Citations

Abstract Alzheimer’s disease (AD) is a multifactorial disease with mixed pathologies. Consequentially, drugs targeting multiple pathological processes may offer synergistic benefits. While histone deacetylase (HDAC) inhibitors have demonstrated efficacy in alleviating AD-related pathologies in animal models, the neuroprotective Wnt/β-catenin signaling pathway remains compromised in AD brain. CI-994 is a class I HDAC inhibitor containing N-(2-aminophenyl)-benzamide. Our recent studies indicate that CI-994 is also an activator of Wnt/β-catenin signaling by stabilizing Wnt co-receptor LRP6. We herein use CI-994 as a scaffold to develop novel potent dual modulators of class I HDACs and Wnt/β-catenin signaling for AD therapy. Our lead compound, W2A-28, selectively inhibits class I HDAC1, 2 and 3 with IC 50 values of 0.51 μM, 0.68 μM, and 0.22 μM, respectively, and shows no inhibitory activities on other HDACs. Furthermore, W2A-28 potently activates Wnt reporter activity with an EC 50 value of 1.61 μM in Wnt-3A-expressing HEK293 cells. As expected, activation of Wnt/β-catenin signaling by W2A-28 is associated with elevated LRP6 protein level. Importantly, W2A-28 displays excellent microsomal stability in both mouse and human liver microsomal stability assays, alongside high permeability and a lack of active efflux in MDR1-MDCKII models. Critically, W2A-28 treatment significantly enhances histone acetylation, activates Wnt/β-catenin signaling, and suppresses tau phosphorylation in AD patient-specific cerebral organoids carrying APOE ε4/ε4 or APOE ε3/ε4 with PSEN1 M146V mutation. Our findings position W2A-28 as a promising multi-target drug candidate for AD therapy.

Abstract Background Alzheimer’s disease (AD) is a multifactorial disease often with mixed pathologies. As such, drugs targeting multiple pathological processes simultaneously could have synergistic therapeutic effects. The histone acetylation homeostasis is greatly impaired in AD, and histone deacetylase (HDAC) inhibitors have been known to alleviate AD‐relevant pathologies in various animal models. In addition, Wnt/β‐catenin signaling is compromised in AD, and restoring Wnt/β‐catenin signaling is an attractive therapeutic strategy for AD treatment. CI‐994 is a class I HDAC inhibitor containing N‐(2‐aminophenyl)‐benzamide. Our recent studies indicate that CI‐994 is also an activator of Wnt/β‐catenin signaling by stabilizing Wnt co‐receptor LRP6. Method We use CI‐994 as a scaffold to develop novel potent dual modulators for class I HDAC inhibition and Wnt/β‐catenin signaling activation, and then determine the therapeutic potential of the lead compound W2A‐28 in AD patient‐specific iPSC‐derived cerebral organoids. Result W2A‐28 inhibits class I HDAC1, 2 and 3 activities with IC 50 values of 512 nM, 675 nM and 217 nM, respectively, with no inhibitory activities on other HDACs and Sirtuin family members. Furthermore, W2A‐28 greatly increases Wnt reporter activity with an EC 50 value of 1.61 µM in Wnt‐3A‐expressing HEK293 cells. As expected, activation of Wnt/β‐catenin signaling by W2A‐28 is associated with elevated Wnt co‐receptor LRP6 protein level by reducing LRP6 degradation. Importantly, W2A‐28 displays excellent aqueous solubility and microsomal stability. Further, W2A‐28 shows high permeability with no active efflux in MDR1‐MDCKII permeability assays and is not a P‐gp substrate. Finally, W2A‐28 significantly reduces Aβ40 and Aβ42 levels and suppresses tau phosphorylation in AD patient‐specific iPSC‐derived cerebral organoids carrying APOE4. Conclusion Our studies suggest that W2A‐28 is a potential drug candidate for the treatment of AD.

Key Points ift140 -deficient zebrafish exhibited phenotypes observed in mammals, including skeletal defects and kidney cysts. Beyond ciliogenesis, ift140 also regulated cell polarity and intracellular microtubule stability, potentially contributing to cystogenesis. Microhomology-mediated end joining–induced mosaic ift140 crispants enabled evaluation of cystogenesis in adult fish and F0-based screening for modifiers of kidney cysts. Background Genetic modifiers are believed to play an important role in the onset and severity of polycystic kidney disease, but identifying these modifiers has been challenging due to the lack of effective methodologies. Methods We generated zebrafish mutants of IFT140 , a skeletal ciliopathy gene and newly identified autosomal dominant polycystic kidney disease gene, to examine skeletal development and kidney cyst formation in larval and juvenile mutants. In addition, we used ift140 crispants, generated through efficient microhomology-mediated end joining–based genome editing, to compare phenotypes with mutants and conduct a pilot genetic modifier screen. Results ift140 mutants developed kidney cysts and bone defects similar to those seen in mammalian models. ift140 crispants recapitulated mutant phenotypes while bypassing the early lethality of the mutants, enabling the analysis of kidney cyst formation in adult fish. In addition to cilia defects, we identified nonciliary phenotypes, including disrupted cell polarity and aberrant cytoplasmic microtubule stabilization in kidney epithelial cells, as potential contributors to ift140 -associated cystogenesis. The ability to detect ift140 -associated kidney cysts with ease allowed us to develop an F0-based genetic screen to identify potential protective modifiers. A pilot screen of 16 genes previously implicated in dysregulated signaling pathways in autosomal dominant polycystic kidney disease revealed both known and novel modifiers, including mtor and ulk1a . We further found that inhibition of mtor and ulk1a reversed both cilia-related and non–cilia-related abnormalities in the kidney. Conclusions By establishing a zebrafish model of ift140 -associated cystic kidney disease, we recapitulated ift140 ’s ciliary role and uncovered a nonciliary function in kidney cystogenesis. Importantly, we demonstrated the feasibility of using ift140 mosaic crispants to evaluate cystogenesis in adult fish and to perform F0-based screening for identifying genetic modifiers of kidney cysts.

ABSTRACT While BAG3 has been identified as a causative gene for dilated cardiomyopathy, the major pathological events in BAG3‐ related cardiomyopathy that could be targeted for therapeutic benefit remain to be discovered. Here, we aim to uncover novel pathological events through genetic studies in a zebrafish bag3 cardiomyopathy model. Given the known cardioprotective effects of mtor inhibition and the fact that transcription factor EB (tfeb ) encodes a direct downstream phosphorylation target of mTOR signaling, we generated a cardiomyocyte‐specific transgenic line overexpressing tfeb ( Tg[cmlc2:tfeb] ). This overexpression was sufficient to restore defective proteostasis and rescue cardiac dysfunction in the bag3 cardiomyopathy model. Importantly, we detected accelerated cardiac senescence in the bag3 cardiomyopathy model, which can be mitigated by Tg(cmlc2:tfeb) . We compared cardiac transcriptomes between the Tg(cmlc2:tfeb) transgenic fish and the mtor xu015/+ mutant and found that inhibition of the fatty acid binding protein a ( fabp7a ) gene exerts therapeutic effects. Consistent with this genetic evidence, we detected elevated fabp7a expression in the bag3 cardiomyopathy model, whereas cardiomyocyte‐specific overexpression of fabp7a induced dysregulated proteostasis, accelerated cardiac senescence, and cardiac dysfunction. To elucidate the functions of Fabp7a in normative cardiac aging, we turned to the African Turquoise Killifish. We noted elevated Fabp7a expression in the hearts of aged killifish, and pharmacological inhibition of Fabp7a mitigated the cardiac aging process. Together, this study uncovered accelerated cardiac senescence as a key pathological event in bag3 cardiomyopathy and reveals that manipulating the mTOR‐Tfeb‐Fabp7a axis can mitigate this pathology and confer cardioprotective effects.

AbstractBackgroundAlzheimer’s disease (AD) is the most common cause of age‐related dementia, and the presence of amyloid‐β (Aβ) plaques and tau‐containing neurofibrillary tangles is associated with the neurodegeneration and cognitive impairment in this incurable disease. Growing evidence shows that epigenetic dysregulation through histone deacetylases (HDACs) plays a critical role in synaptic dysfunction and memory loss in AD, and HDACs have been highlighted as a novel class of anti‐Alzheimer targets. Moreover, restoring Wnt/β‐catenin signaling, which is greatly suppressed in AD brains, is a promising therapeutic strategy for AD. CI‐994 is an orally active class I HDAC inhibitor that has undergone several phase II/III clinical trials on cancer treatment. Importantly, CI‐994 can cross the blood‐brain barrier and is a cognitive enhancer.MethodWe evaluated the therapeutic potential of CI‐994 in human patient‐specific iPSC‐derived neurons and cerebral organoids with APOE ε4/ε4 genotype or carrying MAPT p.P301L mutation.ResultWe found that CI‐994 is not only a potent class I HDAC inhibitor but also a novel modulator of Wnt/β‐catenin signaling, and that activation of Wnt/β‐catenin signaling by CI‐994 is associated with elevated Wnt co‐receptor LRP6 protein level. Moreover, CI‐994 increased synaptic protein levels, enhanced spontaneous synaptic firing and network formation, and decreased tau phosphorylation in iPSC‐derived neurons. Finally, CI‐994 significantly increased histone acetylation, activated Wnt/β‐catenin signaling, and decreased Aβ production and tau phosphorylation in AD patient‐specific iPSC‐derived cerebral organoids.ConclusionOur findings indicate that CI‐994 is a novel potential therapeutic candidate for AD.

Abstract Genetic modifiers are believed to play an important role in the onset and severity of polycystic kidney disease (PKD), but identifying these modifiers has been challenging due to the lack of effective methodologies. In this study, we investigated zebrafish mutants of IFT140 , a newly identified ADPKD gene, and observed phenotypes similar to those seen in mammalian models, including kidney cysts and bone defects. Using efficient microhomology-mediated end joining (MMEJ)-based genome editing technology, we demonstrated that ift140 CRISPRants recapitulate mutant phenotypes while bypassing the early lethality of the mutants to allow for renal cyst analysis in adult fish. In addition to cilia defects, non-cilia phenotypes, such as disrupted cell polarity and aberrant microtubule stabilization in kidney epithelial cells, may also contribute to ift140 -associated cystogenesis. Importantly, the ability to detect ift140 -associated renal cysts with ease allowed us to develop an F0-based genetic screen to identify potential protective modifiers. A pilot screen of sixteen genes previously linked to dysregulated signaling pathways in ADPKD revealed both known and novel modifiers, including mtor and ulk1a . Inhibition of mtor and ulk1a reversed both cilia-related and non-cilia-related abnormalities. In summary, our study underscores the potential of using zebrafish as a model for the efficient discovery of genetic modifiers of kidney cysts.

Aggregated α-synuclein (α-SYN) proteins, encoded by the SNCA gene, are hallmarks of Lewy body disease (LBD), affecting multiple brain regions. However, the specific mechanisms underlying α-SYN pathology in cortical neurons, crucial for LBD-associated dementia, remain unclear. Here, we recapitulated α-SYN pathologies in human induced pluripotent stem cells (iPSCs)–derived cortical organoids generated from patients with LBD with SNCA gene triplication. Single-cell RNA sequencing, combined with functional and molecular validation, identified synaptic and mitochondrial dysfunction in excitatory neurons exhibiting high expression of the SNCA gene, aligning with observations in the cortex of autopsy-confirmed LBD human brains. Furthermore, we screened 1280 Food and Drug Administration–approved drugs and identified four candidates (entacapone, tolcapone, phenazopyridine hydrochloride, and zalcitabine) that inhibited α-SYN seeding activity in real-time quaking-induced conversion assays with human brains, reduced α-SYN aggregation, and alleviated mitochondrial dysfunction in SNCA triplication organoids and excitatory neurons. Our findings establish human cortical LBD models and suggest potential therapeutic drugs targeting α-SYN aggregation for LBD.

AbstractBackgroundThe accumulation of the amyloid‐β (Aβ) aggregates in the brain is a key pathological event in the development of Alzheimer’s disease (AD). The α‐secretase ADAM10 is responsible for cleavage of the amyloid precursor protein (APP) through a non‐amyloidogenic pathway, thereby preventing the generation of pathogenic Aβ in the brain. Secreted frizzled related protein‐1 (sFRP1), an endogenous antagonist of Wnt signaling, has recently been identified as a key negative modulator of ADAM10. Importantly, proteomic studies reveal that unregulated sFRP1 is one of the top Aβ‐correlated proteins in the human AD brains.MethodsWe determined the therapeutic potential of specific sFRP1 WAY316606 in the N2a/APP695swe cells and patient‐specific iPSC‐derived cerebral organoids.ResultsWe herein demonstrated that treatment of sFRP1 inhibitor WAY316606 resulted in augmentation of ADAM10 activity, upregulation of soluble APP‐α (sAPPα) production, and inhibition of Aβ generation in the N2a/APP695swe cells. Moreover, WAY316606 significantly enhanced ADAM10 activity, decreased Aβ production and inhibited tau phosphorylation in human AD patient‐specific iPSC‐derived cerebral organoids.ConclusionOur findings indicate that sFRP1 is a promising therapeutic target for AD.

Background: Anthracyclines are highly effective chemotherapeutics, but their use is limited by Anthracycline-Induced Cardiotoxicity (AIC) with notable decline in ejection fraction (EF). Recovery of cardiac function is slow and often only partial, mandating novel and more efficacious treatment approaches to AIC. Hypothesis: Based on high throughput genetic screening in zebrafish we identified autophagy activation, e.g. via Atg7, a key protein in the induction of autophagy, as a potential novel translational therapeutic approach to AIC. Methods: In a mouse model of cardiotoxicity (4 x weekly 5 mg/kg intravenous doxorubicin treatment) two approaches were tested starting one week after the last treatment: 1) AdenoAssociated Viral vector 9-mediated overexpression of Atg7 in the myocardium (AAV9-ATG7, one time injection) and 2) 3 FDA approved autophagy activating (FAA) drugs (pravastatin (40 mg/kg), spironolactone (20 mg/kg) and empagliflozin (10 mg/kg), by daily gavage). Echocardiograms were performed at baseline, before start of therapy and after 28 days before sacrifice. Results: All three FAA drugs were able to rescue AIC and Atg7 overexpression by AAV9 alone sufficed to rescue from AIC (Figure 1). All three FAA drugs were able to either restore Atg7 levels to baseline (pravastatin and empagliflozin) or 2.5 times above baseline levels (spironolactone) versus more than 60fold above baseline by an AAV9-based approach. Conclusion: Atg7 overexpression and autophagy activation suffices to rescue from AIC and can be recapitulated to different levels by pravastatin, empaglifozin and spironolactone.

The prevalence of Alzheimer’s disease is greater in women, but the underlying mechanisms remain to be elucidated. We herein demonstrated that α-secretase ADAM10 was downregulated and ADAM10 inhibitor sFRP1 was upregulated in 5xFAD mice. While there were no sex effects on ADAM10 protein and sFRP1 mRNA levels, female 5xFAD and age-matched non-transgenic mice exhibited higher levels of sFRP1 protein than corresponding male mice. Importantly, female 5xFAD mice accumulated more Aβ than males, and sFRP1 protein levels were positively associated with Aβ42 levels in 5xFAD mice. Our study suggests that sFRP1 is associated with amyloid pathology in a sex-dependent manner.