The study, published in the Journal of Advanced Research, addresses what the authors describe as a critically understudied area of medicine: tertiary stroke prevention. While primary prevention targets risk factors such as hypertension, diabetes, and smoking in people who have never had a stroke, and secondary prevention aims to stop recurrences in survivors through antiplatelet drugs and surgical interventions, tertiary prevention focuses on mitigating long-term disability and improving quality of life. Despite decades of progress in understanding how to protect neurons during the acute phase of ischemia, no clinically approved pharmacological intervention exists for promoting neural repair in the sub-acute and chronic phases that follow. This gap represents a substantial unmet medical need for the growing population of stroke survivors worldwide.

To investigate the biology of recovery, the researchers used a mouse photothrombosis model, in which cerebral vessels in the cortex are damaged by light-activated clotting to permanently block blood flow and cause a cortical infarction. A key advantage of this model is that it generates no penumbra, the salvageable tissue surrounding the infarct that complicates interpretation in other stroke models. Because the infarct is fixed and permanent, any recovery observed in the surrounding peri-infarct region can be attributed cleanly to spontaneous neural repair rather than to tissue rescue. Using laser speckle imaging, the team confirmed that the model successfully occluded cerebral blood flow and produced prominent cortical infarction visible by Nissl staining at day 14 after surgery.

The first major finding concerned where and when TERT appears in the recovering brain. Western blot analysis of peri-infarct tissue revealed that TERT protein expression rose significantly from day 7 after stroke, peaked at day 14, and then subsided, precisely matching the critical window of post-stroke spontaneous neural repair. To identify which brain cells were producing the enzyme, the researchers performed single-nucleus RNA sequencing on peri-infarct tissue collected at day 14, identifying nine major cell types by unsupervised clustering. TERT was expressed mainly in neurons, and immunofluorescent co-staining confirmed that roughly 78 percent of TERT in the peri-infarct cortex was detected in NeuN-positive neurons rather than in astrocytes, microglia, or endothelial cells. The pattern held in a second, independent stroke model, the middle cerebral artery occlusion model, where TERT was again upregulated at days 7 and 14 and predominantly localized to neurons.

Having established that TERT surges in peri-infarct neurons during recovery, the team asked what the enzyme was actually doing there. Although TERT is classically known for extending telomeres at chromosome ends, recent work has suggested it also regulates gene expression directly. Using Cleavage Under Targets and Tagmentation (CUT&Tag) sequencing, the researchers mapped TERT’s chromatin-binding sites across the whole genome in peri-infarct neurons and, in parallel, mapped the occupancy of Rpb1, a core subunit of RNA polymerase II that marks actively transcribing genes, along with the acetylated histone mark H3K27ac, which is correlated with active transcription. The analysis showed that most TERT-binding peaks were enriched at promoter regions, and that the set of TERT-bound promoters expanded by 27.8 percent in peri-infarct neurons compared with sham controls. Correlation with Rpb1 and H3K27ac signals indicated that TERT can act as both a positive and a negative transcriptional regulator in these neurons.

The identity of the genes whose promoters gained TERT binding was striking. Gene ontology analysis of the upregulated TERT-binding genes ranked terms such as regulation of axon regeneration, filopodium assembly, actin cytoskeleton organization, and positive regulation of neuron projection among the top biological processes, while known telomerase functions such as telomere maintenance appeared as expected positive controls. Even more intriguingly, motif enrichment analysis with HOMER revealed that the sequences bound by TERT best matched the recognition motif of cAMP response element-binding protein, or CREB, a transcription factor long regarded as a critical driver of neural plasticity, neurodevelopment, and repair. Known CREB-target genes involved in neural repair, including GAP43, EVL, NR4A4, and NRP2, were occupied by TERT at their promoters and showed higher transcriptional activity in peri-infarct neurons. When the researchers knocked down TERT in cultured cortical neurons subjected to an ischemia-induced neurite repair assay, the upregulated expression of these CREB-target genes was significantly abrogated, indicating that TERT is required for the transcriptional reprogramming that underlies neural repair.

To test whether boosting TERT could actively enhance recovery, the team delivered the enzyme into the brain using an intravenously injected, brain-tropic AAV PHP.eB vector carrying TERT under a neuron-specific promoter. Mice injected with the TERT overexpression vector showed markedly increased dendritic branching complexity and enriched mushroom and stubby spine morphologies in peri-infarct neurons, as revealed by Golgi-Cox staining and Sholl analysis, along with significantly enhanced axon and dendrite marker intensities measured by immunofluorescence for NEFM and PSD95. Western blotting confirmed upregulation of the repair-associated proteins GAP43, NEFM, and PSD95 in both the stroke-affected and contralateral hemispheres. Crucially, the structural repair translated into function: mice treated with TERT overexpression performed significantly better in the adhesive removal test of sensory function at day 28, and showed improved motor function on the grid-walk test and enhanced sensorimotor integration in the cylinder test from day 7 and day 14 onward, respectively.

Because gene therapy is an impractical near-term option for most patients, the researchers then turned to a pharmacological approach using cycloastragenol (CAG), a known TERT activator that is an active metabolite of Astragaloside IV isolated from the dried roots of Astragalus membranaceus, a legume used in traditional Chinese medicine as the herb Huangqi. The team administered CAG by oral gavage at a well-tolerated dose of 20 mg/kg from day 4 to day 14 after stroke, deliberately covering the recovery window while avoiding any confounding effect in the acute phase. CAG treatment did not change infarct size, confirming it acted on repair rather than tissue salvage, but it significantly increased TERT expression specifically in peri-infarct neurons. Golgi-Cox staining showed enhanced dendritic complexity and basal spine density, biotinylated dextran amine tracing revealed increased axon sprouting, and Western blots confirmed elevated levels of GAP43, NF160, PSD95, and the synaptic markers SYP and SYN. Behaviorally, CAG-treated mice showed significantly improved sensorimotor integration, fewer foot faults on the grid-walk, and shorter adhesive removal times. Conversely, when the researchers knocked down neuronal TERT with an AAV-delivered shRNA, both spontaneous recovery and the benefits of CAG treatment were abolished, demonstrating that endogenous neuronal TERT is necessary for the observed repair effects.

The study also uncovered an unexpected psychiatric benefit. Post-stroke depression affects an estimated 40 to 50 percent of stroke survivors and represents a major unmet therapeutic need. In the forced swim test, CAG-treated stroke mice showed significantly shortened immobility duration, and in the sucrose preference test they consumed significantly more sucrose, both indicating alleviation of depression-like behaviors, while an open-field test ruled out nonspecific psychostimulant effects. Surprisingly, neuronal TERT knockdown did not block the antidepressant effect, even though it abolished CAG-induced upregulation of brain-derived neurotrophic factor (BDNF) in the peri-infarct cortex, suggesting that BDNF is associated with the antidepressant action but that neuronal TERT may not be its key mediator. Using mass spectrometry-based phosphoproteomics, the researchers further traced the signaling pathway by which CAG activates TERT: kinase-substrate enrichment analysis pointed to the death-associated protein kinase DAPK1, and pharmacological inhibition of DAPK1 blocked CAG-induced TERT expression and the phosphorylation of the transcription factor STAT3 at its serine 727 residue, which binds to the mouse Tert promoter to drive transcription.

The translational profile of cycloastragenol adds to the excitement. The US Food and Drug Administration has designated CAG as generally recognized as safe, its oral bioavailability is approximately 25 percent, and its low molecular weight allows it to cross the blood-brain barrier. Clinical trials of CAG for anti-aging purposes have already demonstrated an excellent safety record, including a commercial health maintenance program spanning 7000 person-years of use with no adverse events attributed to the supplement, and preclinical studies across a broad dose range and experimental durations of 4 to 33 weeks have reported an overall favorable safety profile. Compared with AAV-mediated gene therapy, a small molecule offering transient, controllable upregulation of TERT may carry advantages in immunogenicity, cost, and dosing flexibility. The authors caution that important questions remain, including the precise mechanism of CAG’s antidepressant effect, the possible contributions of TERT in non-neuronal brain cells, and detailed pharmacokinetics in brain tissue. Nevertheless, the work provides a proof of concept that reactivating a telomerase enzyme in peri-infarct neurons, whether by gene delivery or by an ancient herbal compound, can rebuild neural circuits and restore function, positioning TERT as a promising therapeutic target for the recovery phase of stroke and potentially for other age-associated neurological diseases.

Subject of Research: The role of telomerase reverse transcriptase (TERT) in neural repair and functional recovery after ischemic stroke

Article Title: TERT is a therapeutic target for tertiary stroke prevention

Article References: TERT is a therapeutic target for tertiary stroke prevention. (n.d.). Original publication

Image Credits: AI Generated

DOI: Not provided

Keywords: TERT, telomerase, ischemic stroke, neural repair, cycloastragenol, CREB, neuroplasticity, tertiary prevention, post-stroke depression, DAPK1, STAT3, gene therapy

A new study published in the Journal of Advanced Research offers a strikingly different approach to attacking tau at its upstream source. Rather than blocking the activity of one of tau’s key dysregulating kinases, the research team designed a small molecule that eliminates the kinase protein itself. The target is death-associated protein kinase 1, or DAPK1, an enzyme that has accumulated an impressive pathogenic dossier in Alzheimer’s research. DAPK1 activates MARK1/2 to trigger abnormal tau phosphorylation, directly phosphorylates tau at disease-relevant residues, and through phosphorylation of SENP1 promotes tau SUMOylation, exacerbating tau pathology and cognitive dysfunction. Genetic knockdown and pharmacological inhibition of DAPK1 have both been shown to reduce tau phosphorylation and mitigate neurodegeneration in cell and animal models. Yet traditional small-molecule kinase inhibitors suffer from well-known drawbacks: short-lived efficacy, compensatory signaling from parallel pathways, and substantial off-target liability.

To overcome these limitations, the team turned to targeted protein degradation, a next-generation pharmacological strategy that hijacks the cell’s own proteostasis machinery. Their specific platform is hydrophobic tagging, or HyT, in which a bifunctional molecule couples a ligand that binds the protein of interest to a bulky hydrophobic moiety, typically adamantane, that mimics the exposed greasy surfaces of misfolded proteins. The cell’s quality-control machinery is fooled into ubiquitinating the tagged protein and destroying it via the ubiquitin-proteasome system. Adamantane-based hydrophobic tags carry distinct advantages over the better-known PROTAC degraders: simpler molecular design, lower molecular weight, favorable ADME characteristics, and high degradation efficiency. The approach has already been used to degrade tau, TDP-43, and mutant huntingtin, but it had never been applied to DAPK1.

From a library of candidate degraders built by conjugating a previously characterized DAPK1-binding ligand to adamantane- or fluorene-based hydrophobic tags through alkylamine linkers of varying length, one compound emerged as the clear leader. Named CJ1, the molecule reduced DAPK1 protein levels in mouse primary cortical neurons to roughly half of control levels at a screening concentration of 1 micromolar, outperforming all other compounds in the series. Molecular docking suggested that CJ1’s DAPK1-binding moiety occupies a distinct hydrophobic pocket on the kinase surface, with favorable electrostatic complementarity at the binding interface. Critically, cytotoxicity testing showed that CJ1 did not compromise the viability of primary neurons even at concentrations up to 20 micromolar, providing a wide margin between functional activity and cellular harm.

The mechanistic characterization of CJ1 was rigorous. In primary cortical neurons, the compound degraded DAPK1 in a dose-dependent fashion, with a half-maximal degradation concentration of approximately 0.18 micromolar, and a time course showing detectable degradation beginning four hours after treatment and sustained suppression lasting up to 48 hours. Quantitative PCR confirmed that DAPK1 mRNA levels were unchanged, indicating a posttranslational mechanism. When cells were pretreated with the proteasome inhibitor MG-132, CJ1-induced degradation was abolished, and ubiquitination assays demonstrated markedly increased polyubiquitinated DAPK1 in the presence of the compound. Together, these experiments established that CJ1 marks DAPK1 for ubiquitin-dependent proteasomal destruction, precisely the event-driven behavior expected of a hydrophobic tagging degrader.

With the mechanism confirmed, the investigators asked whether eliminating DAPK1 would tame tau. In primary cortical neurons, SH-SY5Y neuroblastoma cells, and SH-SY5Y cells expressing the P301L mutant human tau, CJ1 treatment reduced levels of tau phosphorylated at threonine 231, serine 262, and serine 396, along with total tau. These phosphorylation sites are deeply meaningful in Alzheimer’s biology: phosphorylation at Ser262 within the microtubule-binding repeat domain critically weakens tau’s grip on microtubules, Thr231 primes tau for further phosphorylation by GSK3beta and contributes to microtubule destabilization, and the Ser396/404 epitope marks advanced pathological tau. Several control experiments reinforced the specificity of the effect. CJ1 did not alter the levels of the major tau kinases GSK3beta and CDK5, the phosphatase PP2A, or the related kinase DAPK3. In DAPK1 knockout neurons, CJ1 lost its ability to reduce tau or phospho-tau, and a control compound lacking the DAPK1-binding moiety was inert. Overexpressing DAPK1 in cells also rescued the phenotype, strongly arguing that CJ1 acts specifically through DAPK1 degradation rather than through incidental off-target effects.

The in vivo results were the most consequential. A central hurdle for any Alzheimer’s therapeutic is crossing the blood-brain barrier, and liquid chromatography-mass spectrometry confirmed that intact CJ1 was detectable in mouse brain after systemic administration. In a tauopathy model created by injecting an adeno-associated virus carrying human P301L tau into the hippocampal CA3 region, DAPK1 was upregulated and CJ1 treatment lowered it in a dose-dependent manner, with 10 milligrams per kilogram nearly restoring baseline levels. CJ1 reduced human tau accumulation and phosphorylation at Thr231 and the AT8 epitope in the hippocampus. In aged hTau transgenic mice, which express all six human tau isoforms and develop genuine neurofibrillary tangles, five weeks of CJ1 treatment significantly reduced tangle burden in the cortex and lowered total tau and phospho-tau at multiple AD-relevant sites in both sarkosyl-soluble and, importantly, sarkosyl-insoluble fractions, the biochemical signature of aggregated, pathological tau.

Pathology translated into preserved brain structure and function. CJ1-treated tauopathy mice retained more NeuN-positive neurons, showed restored MAP2 immunoreactivity indicating healthier dendrites, and displayed dose-dependent recovery of dendritic spine density on Golgi-Cox staining. Neuroinflammation also receded: astrocytic activation marked by GFAP and microglial activation marked by Iba1 were both suppressed, pro-inflammatory iNOS decreased, and anti-inflammatory Arg-1 increased. Functionally, in the Morris water maze, CJ1-treated mice learned the platform location faster, spent more time in the target quadrant during the probe trial, and crossed the former platform location more often, with all groups swimming at comparable speeds, ruling out motor confounds. Notably, only the higher dose produced cognitive improvement, suggesting that partial biochemical correction of tau pathology is insufficient and that robust suppression is required for behavioral benefit.

Safety data were encouraging. CJ1 showed negligible hemolysis at concentrations up to 800 micrograms per milliliter, serum creatinine, ALT, and AST remained within normal ranges, and histological examination of the heart, liver, lung, and kidney revealed no treatment-related abnormalities. The authors are candid about limitations: the behavioral findings derive from an acute, region-specific model; the selectivity screen covered only a small panel of proteins; and the detailed brain distribution of CJ1 remains unmapped, as do potential differential effects on 3R versus 4R tau isoforms. Nonetheless, the study establishes CJ1 as a first-in-class DAPK1 degrader and provides a compelling proof of concept that hydrophobic tagging can eliminate an upstream driver of tau pathology inside cells, something neither conventional kinase inhibitors, which transiently block catalytic activity, nor tau immunotherapies, which target extracellular aggregates, can accomplish. By abolishing all of DAPK1’s functional domains and scaffolding roles in a catalytic, substoichiometric manner, this degrader strategy offers a mechanistically distinct and potentially more durable route to slowing the neurodegeneration that lies at the heart of Alzheimer’s disease.

Subject of Research: A hydrophobic tagging degrader that selectively eliminates DAPK1 to attenuate tau pathology in Alzheimer's disease

Article Title: Selective degradation of DAPK1 via a novel hydrophobic tagging attenuates tau pathology in Alzheimer’s disease

Article References: Li, R., Wu, X., Yao, J., Chen, J., Shui, X., Zheng, X., Tian, W., Wang, L., Zhou, Y., Zhang, T., Chen, D., Liu, Y., & Lee, T. H. (2026). Selective degradation of DAPK1 via a novel hydrophobic tagging attenuates tau pathology in Alzheimer’s disease. Journal of Advanced Research, 87, 1027-1043. https://doi.org/10.1016/j.jare.2025.12.037

Image Credits: AI Generated

DOI: 10.1016/j.jare.2025.12.037

Keywords: Alzheimer's disease, DAPK1, tau pathology, hydrophobic tagging, targeted protein degradation, neurofibrillary tangles, proteasome, tau phosphorylation, blood-brain barrier, neurodegeneration, drug discovery, tauopathy