Soluble α-synuclein-antibody complexes activate the NLRP3 inflammasome in hiPSC-derived microglia.

Parkinson's disease is characterized by accumulation of α-synuclein (αSyn). Release of oligomeric/fibrillar αSyn from damaged neurons may potentiate neuronal death in part via microglial activation. Heretofore, it remained unknown if oligomeric/fibrillar αSyn could activate the nucleotide-binding oligomerization domain (NOD)-like receptor (NLR) family pyrin domain-containing 3 (NLRP3) inflammasome in human microglia and whether anti-αSyn antibodies could prevent this effect. Here, we show that αSyn activates the NLRP3 inflammasome in human induced pluripotent stem cell (hiPSC)-derived microglia (hiMG) via dual stimulation involving Toll-like receptor 2 (TLR2) engagement and mitochondrial damage. In vitro, hiMG can be activated by mutant (A53T) αSyn secreted from hiPSC-derived A9-dopaminergic neurons. Surprisingly, αSyn-antibody complexes enhanced rather than suppressed inflammasome-mediated interleukin-1ß (IL-1ß) secretion, indicating these complexes are neuroinflammatory in a human context. A further increase in inflammation was observed with addition of oligomerized amyloid-ß peptide (Aß) and its cognate antibody. In vivo, engraftment of hiMG with αSyn in humanized mouse brain resulted in caspase-1 activation and neurotoxicity, which was exacerbated by αSyn antibody. These findings may have important implications for antibody therapies aimed at depleting misfolded/aggregated proteins from the human brain, as they may paradoxically trigger inflammation in human microglia.

Anti-tau scFvs Targeted to the Cytoplasm or Secretory Pathway Variably Modify Pathology and Neurodegenerative Phenotypes.

Immunotherapies designed to treat neurodegenerative tauopathies that primarily engage extracellular tau may have limited efficacy as tau is primarily intracellular. We generated tau-targeting single-chain variable fragments (scFvs) and intrabodies (iBs) from the phosphorylated tau-specific antibodies CP13 and PHF1 and the pan-tau antibody Tau5. Recombinant adeno-associated virus (rAAV) was utilized to express these antibody fragments in homozygous JNPL3 P301L tau mice. Two iBs (CP13i, PHF1i) and one scFv (PHF1s) abrogated tau pathology and delayed time to severe hindlimb paralysis. In a second tauopathy model (rTg4510), CP13i and PHF1i reduced tau pathology, but cognate scFvs did not. These data demonstrate that (1) disease-modifying efficacy does not require antibody effector functions, (2) the intracellular targeting of tau with phosphorylated tau-specific iBs is more effective than extracellular targeting with the scFvs, and (3) robust effects on tau pathology before neurodegeneration only resulted in modest disease modification as assessed by delay of severe motor phenotype.

Intracerebral but Not Peripheral Infection of Live Porphyromonas gingivalis Exacerbates Alzheimer's Disease Like Amyloid Pathology in APP-TgCRND8 Mice.

The impact of oral microbial dysbiosis on Alzheimer's disease (AD) remains controversial. Building off recent studies reporting that various microbes might directly seed or promote amyloid ß (Aß) deposition, we evaluated the effects of periodontal bacteria (Porphyromonas gingivalis, Treponema denticola) and supragingival commensal (Streptococcus gordonii) oral bacterial infection in the APP-transgenic CRND8 (Tg) mice model of AD. We tracked bacterial colonization and dissemination, and monitored effects on gliosis and amyloid deposition. Chronic oral infection did not accelerate Aß deposition in Tg mice but did induce alveolar bone resorption, IgG immune response, and an intracerebral astrogliosis (GFAP: glial fibrillary acidic protein). In contrast, intracerebral inoculation of live but not heat-killed P. gingivalis increased Aß deposition and Iba-1 (ionized calcium-binding adaptor-1) microgliosis after 8 weeks of bacterial infection but not at 4 days. These data show that there may be differential effects of infectious microbes on glial activation and amyloid deposition depending on the species and route of inoculation, and thereby provide an important framework for future studies. Indeed, these studies demonstrate marked effects on amyloid ß deposition only in a fairly non-physiologic setting where live bacteria is injected directly into the brain.

Photodynamic studies reveal rapid formation and appreciable turnover of tau inclusions.

Accumulation of the tau protein in fibrillar intracellular aggregates is a defining feature of multiple neurodegenerative diseases collectively referred to as tauopathies. Despite intensive study of tau, there is limited information on the formation and clearance dynamics of tau inclusions. Using rAAV vectors to mediate expression of Dendra2-tagged human wild-type, P301L and pro-aggregant P301L/S320F tau proteins, with and without the addition of exogenous tau fibrillar seeds, we evaluated tau inclusion dynamics in organotypic brain slice culture (BSC) models using long-term optical pulse labeling methodology. Our studies reveal that tau inclusions typically form in 12-96 h in tauopathy BSC models. Unexpectedly, we demonstrate appreciable turnover of tau within inclusions with an average half-life of ~ 1 week when inclusions are newly formed. When BSCs with inclusions are aged in culture for extended periods, tau inclusions continue to turnover, but their half-lives increase to ~ 2 weeks and ~ 3 weeks after 1 and 2 months in culture, respectively. Individual tau inclusions can be long-lived structures that can persist for months in these BSC models and for even longer in the human brain. However, our data indicate that tau inclusions, are not 'tombstones', but dynamic structures with appreciable turnover. Understanding the cellular processes mediating this inclusion turnover may lead to new therapeutic strategies that could reverse pathological tau inclusion formation.

A C1qTNF3 collagen domain fusion chaperones diverse secreted proteins and anti-Aß scFvs: Applications for gene therapies.

Enhancing production of protein cargoes delivered by gene therapies can improve efficacy by reducing the amount of vector or simply increasing transgene expression levels. We explored the utility of a 126-amino acid collagen domain (CD) derived from the C1qTNF3 protein as a fusion partner to chaperone secreted proteins, extracellular "decoy receptor" domains, and single-chain variable fragments (scFvs). Fusions to the CD domain result in multimerization and enhanced levels of secretion of numerous fusion proteins while maintaining functionality. Efficient creation of bifunctional proteins using the CD domain is also demonstrated. Recombinant adeno-associated viral vector delivery of the CD with a signal peptide resulted in high-level expression with minimal biological impact as assessed by whole-brain transcriptomics. As a proof-of-concept in vivo study, we evaluated three different anti-amyloid Aß scFvs (anti-Aß scFvs), alone or expressed as CD fusions, following viral delivery to neonatal CRND8 mice. The CD fusion increased half-life, expression levels, and improved efficacy for amyloid lowering of a weaker binding anti-Aß scFv. These studies validate the potential utility of this small CD as a fusion partner for secretory cargoes delivered by gene therapy and demonstrate that it is feasible to use this CD fusion to create biotherapeutic molecules with enhanced avidity or bifunctionality.

Optical pulse labeling studies reveal exogenous seeding slows α-synuclein clearance.

The accumulation of α-synuclein (α-syn) in intracellular formations known as Lewy bodies (LBs) is associated with several neurodegenerative diseases including Parkinson's disease and Lewy Body Dementia. There is still limited understanding of how α-syn and LB formation is associated with cellular dysfunction and degeneration in these diseases. To examine the clearance and production dynamics of α-syn we transduced organotypic murine brain slice cultures (BSCs) with recombinant adeno-associated viruses (rAAVs) to express Dendra2-tagged human wild-type (WT) and mutant A53T α-syn, with and without the addition of exogenous α-syn fibrillar seeds and tracked them over several weeks in culture using optical pulse labeling. We found that neurons expressing WT or mutant A53T human α-syn show similar rates of α-syn turnover even when insoluble, phosphorylated Ser129 α-syn has accumulated. Taken together, this data reveals α-syn aggregation and overexpression, pSer129 α-syn, nor the A53T mutation affect α-syn dynamics in this system. Prion-type seeding with exogenous α-syn fibrils significantly slows α-syn turnover, in the absence of toxicity but is associated with the accumulation of anti-p62 immunoreactivity and Thiazin Red positivity. Prion-type induction of α-syn aggregation points towards a potential protein clearance deficit in the presence of fibrillar seeds and the ease of this system to explore precise mechanisms underlying these processes. This system facilitates the exploration of α-syn protein dynamics over long-term culture periods. This platform can further be exploited to provide mechanistic insight on what drives this slowing of α-syn turnover and how therapeutics, other genes or different α-syn mutations may affect α-syn protein dynamics.

LRP1 is a neuronal receptor for α-synuclein uptake and spread.

BACKGROUND: The aggregation and spread of α-synuclein (α-Syn) protein and related neuronal toxicity are the key pathological features of Parkinson's disease (PD) and Lewy body dementia (LBD). Studies have shown that pathological species of α-Syn and tau can spread in a prion-like manner between neurons, although these two proteins have distinct pathological roles and contribute to different neurodegenerative diseases. It is reported that the low-density lipoprotein receptor-related protein 1 (LRP1) regulates the spread of tau proteins; however, the molecular regulatory mechanisms of α-Syn uptake and spread, and whether it is also regulated by LRP1, remain poorly understood. METHODS: We established LRP1 knockout (LRP1-KO) human induced pluripotent stem cells (iPSCs) isogenic lines using a CRISPR/Cas9 strategy and generated iPSC-derived neurons (iPSNs) to test the role of LRP1 in α-Syn uptake. We treated the iPSNs with fluorescently labeled α-Syn protein and measured the internalization of α-Syn using flow cytometry. Three forms of α-Syn species were tested: monomers, oligomers, and pre-formed fibrils (PFFs). To examine whether the lysine residues of α-Syn are involved in LRP1-mediated uptake, we capped the amines of lysines on α-Syn with sulfo-NHS acetate and then measured the internalization. We also tested whether the N-terminus of α-Syn is critical for LRP1-mediated internalization. Lastly, we investigated the role of Lrp1 in regulating α-Syn spread with a neuronal Lrp1 conditional knockout (Lrp1-nKO) mouse model. We generated adeno-associated viruses (AAVs) that allowed for distinguishing the α-Syn expression versus spread and injected them into the hippocampus of six-month-old Lrp1-nKO mice and the littermate wild type (WT) controls. The spread of α-Syn was evaluated three months after the injection. RESULTS: We found that the uptake of both monomeric and oligomeric α-Syn was significantly reduced in iPSNs with LRP1-KO compared with the WT controls. The uptake of α-Syn PFFs was also inhibited in LRP1-KO iPSNs, albeit to a much lesser extent compared to α-Syn monomers and oligomers. The blocking of lysine residues on α-Syn effectively decreased the uptake of α-Syn in iPSNs and the N-terminus of α-Syn was critical for LRP1-mediated α-Syn uptake. Finally, in the Lrp1-nKO mice, the spread of α-Syn was significantly reduced compared with the WT littermates. CONCLUSIONS: We identified LRP1 as a key regulator of α-Syn neuronal uptake, as well as an important mediator of α-Syn spread in the brain. This study provides new knowledge on the physiological and pathological role of LRP1 in α-Syn trafficking and pathology, offering insight for the treatment of synucleinopathies.

Utilizing minimally purified secreted rAAV for rapid and cost-effective manipulation of gene expression in the CNS.

BACKGROUND: Recombinant adeno-associated virus (rAAV) is widely used in the neuroscience field to manipulate gene expression in the nervous system. However, a limitation to the use of rAAV vectors is the time and expense needed to produce them. To overcome this limitation, we evaluated whether unpurified rAAV vectors secreted into the media following scalable PEI transfection of HEK293T cells can be used in lieu of purified rAAV. METHODS: We packaged rAAV2-EGFP vectors in 30 different wild-type and mutant capsids and subsequently collected the media containing secreted rAAV. Genomic titers of each rAAV vector were assessed and the ability of each unpurified virus to transduce primary mixed neuroglial cultures (PNGCs), organotypic brain slice cultures (BSCs) and the mouse brain was evaluated. RESULTS: There was ~ 40-fold wide variance in the average genomic titers of the rAAV2-EGFP vector packaged in the 30 different capsids, ranging from a low ~ 4.7 × 1010 vector genomes (vg)/mL for rAAV2/5-EGFP to a high of ~ 2.0 × 1012 vg/mL for a capsid mutant of rAAV2/8-EGFP. In PNGC studies, we observed a wide range of transduction efficiency among the 30 capsids evaluated, with the rAAV2/6-EGFP vector demonstrating the highest overall transduction efficiency. In BSC studies, we observed robust transduction by wild-type capsid vectors rAAV2/6, 2/8 and 2/9, and by capsid mutants of rAAV2/1, 2/6, and 2/8. In the in vivo somatic brain transgenesis (SBT) studies, we found that intra-cerebroventricular injection of media containing unpurified rAAV2-EGFP vectors packaged with select mutant capsids resulted in abundant EGFP positive neurons and astrocytes in the hippocampus and forebrain of non-transgenic mice. We demonstrate that unpurified rAAV can express transgenes at equivalent levels to lysate-purified rAAV both in vitro and in vivo. We also show that unpurified rAAV is sufficient to drive tau pathology in BSC and neuroinflammation in vivo, recapitulating previous studies using purified rAAV. CONCLUSIONS: Unpurified rAAV vectors secreted into the media can efficiently transduce brain cells in vitro and in vivo, providing a cost-effective way to manipulate gene expression. The use of unpurified virus will greatly reduce costs of exploratory studies and further increase the utility of rAAV vectors for standard laboratory use.

Unique α-synuclein pathology within the amygdala in Lewy body dementia: implications for disease initiation and progression.

The protein α-synuclein (αsyn) forms pathologic aggregates in a number of neurodegenerative diseases including Lewy body dementia (LBD) and Parkinson's disease (PD). It is unclear why diseases such as LBD may develop widespread αsyn pathology, while in Alzheimer's disease with amygdala restricted Lewy bodies (AD/ALB) the αsyn aggregates remain localized. The amygdala contains αsyn aggregates in both LBD and in AD/ALB; to understand why αsyn pathology continues to progress in LBD but not in AD/ALB, tissue from the amygdala and other regions were obtained from 14 cases of LBD, 9 cases of AD/ALB, and 4 controls for immunohistochemical and biochemical characterization. Utilizing a panel of previously characterized αsyn antibodies, numerous unique pathologies differentiating LBD and AD/ALB were revealed; particularly the presence of dense neuropil αsyn aggregates, astrocytic αsyn, and αsyn-containing dystrophic neurites within senile plaques. Within LBD, these unique pathologies were predominantly present within the amygdala. Biochemically, the amygdala in LBD prominently contained specific carboxy-truncated forms of αsyn which are highly prone to aggregate, suggesting that the amygdala may be prone to initiate development of αsyn pathology. Similar to carboxy-truncated αsyn, it was demonstrated herein that the presence of aggregation prone A53T αsyn is sufficient to drive misfolding of wild-type αsyn in human disease. Overall, this study identifies within the amygdala in LBD the presence of unique strain-like variation in αsyn pathology that may be a determinant of disease progression.

An anti-CRF antibody suppresses the HPA axis and reverses stress-induced phenotypes.

Hypothalamic-pituitary-adrenal (HPA) axis dysfunction contributes to numerous human diseases and disorders. We developed a high-affinity monoclonal antibody, CTRND05, targeting corticotropin-releasing factor (CRF). In mice, CTRND05 blocks stress-induced corticosterone increases, counteracts effects of chronic variable stress, and induces other phenotypes consistent with suppression of the HPA axis. CTRND05 induces skeletal muscle hypertrophy and increases lean body mass, effects not previously reported with small-molecule HPA-targeting pharmacologic agents. Multiorgan transcriptomics demonstrates broad HPA axis target engagement through altering levels of known HPA-responsive transcripts such as Fkbp5 and Myostatin and reveals novel HPA-responsive pathways such as the Apelin-Apelin receptor system. These studies demonstrate the therapeutic potential of CTRND05 as a suppressor of the HPA axis and serve as an exemplar of a potentially broader approach to target neuropeptides with immunotherapies, as both pharmacologic tools and novel therapeutics.

Generation and characterization of new monoclonal antibodies targeting the PHF1 and AT8 epitopes on human tau.

Tauopathies are a group of neurodegenerative disorders, including Alzheimer's disease, defined by the presence of brain pathological inclusions comprised of abnormally aggregated and highly phosphorylated tau protein. The abundance of brain tau aggregates correlates with disease severity and select phospho-tau epitopes increase at early stages of disease. We generated and characterized a series of novel monoclonal antibodies directed to tau phosphorylated at several of these phospho-epitopes, including Ser396/Ser404, Ser404 and Thr205. We also generated phosphorylation independent antibodies against amino acid residues 193-211. We show that most of these antibodies are highly specific for tau and strongly recognize pathological inclusions in human brains and in a transgenic mouse model of tauopathy. They also reveal epitope-specific differences in the biochemical properties of Alzheimer's disease sarkosyl-insoluble tau. These new reagents will be useful for investigating the progression of tau pathology and further as tools to target the cellular transmission of tau pathology.