Karen E. Duff

The accumulation of pathological four-repeat (4R) tau is central to several frontotemporal dementia (FTD) subtypes, but human neuronal models amenable to high-throughput screening of 4R tau–targeting therapies remain very limited. To address this, we developed induced pluripotent stem cell (iPSC)–derived i ³ Neuron (i ³ N) lines expressing >75% 4R tau, driven by FTD splice-shifting mutations (Ser ³⁰⁵ Asn; S305N or S305N/IVS10 + 3). These neurons develop hyperphosphorylated tau and demonstrate somatodendritic mislocalization. These i ³ N neurons develop endogenous seed-competent tau and present pentameric formyl thiophene acetic acid–(pFTAA)-positive tau assemblies after 28 days in culture. For scalable screening, we CRISPR-engineered an HiBiT luminescence tag at the endogenous MAPT locus into the S305N/IVS10 + 3 iPSC line, enabling precise quantification of tau levels and pharmacological responses. The model responded predictably to compounds affecting tau clearance, demonstrating its suitability for drug discovery. Overall, this i ³ N platform recapitulates key features of 4R tauopathy and provides a robust system to identify therapeutic modulators of pathological tau.

Transcriptomics has transformed our understanding of the brain, but assigning transcriptomic identities to neurons recorded in vivo remains challenging at scale. Existing platforms can pair transcriptomic identity with two-photon calcium imaging in small populations of approximately 100 neurons, but they require recorded cells to be sparse and therefore cannot be applied to large population recordings. Here, we present coppaFISH 3D, a spatially resolved transcriptomics method, and CASTalign, an in silico alignment framework, which together enable transcriptomic identification of thousands of simultaneously recorded cells. coppaFISH 3D detects hundreds of genes in thick 50μm fixed sections while preserving tissue integrity, enabling both 3D registration to in vivo imaging and integration with immunofluorescence labelling. The platform is fully powered by open chemistry and open source software, runs on commodity hardware, and can be performed at very low cost per section. It therefore enables transcriptomic identification of recorded neurons at scale, making it possible to study how transcriptomic identity shapes activity in neural populations.

Neurofibrillary tangles composed of hyperphosphorylated tau are a defining pathological hallmark of Alzheimer’s disease (AD); however, the pathways and mechanisms associated with the transition from physiological tau to tangle pathology remain unclear. Here, we integrate laser microdissection of post-mortem, fixed human AD brain tissue labelled with an antibody recognizing tangle-associated phospho-tau (AT8) with mass spectrometry–based proteomics, applied to individual neurons and to small neuronal pools. This approach identified ∼2,000 and ∼5,000 proteins, respectively, and enabled direct detection of disease-associated tau phosphorylation sites without prior enrichment. A layered analysis of the proteome of tangle-positive and tangle-negative neurons revealed heterogeneous disease-associated states. Pseudotime analysis, combined with an AI-driven analytical framework, indicates that neurons do not segregate into discrete classes but instead organize along a continuum of proteomic changes that correlate with tau abundance. This organization enabled the construction of a trajectory of pathological neuronal responses that can be resolved within an individual brain. Early stages of this trajectory are characterized by coordinated remodeling of proteostasis networks, including reduced proteasome component abundance and increased lysosomal acidification machinery, followed by disruption of synaptic pathways. Notably, despite extensive proteomic remodeling, neurons bearing tangles show little evidence of activated cell-death programs, suggesting prolonged molecular adaptation rather than acute degeneration. Together, these findings establish a framework for single-cell–resolved proteome analysis of human brain disease in situ and define a continuum of neuronal states underlying tau pathogenesis, revealing early vulnerabilities and adaptive responses during AD progression.

Neural regeneration therapies aim to treat neurodegeneration by promoting the proliferation and maturation of exogenous or endogenous neural progenitor cells (NPCs). However, their efficacy has been limited. Deep brain stimulation (DBS) via implanted electrodes has been shown to promote neurogenesis in vitro and in vivo. Still, its invasiveness precludes deployment in research and widespread clinical use. Temporal interference (TI) has emerged as a strategy for non‐invasive, high‐precision DBS using multiple kHz‐range electric fields to target the deep brain. Here, we validate the potential of TI stimulation for neural regeneration augmentation in the central nervous system (CNS). First, we showed that TI stimulation modulated at the theta‐band frequency enhances the maturation of embryonic neural progenitor cells in vitro. We then demonstrate that theta‐band TI stimulation targeting the hippocampus enhances endogenous hippocampal neurogenesis in an in vivo mouse model of Alzheimer's disease‐like amyloidosis. By uncovering frequency‐specific control of stem cell fate, we propose a clinically relevant regeneration strategy that avoids pharmacological or genetic manipulation. Our results enable focal, non‐invasive augmentation of deep‐brain neural regeneration via electrical stimulation.

Tau pathology underlies a broad spectrum of neurodegenerative disorders, collectively termed tauopathies, yet these diseases exhibit striking heterogeneity in their biological mechanisms and clinical outcomes. The basis for this heterogeneity remains poorly understood. Here, we address this question using knock-in mouse models expressing two distinct frontotemporal dementia-associated tau mutations to define how different tau variants drive divergent pathogenic programs in vivo. We find that the two mutations give rise to fundamentally different trajectories of tau pathogenesis. One trajectory is marked by progressive tau hyperphosphorylation and cytoskeletal destabilization occurring in the absence of detectable tau seed formation. In contrast, an alternative trajectory is characterized by tau hypophosphorylation, early seed formation, and alterations in nucleotide metabolism and chromatin organization, without overt cytoskeletal disruption. With aging, tau in this latter pathway transitions to a hyperphosphorylated state and forms mature fibrillar aggregates. Genetic enhancement of β-amyloid selectively accelerates fibril formation, particularly in the model exhibiting early seeding. Together, these findings demonstrate that distinct tau mutations can engage separable pathogenic mechanisms, providing a biological framework for the heterogeneity observed across tauopathies and highlighting the need for mechanism-informed therapeutic strategies and patient stratification.

Tau pathology underlies a broad spectrum of neurodegenerative disorders, collectively termed tauopathies, yet these diseases exhibit striking heterogeneity in their biological mechanisms and clinical outcomes. The basis for this heterogeneity remains poorly understood. Here, we address this question using knock-in mouse models expressing two distinct frontotemporal dementia-associated tau mutations to define how different tau variants drive divergent pathogenic programs in vivo . We find that the two mutations give rise to fundamentally different trajectories of tau pathogenesis. One trajectory is marked by progressive tau hyperphosphorylation and cytoskeletal destabilization occurring in the absence of detectable tau seed formation. In contrast, an alternative trajectory is characterized by tau hypophosphorylation, early seed formation, and alterations in nucleotide metabolism and chromatin organization, without overt cytoskeletal disruption. With aging, tau in this latter pathway transitions to a hyperphosphorylated state and forms mature fibrillar aggregates. Genetic enhancement of β-amyloid selectively accelerates fibril formation, particularly in the model exhibiting early seeding. Together, these findings demonstrate that distinct tau mutations can engage separable pathogenic mechanisms, providing a biological framework for the heterogeneity observed across tauopathies, and highlighting the need for mechanism-informed therapeutic strategies and patient stratification. Abstract Figure: Graphical Schematic representation comparing two trajectories of tau pathology. The S305N mutation promotes a 4R isoform shift, cytoskeletal damage and synapse loss, and accumulation of soluble hyperphosphorylated tau. Tau remains soluble even at old ages. In contrast, the P301S mutation generates hypophosphorylated, seed-competent tau that forms fibrils. The effect of amyloid is slow in the S305N, but results in accelerated acceleration of pathology in the P301S. Figure made with Biorender.com .

Background: Age is the greatest risk factor for Alzheimer's disease (AD) and aging is associated with decline in multiple pathways including cellular proteostasis efficiency, which has been shown to precipitate the accumulation of pathological proteins including tau. We are interested in whether there is an intersection between neuronal populations that are selectively vulnerable to age‐related proteostasis deficits and those that are vulnerable to tauopathy. These data may help explain the phenomenon of primary age‐related tauopathy (PART) (doi:10.1007/s00401‐014‐1349‐0) and provide a framework to study the relationship between PART and AD. Method: To test this, we have used a range of spatial biology and computational techniques, including spatial cell‐typing and multi‐plex imaging in aged mice, and compared vulnerable neuron populations to those affected by tauopathy in new, targeted mouse models of tauopathy ( https://doi.org/10.1038/s41593‐024‐01829‐7 ). Result: During healthy aging, we have demonstrated that supragranular neurons from the entorhinal cortex uniquely accumulate the autophagy adaptor protein p62. Using a new spatial imaging technique, coppaFISH‐3D, we identified that Layer2/3 intra‐telencephalic projecting neurons of the entorhinal cortex (L2/3 IT ENT) are more likely to accumulate p62 than other L2/3 IT neurons. Those neurons are thought to be amongst the earliest ones affected by tau pathology. Building on a single‐nuclei multiome dataset we showed that a gene network including synapse and calcium related genes was enriched in L2/3 IT ENT to be profoundly downregulated with aging, suggesting a functional impairment of those neurons. Such downregulation was not seen in neighbouring L2/3 IT neurons. ‘Protected’ neurons had higher expression of a proteostasis‐related module, containing chaperone, lysosomal, and proteasome genes was observed. L2/3 IT ENT neurons fail to boost a proteostasis response potentially explaining p62 accumulation during ageing. We then assessed vulnerability to tau accumulation (including those phosphorylated at different epitopes) in the entorhinal cortex of a new MAPT KI mouse in situ , classifying affected neurons into fine transcriptomic subtypes. Affected neurons in the EC mapped to L2/3 IT ENT neurons. Conclusion: These data strongly support the idea that neuronal populations that are vulnerable to declining proteostasis in older age are also vulnerable to accumulating tau.

Neural regeneration therapies aim to treat neurodegeneration by promoting the proliferation and maturation of exogenous or endogenous neural progenitor cells (NPCs). However, their efficacy has been limited. Deep brain stimulation (DBS) via implanted electrodes has been shown to promote neurogenesis. However, its invasiveness precludes deployment in research and widespread clinical use. Temporal interference (TI) has emerged as a strategy for non-invasive, high-precision DBS using multiple kHz-range electric fields, with a frequency difference within the range of neural activity. Here, we validate the potential of TI stimulation for neural regeneration augmentation. We demonstrate that TI stimulation with a theta-band frequency difference enhances the maturation of embryonic neural progenitor cells in vitro . We then demonstrate that theta-band TI stimulation targeting the hippocampus enhances endogenous hippocampal neurogenesis in an in vivo mouse model of Alzheimer’s disease. By uncovering frequency-specific control of stem cell fate, we propose a clinically relevant regeneration strategy which avoids pharmacological or genetic manipulation. Our results demonstrate focal, non-invasive augmentation of deep-brain neural regeneration. GRAPHICAL

Preservation of synapses is crucial for healthy cognitive ageing, and synapse loss is one of the closest anatomical correlates of cognitive decline in Alzheimer disease, dementia with Lewy bodies and frontotemporal dementia. In these conditions, some synapses seem particularly vulnerable to degeneration whereas others are resilient and remain preserved. Evidence has highlighted that vulnerability and resilience are intrinsically distinct phenomena linked to specific brain structural and/or functional signatures, yet the key features of vulnerable and resilient synapses in the dementias remain incompletely understood. Defining the characteristics of vulnerable and resilient synapses in each form of dementia could offer novel insight into the mechanisms of synapse preservation and of synapse loss that underlies cognitive decline, thereby facilitating the discovery of targeted biomarkers and disease-modifying therapies. In this Review, we consider the concepts of synapse vulnerability and resilience, and provide an overview of our current understanding of the associations between synaptic protein changes, neuropathology and cognitive decline. We also consider how understanding of the underlying mechanisms could identify novel strategies to mitigate the cognitive dysfunction associated with dementias.

Tau pathology is a hallmark of several neurodegenerative diseases, including frontotemporal dementia and Alzheimer’s disease. However, the sequence of events and the form of tau that confers toxicity are still unclear, due in large part to the lack of physiological models of tauopathy initiation and progression in which to test hypotheses. We have developed a series of targeted mice expressing frontotemporal-dementia-causing mutations in the humanized MAPT gene to investigate the earliest stages of tauopathy. MAPT ^Int10+3G>A and MAPT ^{S305N;Int10+3G>A} lines show abundant hyperphosphorylated tau in the hippocampus and entorhinal cortex, but they do not develop seed-competent fibrillar structures. Accumulation of hyperphosphorylated tau was accompanied by neurite degeneration, loss of viable synapses and indicators of behavioral abnormalities. Our results demonstrate that neuronal toxicity can occur in the absence of fibrillar, higher-order structures and that tau hyperphosphorylation is probably involved in the earliest etiological events in tauopathies showing isoform ratio imbalance.