Yijen Lin Wu

Cardiomyopathy is an important manifestation in patients with fatty acid oxidation disorders and represents a major cause of morbidity and early mortality in mitochondrial trifunctional protein (TFP) deficiency. Although a mouse model carrying the TFP β-subunit p.Met404Lys mutation (βTFP-deficient mice) has been described, cardiac involvement in this model has not been systematically characterized. Here, we combined cardiac histology and multiparametric cardiac magnetic resonance imaging (CMR) to define myocardial structure, function and tissue characteristics in this mouse model. Histological analysis with automated whole-slide collagen quantification revealed myocardial fibrosis with collagen deposition in mutant hearts, and CMR demonstrated increased myocardial extracellular volume in both male and female homozygous mutants. Homozygous males showed reduced ejection fraction, impaired systolic strain and increased left ventricular end-systolic volume, indicating systolic dysfunction. Male mice were more severely affected than females and exhibited reduced survival. Together, these findings demonstrate that βTFP-deficient mice develop fibrotic cardiomyopathy with systolic dysfunction, reproducing important cardiac features observed in human TFP deficiency. This work establishes the model as a relevant platform for investigating disease mechanisms and therapeutic strategies for cardiomyopathy in TFP deficiency.

Fetal Alcohol Spectrum Disorder, caused by prenatal alcohol exposure, remains a major public health concern, affecting up to 1 in 20 U.S. schoolchildren. Pregnancy is typically not recognized until six weeks of gestation, alcohol exposure often occurs unknowingly during the peri-conceptional period, a highly vulnerable developmental window of oocyte maturation. The placenta is increasingly recognized as a mediator of alcohol-induced neurodevelopmental risk through the placenta–brain axis. We characterize peri-conceptional alcohol exposure on placental development in a mouse PCA model mimicking human drinking patterns. Single-cell transcriptomic analyses on late gestation placentas revealed disruption of extracellular matrix and collagen-remodeling pathways within the labyrinth; processes essential for placental vascular development. Mitochondria isolated from E17.5 placentas showed compromised respiration. We assess placental vascular functions with non-invasive dynamic contrast-enhanced MRI, enabling analysis across placental compartments for longitudinal evaluation of perfusion, substrate delivery, and contractile function. We demonstrate that PCA exposure arrests placental vascularization, perfusion, and morphological maturation. PCA exposure before implantation altered long-term placental programming, resulting in impaired placental perfusion and mitochondrial functions across gestation. These findings implicate placental dysfunction as a critical mechanism in Fetal Alcohol Spectrum Disorder pathogenesis and identify extracellular matrix remodeling and mitochondria as potential therapeutic targets following PCA exposure.

MALT1 protease is an intracellular signaling molecule that promotes tumor progression via cancer cell-intrinsic and cancer cell-extrinsic mechanisms. MALT1 has been mostly studied in lymphocytes, and little is known about its role in tumor-associated macrophages. We show that MALT1 is expressed in glioblastoma (GBM)-associated macrophages. Mechanistically, GBM tumor cells induce a MALT1-NF-κB signaling axis in macrophages, leading to enhanced macrophage migration and polarization toward an immunosuppressive (‘M2-like’) phenotype. Inactivation of MALT1 protease promotes transcriptional reprogramming that reduces migration and restores a macrophage anti-tumor ‘M1-like’ phenotype. Preclinical in vivo analysis shows that MALT1 inhibitor treatment results in immuno-reactivity of GBM-associated macrophages and reduced GBM tumor growth. The addition of MALT1 inhibitor to temozolomide reduces immunosuppression in the tumor microenvironment, indicating that pharmacological inhibition of MALT1 protease may enhance the efficacy of chemotherapeutic. Thus, our findings suggest that MALT1 protease inhibition represents a promising macrophage-targeted immunotherapeutic strategy for the treatment of GBM.

Heart muscle growth and regeneration require the proliferation of cardiomyocytes. Rapid pulsatile increases in cytosolic Ca2+ concentration, called calcium transients (CaTs), trigger cardiomyocyte contractions, but how cardiomyocytes adapt Ca2+ signaling during proliferation is largely unknown. Here, we show that cardiomyocyte proliferation requires changes in Ca2+ signaling. Cardiomyocytes undergo a sequence of CaT changes during M phase: CaT amplitudes begin to decline in prometaphase, reach a minimum in metaphase, rise during anaphase, and return to the original state in daughter cardiomyocytes. Spindle poles show decreased Ca2+ levels during prometaphase and metaphase. Localized reduction of Ca2+ levels at spindle poles is mediated by dynein 1–dependent SERCA2a accumulation. Active cyclin-dependent kinase 1 (CDK1) induces both the decrease in CaT amplitudes and the accumulation of SERCA2a at the spindle poles, whereas CDK1 inhibition reverses these effects. Forcing an increase in cytosolic Ca2+ levels by blocking SERCA2a during prometaphase and metaphase disrupts mitosis and produces binucleated cardiomyocytes, underscoring the essential role of Ca2+ signaling changes for cardiomyocyte proliferation.

Mitochondria are essential for cellular energy production and are particularly critical for brain development and function. Neurons rely predominantly on oxidative phosphorylation for energy production, rendering the brain highly vulnerable to mitochondrial dysfunction. Consequently, impaired mitochondrial function contributes to a broad spectrum of neurological and systemic disorders, making mitochondria attractive therapeutic targets. Despite this importance, there is currently no non-invasive, spatially resolved method to assess mitochondrial function in the intact living brain. Here, we establish a non-invasive functional MRI approach—4D Oxy-wavelet MRI—to probe in vivo mitochondrial electron transport chain (ETC) function in a spatially specific manner across the lifespan, from fetal to adult brains. This method employs a low-rank k-t sub-Nyquist acquisition strategy to achieve simultaneous structural and functional imaging with high spatial (78 μm) and temporal (≈14 ms) resolution, enabling motion-robust imaging in multi-fetal mouse pregnancies. Mitochondrial ETC function is interrogated by measuring oxygen homeostasis responses to brief hypoxic challenges, analyzed using computational time-frequency wavelet profiling. We validate this approach in mouse models of mitochondrial respiratory chain disease and late-onset Alzheimer's disease, from in utero fetuses to adults, and demonstrate reproducibility and specificity using pharmacological hyperemia and ETC complex I inhibition. We further show parallel wavelet responses in placenta and fetal brain, enabling multi-organ interrogation of the placenta–brain axis. Finally, we present first-in-human feasibility data, supporting translational potential for non-invasive assessment of mitochondrial function in living brains across the lifespan.

Cyclin-Dependent Kinase-Like 5 (CDKL5) Deficient Disorder (CDD) is a rare X-linked developmental and epileptic encephalopathy characterized by early-onset refractory epilepsy, severe neurodevelopmental impairment, and lifelong disability. Although more than thirty anti-seizure medications are available, most CDD patients remain pharmaco-resistant. Gene-based therapies are emerging, but therapeutic development is hindered by marked clinical heterogeneity, small patient populations, and the lack of robust, translatable brain-based biomarkers for clinical trials. Genetically engineered Cdkl5 mouse models recapitulate many cognitive, behavioral, and molecular features of CDD, yet their utility is limited by the absence of overt seizures, precluding seizure-based outcome measures. Here, we establish high-definition brain network (HDBN) biomarkers using advanced diffusion MRI tractography combined with graph-theoretical analysis to quantify whole-brain network organization in Cdkl5 knockout mice. Diffusion MRI enables non-invasive mapping of axonal connectivity by leveraging anisotropic water diffusion, while high-angular-resolution acquisition overcomes key limitations of conventional diffusion tensor imaging in regions with complex fiber architecture. We demonstrate that Cdkl5 knockout mice exhibit reproducible and region-specific disruptions in brain network organization, prominently affecting the somatosensory and somatomotor cortex, hippocampus, hypothalamus, amygdala, and superior colliculus—regions implicated in cognition, learning and memory, homeostasis, anxiety, and visual–motor function. In contrast, networks within the entorhinal cortex remain largely preserved. These findings identify HDBN metrics as sensitive, non-invasive biomarkers that capture clinically relevant circuit-level abnormalities in CDD. Because diffusion MRI–based network analyses are directly translatable across species, HDBN biomarkers provide a unified framework for therapeutic evaluation in mouse models, large animals, and human clinical trials, enabling longitudinal monitoring of disease progression and treatment response.

Cyclin-Dependent Kinase-Like 5 (CDKL5) Deficient Disorder (CDD) is a rare X-linked developmental and epileptic encephalopathy characterized by early-onset refractory epilepsy, severe neurodevelopmental impairment, and lifelong disability. Although more than thirty anti-seizure medications are available, most CDD patients remain pharmaco-resistant. Gene-based therapies are emerging, but therapeutic development is hindered by marked clinical heterogeneity, small patient populations, and the lack of robust, translatable brain-based biomarkers for clinical trials. Genetically engineered Cdkl5 mouse models recapitulate many cognitive, behavioral, and molecular features of CDD, yet their utility is limited by the absence of overt seizures, precluding seizure-based outcome measures. Here, we establish high-definition brain network (HDBN) biomarkers using advanced diffusion MRI tractography combined with graph-theoretical analysis to quantify whole-brain network organization in Cdkl5 knockout mice. Diffusion MRI enables non-invasive mapping of axonal connectivity by leveraging anisotropic water diffusion, while high-angular-resolution acquisition overcomes key limitations of conventional diffusion tensor imaging in regions with complex fiber architecture. We demonstrate that Cdkl5 knockout mice exhibit reproducible and region-specific disruptions in brain network organization, prominently affecting the somatosensory and somatomotor cortex, hippocampus, hypothalamus, amygdala, and superior colliculus—regions implicated in cognition, learning and memory, homeostasis, anxiety, and visual-motor function. In contrast, networks within the entorhinal cortex remain largely preserved. These findings identify HDBN metrics as sensitive, non-invasive biomarkers that capture clinically relevant circuit-level abnormalities in CDD. Because diffusion MRI–based network analyses are directly translatable across species, HDBN biomarkers provide a unified framework for therapeutic evaluation in mouse models, large animals, and human clinical trials, enabling longitudinal monitoring of disease progression and treatment response.

Developmental and Epileptic Encephalopathies (DEEs) comprise a heterogeneous group of severe neurological disorders with early onset refractory epilepsy and profound neurodevelopmental impairments. Despite the availability of more than thirty anti-seizure medications, most individuals with DEE remain refractory, experiencing persistent seizures that impose substantial burdens on patients, caregivers, and health care systems. DEEs are predominantly genetic, largely caused by de novo mutations, although non-genetic factors also contribute. Crucially, developmental deficits in DEE arise from both the underlying etiology and the disruptive effects of epileptic activity on the developing brain, distinguishing DEE from epilepsies without major neurodevelopmental involvement. Seizures during critical developmental windows exert acute and enduring effects on neurogenesis, synaptogenesis, circuit refinement, pruning, and brain maturation, which can greatly disrupt neurodevelopment. Mitochondria emerge as central regulators at the intersection of neurodevelopment and epilepsy. Mitochondrial dysfunction, whether from primary genetic mutations or seizure-induced injury, impairs bioenergetics, calcium homeostasis, redox states, and metabolic signaling, lowers seizure thresholds, and increases neuronal vulnerability. Moreover, mitochondrial metabolism directly interfaces with epigenetic regulation, linking energy state to long-term gene expression, altering developmental programming. This review synthesizes current knowledge on mitochondrial contributions to DEE pathogenesis, clinical diagnosis, and highlights emerging mitochondrial-targeted therapeutic strategies.

Background: Interstitial myocardial fibrosis is a crucial pathological feature of many cardiovascular disorders. Myocardial fibrosis resulting in extracellular volume (ECV) expansion can be quantified via cardiac MRI (CMR) with T₁ mapping before and after minimally invasive gadolinium (Gd) contrast agent administration. However, longitudinal repetitive ECV measurements are challenging in rodents due to the prolonged scan time with cardiac and respiratory gating that is required for conventional T₁ mapping and the invasive nature of the rodent intravenous lines. Methods: To address these challenges, the objective of this study is to establish a fast, free-breathing, and gating-free ECV procedure using a minimally invasive subcutaneous catheter for in-scanner Gd administration that can allow longitudinal repetitive ECV evaluations in rodent models. This is achieved by the (1) IntraGate sequence for free-breathing, gating-free cardiac imaging; (2) minimally invasive subcutaneous in-scanner Gd administration; and (3) fast T₁ mapping with a varied flip angle (VFA) in conjunction with (4) triple jugular vein blood T₁ normalization. Additionally, full cine CMR (multi-slice short-axis, long-axis 2-chamber, and long-axis 4-chamber) was acquired during the waiting period to assess comprehensive cardiac function and strain. Results: We successfully established a minimally invasive fast ECV quantification protocol to enable longitudinal repetitive ECV quantifications in rodents. Minimally invasive subcutaneous Gd bolus administration induced a reasonable dynamic contrast enhancement (DCE) time course, reaching a steady state in ~20 min for stable T₁ quantification. The free-breathing gating-free VFA T₁ quantification scheme allows for rapid cardiac (~2.5 min) and jugular vein (49 s) T₁ quantification with no motion artifacts. The triple jugular vein T₁ acquisitions (1 pre-contrast and 2 post-contrast) immediately flanking the heart T₁ acquisitions enable accurate myocardial ECV quantification. Our data demonstrated that left-ventricular myocardial ECV quantification was highly reproducible with repeated scans, and the ECV values (0.25) are comparable to reported ranges among humans and rodents. This protocol was successfully applied to the ischemia–reperfusion injury model to detect myocardial fibrosis, which was validated by histopathology. Conclusions: We established a simple, fast, minimally invasive, and robust CMR protocol in rodents that can enable longitudinal repetitive ECV quantification for cardiovascular disease progression. It can be used to monitor disease regression with interventions.

Background: Interstitial myocardial fibrosis is a crucial pathological feature of many cardiovascular disorders. Myocardial fibrosis resulting in extracellular volume (ECV) expansion can be non-invasively quantified by cardiac MRI (CMR) with T1 mapping before and after gadolinium (Gd) contrast agent administration. However, longitudinal repetitive ECV measurements are challenging in rodents due to the prolonged scan time with cardiac and respiratory gating that is required for conventional T1 mapping and the invasive nature of the rodent intravenous lines. Methods: To address these challenges, the objective of this study is to establish a fast, free- breathing, and gating-free ECV procedure with a non-invasive subcutaneous catheter for in- scanner Gd administration that can allow longitudinal repetitive ECV evaluations in rodent models. This is achieved by (1) IntraGate sequence for free-breathing gating-free cardiac imaging, (2) non-invasive subcutaneous in-scanner Gd administration, and (3) fast T1 mapping with varied flip angle (VFA) in conjunction with (4) triple jugular vein blood T1 normalization. Additionally, full cine CMR (multi-slice short-axis, long-axis 2-chamber, and long-axis 4- chamber) was acquired during the waiting period for comprehensive systolic cardiac functional and strain analysis. Results: We have successfully established a non-invasive fast ECV quantification protocol to enable longitudinal repetitive ECV quantifications in rodents. Non-invasive subcutaneous Gd bolus administration induced reasonable dynamic contrast enhancement (DCE) time course reaching a steady state in ∼ 20 min for stable T1 quantification. The free-breathing gating-free VFA T1 quantification scheme allows for rapid cardiac (∼2.5 min) and jugular vein (49 sec) T1 quantification with no motion artifacts. The triple jugular vein T1 acquisitions (1 pre-contrast and 2 post-contrast) immediately flanking the heart T1 acquisitions enable accurate myocardial ECV quantification. Our data demonstrated that left-ventricular myocardial ECV quantifications were highly reproducible with repeated scans, and the ECV values (0.25) are comparable to reported ranges among humans and rodents. This protocol was successfully applied to ischemic reperfusion injury model to detect myocardial fibrosis that was validated with histopathology. Conclusion: We have established a simple, fast, non-invasive, and robust CMR protocol in rodents that can enable longitudinal repetitive ECV quantifications for cardiovascular disease progression. It can be used to monitor disease regression with interventions. Graphical