Biology subjects

Kalume, F.

Publications and source records attributed to Kalume, F..

5 recordsLinked to original sources

bioRxiv2026

Optimizing Lentiviral Vector-Based Delivery of SCN1A transgenes to Mammalian Cells

The SCN1A gene encodes NaV1.1, a voltage-gated sodium channel protein that is necessary for neuronal excitability and whose loss-of-function mutations cause Dravet syndrome, a treatment-resistant childhood onset epilepsy. Gene replacement strategies for this syndrome are challenged by the large size of SCN1A and difficulty achieving stable cellular expression. Lentiviral vectors (LVVs) offer sufficient packaging capacity and genomic integration for defective SCN1A gene replacement. Here, we evaluated LVV-mediated delivery of different engineered SCN1A transgene sequences in human cells. LVV-transduced cells expressed full-length NaV1.1 protein that trafficked to the membrane and produced functional sodium currents. However, SCN1A transgene expression declined over time despite stable vector copy number, indicating post-integration regulatory limitations. Expression efficiency varied by SCN1A transgene sequence, with a codon-optimized variant showing higher expression despite lower LVV copy number. Treatment with sodium butyrate, a histone deacetylase inhibitor, significantly enhanced SCN1A transgene expression and partially rescued expression decay in a sequence-dependent manner. Incorporation of a ubiquitous chromatin opening element (UCOE) upstream of the promoter to maintain expression resulted in a trend of increased expression and increased responsiveness to butyrate. These findings demonstrate that sequence-specific and epigenetic factors may influence expression of large transgenes following lentiviral delivery, highlighting key challenges and design considerations for therapeutic SCN1A transgene expression.

synthetic biology↗

bioRxiv2024

Ndufs4 inactivation in glutamatergic neurons reveals swallow-breathing discoordination in a mouse model of Leigh Syndrome

Swallowing, both nutritive and non-nutritive, is highly dysfunctional in children with Leigh Syndrome (LS) and contributes to the need for both gastrostomy and tracheostomy tube placement. Without these interventions aspiration of food, liquid, and mucus occur resulting in repeated bouts of respiratory infection. No study has investigated whether mouse models of LS, a neurometabolic disorder, exhibit dysfunctions in neuromuscular activity of swallow and breathing integration. We used a genetic mouse model of LS in which the NDUFS4 gene is knocked out (KO) specifically in Vglut2 or Gad2 neurons. We found increased variability of the swallow motor pattern, disruption in breathing regeneration post swallow, and water-induced apneas only in Vglut2 KO mice. These physiological changes likely contribute to weight loss and premature death seen in this mouse model. Following chronic hypoxia (CH) exposure, swallow motor pattern, breathing regeneration, weight, and life expectancy were not changed in the Vglut2-Ndufs4-KO CH mice compared to control, indicating a rescue of phenotypes. These findings show that like patients with LS, Ndufs4 mouse models of LS exhibit swallow impairments as well as swallow-breathing dyscoordination alongside the other phenotypic traits described in previous studies. Understanding this aspect of LS will open roads for the development of future more efficacious therapeutic intervention for this illness.

neuroscience↗

bioRxiv2021

Suppression of PIK3CA-driven epileptiform activity by acute pathway control

Patients harboring mutations in the PI3K-AKT-MTOR signaling pathway often develop a spectrum of neurodevelopmental disorders including epilepsy. A significant proportion of them remain unresponsive to conventional anti-seizure medications. Understanding mutation-specific pathophysiology is thus critical for molecularly targeted therapies. We previously determined that mouse models expressing patient-related activating mutation in PIK3CA are epileptic and acutely treatable with PI3K inhibition, irrespective of dysmorphology (Roy et al. 2015). Using the same mutant model, we have now identified physiological mechanisms underlying the dysregulated neuronal excitability and its acute attenuation. We show that Pik3ca-driven hyperexcitability in hippocampal pyramidal neurons is mediated by changes in multiple non-synaptic, cell-intrinsic properties. These are distinct from mechanisms driving epilepsy in TSC/RHEB models. Further, we report that acute inhibition of PI3K or AKT, but not MTOR, suppresses the intrinsic epileptiform nature of the mutant neurons. These data represent an important step towards precision therapeutics against intractable epilepsy, using pathway drugs originally developed as anti-cancer agents.

neuroscience↗

bioRxiv2020

Sleep Identification Enabled by Supervised Training Algorithms (SIESTA): An open-source platform for automatic sleep staging of rodent polysomnographic data

The temporal distribution of sleep stages is critical for the study of sleep function, regulation, and disorders in higher vertebrates. This temporal distribution is typically determined polysomnographically. In laboratory rodents, scoring of electrocorticography (ECoG) and electromyography (EMG) recordings is usually performed manually, where 5-10 second epochs are categorized as one of three specific stages: wakefulness, rapid-eye-movement (REM) sleep and non-REM (NREM) sleep. This process is laborious, time-consuming, and particularly impractical for large experimental cohorts with recordings lasting longer than 24 hours. To circumvent this problem, we developed an open-source Python toolkit, Sleep Identification Enabled by Supervised Training Algorithms (SIESTA), that automates the detection of these three main behavioral stages in mice. Our supervised machine learning algorithm extracts features from the ECoG and EMG signals, then automatically scores recordings with a hierarchical classifier based on Bagging Random Forest approaches. We evaluated this approach on data collected from wild-type mice housed under both normal and different lighting conditions, as well as from a mutant mouse line with abnormal sleep phenotypes. To validate its performance on test data, we compared SIESTA with manually scored data and obtained F1 scores of 0.92 for wakefulness, 0.81 for REM, and 0.93 for NREM. SIESTA has a user-friendly interface that can be used without coding expertise. To our knowledge, this is the first time that such a strategy has been developed using all open-source and freely available resources, and our aim is that SIESTA becomes a useful tool that facilitates further research of sleep in rodent models.

neuroscience↗

bioRxiv2019

Defined neuronal populations drive fatal phenotype in Leigh Syndrome

Dysfunctions of the mitochondrial energy-generating machinery cause a series of progressive, untreatable and usually fatal diseases collectively known as mitochondrial disease. High energy-requiring organs such as the brain are especially affected, leading to developmental delay, ataxia, respiratory failure, hypotonia, seizures and premature death. While neural affectation is a critical component of the pathology, only discrete neuronal populations are susceptible. However, their molecular identity and their contribution to the disease remain unknown. Mice lacking the mitochondrial Complex I subunit NDUFS4 (Ndufs4KO mice) recapitulate the classical signs of Leigh Syndrome (LS), the most common presentation of mitochondrial disease with predominant CNS affectation. Here, we identify the critical role of two genetically-defined neuronal populations driving the fatal phenotype in Ndufs4KO mice. Selective inactivation of Ndufs4 in Vglut2-expressing glutamatergic neurons causes brainstem inflammation, motor and respiratory deficits, and early death. On the other hand, Ndufs4 deletion in GABAergic neurons leads to basal ganglia inflammation without motor or respiratory involvement, but accompanied by severe refractory epileptic seizures preceding premature death. These results provide novel insight in the cell type-specific contribution to LS pathology and open new avenues to understand the underlying cellular mechanisms of mitochondrial disease.

neuroscience↗