Reeves, Cristian A Lasagna

Abstract Background Progressive supranuclear palsy (PSP) is a devastating neurodegenerative disorder with rapid progression to death. Multiple efforts to develop a disease‐modifying therapy for PSP have yet to succeed. We previously reported the discovery of three potential therapeutic targets for PSP. These three genes, DDR2, KANK2, and STOM, are consistently upregulated in the brains of PSP patients and mouse models of tauopathy. Reducing their expression in a drosophila model significantly rescued the tau‐induced eye degeneration and tau seeding activity. To translate these targets into the clinic, we are developing antisense oligonucleotides (ASO) as potential therapies. Method We screened candidate ASOs in neuroglioma cell line H4. ASO hits were validated for their potency and safety. We also used iPSC‐derived neurons from PSP patients to test the potency and potential toxicity of our ASOs. To investigate target engagement, therapeutic efficacy and preliminary pharmacodynamic properties, we treated iPSC‐derived midbrain organoids with the ASO leads. Lastly, we will assess the off‐target effects of the ASOs by investigating the levels of mRNA transcripts that share similar sequences as our targets using RNAseq. Result Our in vitro screening identified ASO leads that significantly reduce the target gene expressions at mRNA and protein levels in a dose‐dependent manner. The ASO hits also exhibit low cellular toxicity in the H4 cell line. Upon treating iPSC neurons with ASO hits, we were able to validate the dose‐dependent reduction of target mRNAs. Cell viability analysis indicated low toxicity, suggesting that these ASOs are potent and safe. In midbrain organoid model, DDR2‐targeting ASO showed time‐dependent target engagement and were prioritized for further development. Conclusion We developed candidate therapeutics for PSP using ASOs that silence novel therapeutic targets discovered in our systems based cross‐species screening. We showed that our ASOs have low toxicity and confirmed target engagement at both mRNA and protein levels in vitro. The results suggest a favorable preclinical profile of our ASO leads that warrants additional investigations. Importantly, PSP shares congruent pathophysiological changes with other tauopathies, such as Alzheimer’s disease. These common perturbed pathways indicate that our ASOs can be potentially repurposed for multiple neurodegenerative tauopathies.

Abstract Background Neurodegenerative tauopathies, characterized by the aggregation of misfolded tau protein, poses a significant clinical and scientific challenge. Tau interaction with other proteins affects the formation of ‘tau‐seed’ but less is known about its effects on tau‐seed properties. Our previous study revealed that Bassoon (BSN), a presynaptic protein, interacts with tau‐seed, exacerbating its toxicity. A parallel study also associated missense mutations in BSN with tau aggregation in patients, prompting an investigation into the influence of BSN and its mutations in neurodegeneration for potential therapeutic insights. Method We generated a knock‐in mouse model (BSNKI) harboring the disease‐associated p.Pro3866Ala mutation in endogenous Bsn. Cognitive and motor abilities were evaluated throughout the lifespan of heterozygous and homozygous BSNKI mice, along with analyses of BSN and tau patterns, gliosis, and gene expression changes in their brains. Additionally, we conducted single‐nucleic RNA sequencing to identify changes at the cellular level. Result BSNKI mice displayed progressive motor and memory impairments on the rotarod and fear conditioning assays, respectively. At 10 months, their brains displayed somatic BSN and pathological tau accumulation, with the gene expression changes indicating alterations of microglia, classical complement cascade, and synapse pruning pathways, as well as dysregulation of protein quality control pathways. Neuropathological data suggests that the BSN P3866A mutation instigates robust microglia activation, increased microglia‐mediated synapse engulfment, and a co‐deposition of proteasomal subunits with BSN. Furthermore, snRNA‐seq data suggest changes in neuronal populations and cell signaling. Conclusion Our BSNKI mouse model revealed the contributions of BSN P3866A mutation to tau pathogenesis and neurodegeneration. BSNKI animals displayed progressive motor and memory impairments, deposits of endogenous tau and BSN, as well as changes in gene expression and neuronal populations. Notably, BSN likely plays a dual role, promoting tau aggregation and sequestering protein degradation molecules, leading to tau and protein accumulation at the soma and synapse, which in turn could trigger neuroinflammation and other cellular changes. These findings propose BSN as a promising therapeutic target for tauopathies, underscoring the need for further exploration to elucidate underlying mechanisms and therapeutic implications.

AbstractBackgroundTau aggregates, a hallmark of Alzheimer’s disease (AD) and other tauopathies, spread throughout the brain, contributing to neurodegeneration. How this propagation occurs remains elusive. Previous research suggests that tau‐seed interactors play a crucial role. Based on this, the study aimed to identify novel tau‐seed interactors in AD brains and validate their impact in vivo.MethodAD and control brain extracts were separated in fractions by Size Exclusion Chromatography. Fractions with the highest tau seeding activity, measured using a tai‐biosensor cell line, were analyzed by mass spectrometry to identify interacting proteins. Bioinformatic tools dissected enriched pathways, identifying interactors that were validated in a Drosophila tauopathy model by genetically interfering with their homologs and assessing tau accumulation and eye degeneration.ResultsTau seeding activity was concentrated in high molecular weight fractions containing only a small portion of total tau in the AD brains. Compared to controls, AD brains revealed a distinct interactome for tau‐seeds, enriched in proteins associated with synaptic and mitochondrial pathways. Notably, Drosophila screening confirmed that several novel interactors significantly reduced tau accumulation and eye degeneration, suggesting their potential therapeutic relevance.ConclusionThis study sheds light on tau propagation mechanisms in AD by identifying novel tau‐seed interactors. These interactors, particularly those involved in synaptic and mitochondrial pathways, offer promising targets for therapeutic interventions aimed at decreasing tau spread and potentially preventing neurodegeneration in tauopathies. The findings add to the growing evidence that targeting tau‐seed interactors, like previously identified BSN, could represent a novel strategy for treating these debilitating conditions.

AbstractBackgroundTau aggregation is the major cause of several neurodegenerative tauopathies. Tau interaction with other proteins affects the formation of tau aggregates with seeding activity but less is known about its effects on tau‐seed properties. Our previous study revealed that Bassoon (BSN), a presynaptic protein, interacts with tau‐seed, exacerbating its toxicity in vivo. Bsndownregulation reduced tau spreading and overall pathology. Intriguingly, a parallel study associated missense mutations in BSN with tau aggregation in patients, prompting an investigation into the influence of genetic mutations in BSN on tau pathology for potential therapeutic insights.MethodWe generated a knock‐in mouse model (BSNKI) harboring the disease‐associated p.Pro3866Ala mutation in endogenous Bsn. Cognitive and motor abilities were assessed in aged heterozygous and homozygous BSNKI mice, followed by analyses of BSN and tau patterns, gliosis, and gene expression changes in their brains. Additionally, we validated our findings in a human BSN mutation carrier.ResultAt 10 months, BSNKI mice displayed motor impairments on the rotarod, and grip strength assays compared to WT mice. Their brains displayed somatic BSN and pathological tau accumulation, with the gene expression changes indicating alterations in microglia activation, protein degradation pathways, complement activation, and synapse pruning. We also observed an accumulation of pathological tau at the synapses and synapse engulfment by microglia. Histopathological analyses revealed robust microglia activation and co‐deposition of proteasomal subunits with BSN. The human BSN mutation carrier displayed inclusions of BSN and tau pathology similar to observations in the BSNKI model.ConclusionOur BSNKI mouse model, reflecting a disease‐associated BSN mutation, revealed motor impairments and pathological tau and BSN deposits, mirroring observations in BSN mutation carriers. Notably, BSN seems to play a dual role, promoting tau aggregation and sequestering protein degradation molecules, leading to tau and protein accumulation at the soma and synapse, triggering microgliosis and neuroinflammation. These findings propose BSN as a promising therapeutic target for tauopathies, underscoring the need for further exploration to elucidate underlying mechanisms and therapeutic implications.

AbstractBackgroundTau aggregation is the underlying cause of several neurodegenerative tauopathies. Formation of tau aggregates with seeding activity involves interactions with other proteins, but with unknown effects on tau‐seed properties. Our previous study found that Bassoon (BSN), a presynaptic protein, interacts with tau‐seed, exacerbating its toxicity in vivo. Bsn downregulation reduced tau spreading and overall pathology. A previous study also linked missense mutations in BSN to the aggregation of 3‐ and 4‐repeat tau in patients. However, it is unknown how genetic mutations in BSN influence tau pathology in vivo.MethodWe developed a knock‐in mouse model harboring a mutation in endogenous BSN (p.Pro3882Ala), equivalent to p.Pro3866Ala BSN mutation identified in patients. We evaluated the cognitive and motor abilities of aged heterozygous and homozygous BSNKI mice and also we analyzed BSN and tau patterns in the brains of these mice. Additionally, we examined BSN and tau patterns in the BSN mutation carrier.ResultAt 10 months, BSNKI mice did not exhibit anxiety‐related behavior and had similar activity as WT littermates. They also displayed similar cognitive abilities as the WT mice. Interestingly, the BSNKI mice displayed lower latency to fall off the rotarod and reduced grip strength compared to the WT mice. We also observed an increase in the accumulation of pathological tau and BSN deposits in the brains of the BSNKI mice. The BSN mutation carrier displayed inclusions of BSN and tau pathology.ConclusionWe developed a mouse model with a knock‐in mutation in the Bassoon gene, allowing us to evaluate the effects of this disease‐associated mutation in vivo. Our results have shown that the mice with Bsn mutation have impairments in motor coordination, and postural instability, similar to BSN mutation carriers and show pathological tau and BSN deposits in the brain. We found inclusions of BSN and tau pathology in BSN mutation the carrier that resembled certain pathological features of AD and PSP. These findings suggest that BSN plays a role in tau pathology and targeting it may represent a promising therapeutic strategy for tauopathies. Further research is needed to fully understand our findings' underlying mechanisms and potential therapeutic implications.