau knock-in (MAPT KI) mouse was developed that may overcome many limitations associated with current animal models used to study tau. In MAPT KI mice, the entire murine Mapt gene was replaced with the human MAPT gene under control of the endogenous Mapt promoter. This model represents an ideal in vivo platform to study the function and dysfunction of human tau protein. Accordingly, a detailed understanding of the effects MAPT KI has on structure and function of the CNS is warranted. Here, we provide a detailed behavioral and neuropathological assessment of MAPT KI mice. We compared MAPT KI to wild-type (WT) C57BL/6j mice in behavioral assessments of anxiety, attention, working memory, spatial memory, and motor performance from 6 to 24 months (m) of age. Using immunohistological and biochemical assays, we quantified markers of glia (microglia, astrocytes and oligodendrocytes), synaptic integrity, neuronal integrity and the cytoskeleton. Finally, we quantified levels of total tau, tau isoforms, tau phosphorylation, and tau conformations. MAPT KI mice show normal cognitive and locomotor behavior at all ages, and resilience to mild age-associated locomotor deficits observed in WT mice. Markers of neuronal and synaptic integrity are unchanged in MAPT KI mice with advancing age. Glial markers are largely unchanged in MAPT KI mice, but glial fibrillary acidic protein is increased in the hippocampus of WT and MAPT KI mice at 24 m. MAPT KI mice express all 6 human tau isoforms and levels of tau remain stable throughout adulthood. Hippocampal tau in MAPT KI and WT mice is phosphorylated at serine 396/404 (PHF1) and murine tau in WT animals displays more PHF1 phosphorylation at 6 and 12 m. Lastly, we extended previous reports showing that MAPT KI mice do not display overt pathology. No evidence of other tau phosphorylation residues (AT8, pS422) or abnormal conformations (TNT2 or TOC1) associated with pathogenic tau were detected. The lack of overt pathological changes in MAPT KI mice make this an ideal platform for future investigations into the function and dysfunction of tau protein in vivo

Continuous Collection of Adeno-Associated Virus from Producer Cell Medium Significantly Increases Total Viral Yield

The ability to efficiently produce large amounts of high-titer recombinant adeno-associated virus (AAV) is a prerequisite to the continued success of AAV as a gene therapy tool targeted toward large-animal preclinical studies or human clinical therapeutics. Current manufacturing procedures necessitate laborious and time-consuming purification procedures to obtain AAV particles of sufficient titer and purity for these demanding biomedical applications. The finding that AAV can be harvested and purified from producer cell medium may represent an efficient alternative to purifying AAV from cellular lysates. Here we sought to determine the maximum duration of time, and frequency within which AAV can be harvested from producer cell medium, in order to maximize the yield obtained from a single transfection preparation. Human embryonic kidney 293T cells were transfected with polyethylenimine to produce AAV2/5 expressing green fluorescent protein (GFP), and cellular medium was harvested every 2 days until a maximum duration of 19 days posttransfection. AAV2/5-GFP was released into producer cell medium at a steady state until 7 days posttransfection, at which time titers dropped dramatically. Harvesting medium every two days resulted in the maximum yield of AAV from a single preparation, and the cumulative yield of AAV harvested from the producer cell medium was 4-fold higher than the yield obtained from a traditional purification of AAV from cellular lysates. The AAV2/5 harvested from medium within the 7-day collection time-course mediated high levels of transduction in vivo, comparable to AAV2/5 harvested from cellular lysates. AAV purified from cell lysates showed increasing amounts of empty particles at 5 and 7 days posttransfection, whereas AAV purified from cell medium did not show an increase in the amount of empty particles throughout the 7-day time course. Finally, we extended these findings to AAV2/9, demonstrating that a comparable ratio of AAV2/9 particles are also released for up to 7 days posttransfection

Continuous collection of adeno-associated virus from producer cell medium significantly increases total viral yield

The ability to efficiently produce large amounts of high-titer recombinant adeno-associated virus (AAV) is a prerequisite to the continued success of AAV as a gene therapy tool targeted toward large-animal preclinical studies or human clinical therapeutics. Current manufacturing procedures necessitate laborious and time-consuming purification procedures to obtain AAV particles of sufficient titer and purity for these demanding biomedical applications. The finding that AAV can be harvested and purified from producer cell medium may represent an efficient alternative to purifying AAV from cellular lysates. Here we sought to determine the maximum duration of time, and frequency within which AAV can be harvested from producer cell medium, in order to maximize the yield obtained from a single transfection preparation. Human embryonic kidney 293T cells were transfected with polyethylenimine to produce AAV2/5 expressing green fluorescent protein (GFP), and cellular medium was harvested every 2 days until a maximum duration of 19 days posttransfection. AAV2/5-GFP was released into producer cell medium at a steady state until 7 days posttransfection, at which time titers dropped dramatically. Harvesting medium every two days resulted in the maximum yield of AAV from a single preparation, and the cumulative yield of AAV harvested from the producer cell medium was 4-fold higher than the yield obtained from a traditional purification of AAV from cellular lysates. The AAV2/5 harvested from medium within the 7-day collection time-course mediated high levels of transduction in vivo, comparable to AAV2/5 harvested from cellular lysates. AAV purified from cell lysates showed increasing amounts of empty particles at 5 and 7 days posttransfection, whereas AAV purified from cell medium did not show an increase in the amount of empty particles throughout the 7-day time course. Finally, we extended these findings to AAV2/9, demonstrating that a comparable ratio of AAV2/9 particles are also released for up to 7 days posttransfection

Multimodal production of adeno-associated virus

Adeno-associated virus (AAV) is an increasingly popular tool in the research laboratory, and use of this viral vector clinically is occurring at an accelerated pace. Nevertheless, despite its popularity, AAV is a relatively cumbersome virus to produce; however, significant efforts have been invested to develop, optimize, and simplify methodology that allows the generation of high-quality AAV with significantly increased production yields. Here we describe multiple modalities for production and purification of AAV particles produced in HEK293 cell cultures using an iodixanol density gradient. We include two methods adapted for harvesting virus from the culture media: Tangential flow filtration (TFF) and polyethylene glycol precipitation (PEGylation). Moreover, we also describe the protocol for anion exchange chromatography, which can be used after the iodixanol gradient as an additional purification step. Last, we provide various protocols for determining virus titer

Silencing Alpha Synuclein in Mature Nigral Neurons Results in Rapid Neuroinflammation and Subsequent Toxicity

Human studies and preclinical models of Parkinson\u27s disease implicate the involvement of both the innate and adaptive immune systems in disease progression. Further, pro-inflammatory markers are highly enriched near neurons containing pathological forms of alpha synuclein (α-syn), and α-syn overexpression recapitulates neuroinflammatory changes in models of Parkinson\u27s disease. These data suggest that α-syn may initiate a pathological inflammatory response, however the mechanism by which α-syn initiates neuroinflammation is poorly understood. Silencing endogenous α-syn results in a similar pattern of nigral degeneration observed following α-syn overexpression. Here we aimed to test the hypothesis that loss of α-syn function within nigrostriatal neurons results in neuronal dysfunction, which subsequently stimulates neuroinflammation. Adeno-associated virus (AAV) expressing an short hairpin RNA (shRNA) targeting endogenous α-syn was unilaterally injected into the substantia nigra pars compacta (SNc) of adult rats, after which nigrostriatal pathology and indices of neuroinflammation were examined at 7, 10, 14 and 21 days post-surgery. Removing endogenous α-syn from nigrostriatal neurons resulted in a rapid up-regulation of the major histocompatibility complex class 1 (MHC-1) within transduced nigral neurons. Nigral MHC-1 expression occurred prior to any overt cell death and coincided with the recruitment of reactive microglia and T-cells to affected neurons. Following the induction of neuroinflammation, α-syn knockdown resulted in a 50% loss of nigrostriatal neurons in the SNc and a corresponding loss of nigrostriatal terminals and dopamine (DA) concentrations within the striatum. Expression of a control shRNA did not elicit any pathological changes. Silencing α-syn within glutamatergic neurons of the cerebellum did not elicit inflammation or cell death, suggesting that toxicity initiated by α-syn silencing is specific to DA neurons. These data provide evidence that loss of α-syn function within nigrostriatal neurons initiates a neuronal-mediated neuroinflammatory cascade, involving both the innate and adaptive immune systems, which ultimately results in the death of affected neurons

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Human studies and preclinical models of Parkinson\u27s disease implicate the involvement of both the innate and adaptive immune systems in disease progression. Further, pro-inflammatory markers are highly enriched near neurons containing pathological forms of alpha synuclein (α-syn), and α-syn overexpression recapitulates neuroinflammatory changes in models of Parkinson\u27s disease. These data suggest that α-syn may initiate a pathological inflammatory response, however the mechanism by which α-syn initiates neuroinflammation is poorly understood. Silencing endogenous α-syn results in a similar pattern of nigral degeneration observed following α-syn overexpression. Here we aimed to test the hypothesis that loss of α-syn function within nigrostriatal neurons results in neuronal dysfunction, which subsequently stimulates neuroinflammation. Adeno-associated virus (AAV) expressing an short hairpin RNA (shRNA) targeting endogenous α-syn was unilaterally injected into the substantia nigra pars compacta (SNc) of adult rats, after which nigrostriatal pathology and indices of neuroinflammation were examined at 7, 10, 14 and 21 days post-surgery. Removing endogenous α-syn from nigrostriatal neurons resulted in a rapid up-regulation of the major histocompatibility complex class 1 (MHC-1) within transduced nigral neurons. Nigral MHC-1 expression occurred prior to any overt cell death and coincided with the recruitment of reactive microglia and T-cells to affected neurons. Following the induction of neuroinflammation, α-syn knockdown resulted in a 50% loss of nigrostriatal neurons in the SNc and a corresponding loss of nigrostriatal terminals and dopamine (DA) concentrations within the striatum. Expression of a control shRNA did not elicit any pathological changes. Silencing α-syn within glutamatergic neurons of the cerebellum did not elicit inflammation or cell death, suggesting that toxicity initiated by α-syn silencing is specific to DA neurons. These data provide evidence that loss of α-syn function within nigrostriatal neurons initiates a neuronal-mediated neuroinflammatory cascade, involving both the innate and adaptive immune systems, which ultimately results in the death of affected neurons

Regulation of dopamine neurotransmission from serotonergic neurons by ectopic expression of the dopamine D2 autoreceptor blocks levodopa-induced dyskinesia

Abstract Levodopa-induced dyskinesias (LID) are a prevalent side effect of chronic treatment with levodopa (L-DOPA) for the motor symptoms of Parkinson’s disease (PD). It has long been hypothesized that serotonergic neurons of the dorsal raphe nucleus (DRN) are capable of L-DOPA uptake and dysregulated release of dopamine (DA), and that this “false neurotransmission” phenomenon is a main contributor to LID development. Indeed, many preclinical studies have demonstrated LID management with serotonin receptor agonist treatment, but unfortunately, promising preclinical data has not been translated in large-scale clinical trials. Importantly, while there is an abundance of convincing clinical and preclinical evidence supporting a role of maladaptive serotonergic neurotransmission in LID expression, there is no direct evidence that dysregulated DA release from serotonergic neurons impacts LID formation. In this study, we ectopically expressed the DA autoreceptor D2Rs (or GFP) in the DRN of 6-hydroxydopamine (6-OHDA) lesioned rats. No negative impact on the therapeutic efficacy of L-DOPA was seen with rAAV-D2Rs therapy. However, D2Rs treated animals, when subjected to a LID-inducing dose regimen of L-DOPA, remained completely resistant to LID, even at high doses. Moreover, the same subjects remained resistant to LID formation when treated with direct DA receptor agonists, suggesting D2Rs activity in the DRN blocked dyskinesogenic L-DOPA priming of striatal neurons. In vivo microdialysis confirmed that DA efflux in the striatum was reduced with rAAV-D2Rs treatment, providing explicit evidence that abnormal DA release from DRN neurons can affect LID. This is the first direct evidence of dopaminergic neurotransmission in DRN neurons and its modulation with rAAV-D2Rs gene therapy confirms the serotonin hypothesis in LID, demonstrating that regulation of serotonergic neurons achieved with a gene therapy approach offers a novel and potent antidyskinetic therapy

Regulation of dopamine neurotransmission from serotonergic neurons by ectopic expression of the dopamine D2 autoreceptor blocks levodopa-induced dyskinesia

Levodopa-induced dyskinesias (LID) are a prevalent side effect of chronic treatment with levodopa (L-DOPA) for the motor symptoms of Parkinson\u27s disease (PD). It has long been hypothesized that serotonergic neurons of the dorsal raphe nucleus (DRN) are capable of L-DOPA uptake and dysregulated release of dopamine (DA), and that this false neurotransmission phenomenon is a main contributor to LID development. Indeed, many preclinical studies have demonstrated LID management with serotonin receptor agonist treatment, but unfortunately, promising preclinical data has not been translated in large-scale clinical trials. Importantly, while there is an abundance of convincing clinical and preclinical evidence supporting a role of maladaptive serotonergic neurotransmission in LID expression, there is no direct evidence that dysregulated DA release from serotonergic neurons impacts LID formation. In this study, we ectopically expressed the DA autoreceptor D2R (or GFP) in the DRN of 6-hydroxydopamine (6-OHDA) lesioned rats. No negative impact on the therapeutic efficacy of L-DOPA was seen with rAAV-D2R therapy. However, D2R treated animals, when subjected to a LID-inducing dose regimen of L-DOPA, remained completely resistant to LID, even at high doses. Moreover, the same subjects remained resistant to LID formation when treated with direct DA receptor agonists, suggesting D2R activity in the DRN blocked dyskinesogenic L-DOPA priming of striatal neurons. In vivo microdialysis confirmed that DA efflux in the striatum was reduced with rAAV-D2R treatment, providing explicit evidence that abnormal DA release from DRN neurons can affect LID. This is the first direct evidence of dopaminergic neurotransmission in DRN neurons and its modulation with rAAV-D2R gene therapy confirms the serotonin hypothesis in LID, demonstrating that regulation of serotonergic neurons achieved with a gene therapy approach offers a novel and potent antidyskinetic therapy

Impact of age and vector construct on striatal and nigral transgene expression

Therapeutic protein delivery using viral vectors has shown promise in preclinical models of Parkinson's disease (PD) but clinical trial success remains elusive. This may partially be due to a failure to include advanced age as a covariate despite aging being the primary risk factor for PD. We investigated transgene expression following intracerebral injections of recombinant adeno-associated virus pseudotypes 2/2 (rAAV2/2), 2/5 (rAAV2/5), 2/9 (rAAV2/9), and lentivirus (LV) expressing green fluorescent protein (GFP) in aged versus young adult rats. Both rAAV2/2 and rAAV2/5 yielded lower GFP expression following injection to either the aged substantia nigra or striatum. rAAV2/9-mediated GFP expression was deficient in the aged striatonigral system but displayed identical transgene expression between ages in the nigrostriatal system. Young and aged rats displayed equivalent GFP levels following LV injection to the striatonigral system but LV-delivered GFP was deficient in delivering GFP to the aged nigrostriatal system. Notably, age-related transgene expression deficiencies revealed by protein quantitation were poorly predicted by GFP-immunoreactive cell counts. Further, in situ hybridization for the viral CβA promoter revealed surprisingly limited tropism for astrocytes compared to neurons. Our results demonstrate that aging is a critical covariate to consider when designing gene therapy approaches for PD

Silencing Alpha Synuclein in Mature Nigral Neurons Results in Rapid Neuroinflammation and Subsequent Toxicity

Human studies and preclinical models of Parkinson’s disease implicate the involvement of both the innate and adaptive immune systems in disease progression. Further, pro-inflammatory markers are highly enriched near neurons containing pathological forms of alpha synuclein (α-syn), and α-syn overexpression recapitulates neuroinflammatory changes in models of Parkinson’s disease. These data suggest that α-syn may initiate a pathological inflammatory response, however the mechanism by which α-syn initiates neuroinflammation is poorly understood. Silencing endogenous α-syn results in a similar pattern of nigral degeneration observed following α-syn overexpression. Here we aimed to test the hypothesis that loss of α-syn function within nigrostriatal neurons results in neuronal dysfunction, which subsequently stimulates neuroinflammation. Adeno-associated virus (AAV) expressing an short hairpin RNA (shRNA) targeting endogenous α-syn was unilaterally injected into the substantia nigra pars compacta (SNc) of adult rats, after which nigrostriatal pathology and indices of neuroinflammation were examined at 7, 10, 14 and 21 days post-surgery. Removing endogenous α-syn from nigrostriatal neurons resulted in a rapid up-regulation of the major histocompatibility complex class 1 (MHC-1) within transduced nigral neurons. Nigral MHC-1 expression occurred prior to any overt cell death and coincided with the recruitment of reactive microglia and T-cells to affected neurons. Following the induction of neuroinflammation, α-syn knockdown resulted in a 50% loss of nigrostriatal neurons in the SNc and a corresponding loss of nigrostriatal terminals and dopamine (DA) concentrations within the striatum. Expression of a control shRNA did not elicit any pathological changes. Silencing α-syn within glutamatergic neurons of the cerebellum did not elicit inflammation or cell death, suggesting that toxicity initiated by α-syn silencing is specific to DA neurons. These data provide evidence that loss of α-syn function within nigrostriatal neurons initiates a neuronal-mediated neuroinflammatory cascade, involving both the innate and adaptive immune systems, which ultimately results in the death of affected neurons