Benjamin D. Philpot

Understanding how neural circuits transition from seizure-resistant to seizure-prone is essential for developing improved epilepsy therapies. Here, we study this process by leveraging the heightened susceptibility to seizure kindling of Angelman syndrome (AS) model mice, which lack the maternal Ube3a ( mUbe3a ) allele. We identify parvalbumin-expressing (PV+) interneurons as critical gatekeepers; selective mUbe3a deletion in PV+ neurons phenocopies enhanced AS epileptogenesis, whereas restoring UBE3A broadly in GABAergic neurons confers seizure resistance. Further, pathological remodeling of the extracellular matrix in the dentate gyrus faithfully tracks with post-kindling seizure susceptibility, highlighting this region’s particular relevance to enhanced epileptogenesis. Mechanistically, we uncover a ‘two-hit’ electrophysiologic phenomenon in AS model mice: kindling fails to recruit compensatory inhibition onto dentate granule cells and instead drives their maladaptive intrinsic hyperexcitability. Together, these findings link cell type-specific inhibitory dysfunction and altered homeostatic plasticity to epileptogenesis, suggesting future circuit-based treatment strategies.

Blood-oxygenation-level-dependent (BOLD) functional magnetic resonance imaging (fMRI) has advanced brain mapping across species. This widely used technique, however, faces challenges such as acoustic noise, electromagnetic interference, motion artifacts, magnetic-field inhomogeneity and limitations in sensitivity and specificity. Here we introduce Steady-state On-the-Ramp Detection of INduction-decay with Oversampling (SORDINO), an improved zero echo time (ZTE)-based fMRI technique that maintains ultra-low slew rate and acquires data exclusively during gradient direction changes. At 9.4 T, SORDINO outperforms conventional gradient-recalled-echo-based echo-planar imaging and ZTE in metrics crucial for fMRI while being silent, sensitive, specific and resistant to motion and susceptibility artifacts. We showcase SORDINO’s superior compatibility with multimodal experiments and reveal T₁-based contrast mechanisms distinct from BOLD that are expected to generalize across ZTE-based fMRI techniques. We further demonstrate SORDINO brain-wide activity and functional connectivity mapping in awake, behaving mice, overcoming stress-related, motion-related and sensitivity-related limitations that remain common barriers in current animal fMRI studies.

The transcription factor 4 ( TCF4 ) gene is essential for brain development, and its disruption causes Pitt-Hopkins syndrome. Common genetic variants in TCF4 also confer risk for schizophrenia and related psychiatric disorders. While the developmental roles of TCF4 are well established, its postnatal functions remain poorly defined, particularly during the prolonged maturation of the primate neocortex, when many neuropsychiatric symptoms first emerge. Here, we mapped cell-type-specific TCF4 expression across postnatal development in rhesus macaque neocortex using immunofluorescence, and in situ hybridization. We also re-analyzed public bulk-tissue and single-nucleus transcriptomic datasets spanning 16 brain regions in both humans and macaques. TCF4 was predominantly neuronal, with minimal expression in microglia, oligodendrocytes, and astrocytes. In late gestation, TCF4 was broadly expressed in excitatory and inhibitory neurons, but expression declined with maturation and became selectively enriched in inhibitory populations. By young adulthood, nuclear TCF4 levels were highest in somatostatin-positive and vasoactive intestinal peptide-positive interneurons, intermediate in parvalbumin interneurons (~6-fold above non-GABAergic cells), and lowest in cholecystokinin interneurons (~4-fold above non-GABAergic cells). This interneuron-subtype hierarchy was independently reproduced by single-nucleus RNA sequencing across all neocortical regions. Together, these findings position mature GABAergic interneurons as the principal site of TCF4 function in the primate neocortex, providing a cellular framework for linking TCF4 dysfunction to cortical circuit imbalance in Pitt-Hopkins syndrome and psychiatric disease.

Unsilencing of paternal UBE3A is presently the leading therapeutic strategy for Angelman syndrome, a neurodevelopmental disorder caused by mutation or deletion of maternal UBE3A. Previous work identified (S)-PHA533533 ((S)-3) as a small molecule unsilencer of paternal UBE3A that disrupts UBE3A-ATS, the long noncoding antisense transcript that suppresses paternal UBE3A expression in mature neurons. Initial studies demonstrated that four (S)-3 analogs possess unsilencing behavior, which spurred further interest in the 3-aminopyrazole phenylacetamide (APPA) scaffold. Here, we leveraged mouse primary neurons harboring a sensitive reporter of paternal Ube3a expression to establish structure-activity relationships between APPAs, unsilencing behavior, and undesired cellular toxicity. We uncovered compounds with improved potency and less cytotoxicity than hit (S)-3 and report a concise set of identification efforts. We demonstrate that the most promising APPAs are 3' biased in their transcriptional disruption of Ube3a-ATS. Overall, this work provides a basis for additional development of APPAs.

Recent progress in the development of genetic therapies promises that impactful treatments for single-gene neurodevelopmental disorders are imminent. But can derailed neurodevelopmental processes be mended after broken genes are replaced or otherwise restored? The results of ongoing clinical trials for Angelman syndrome will soon yield answers to this pressing question, yet the trials face significant obstacles. Here we identify insights needed to aid the quest for a disease-modifying Angelman syndrome therapy, which could serve as a roadmap for the expeditious development of genetic therapies for other single-gene neurodevelopmental disorders.

Rare germline and somatic variants in SLC35A2 cause a spectrum of severe glycosylation disorders that commonly present with epilepsy. SLC35A2 encodes the Golgi transporter for UDP-galactose, but how its deficiency leads to severe neurodevelopmental disorders is unknown. Using a mouse model deficient for Slc35a2 in the forebrain, we identified a specific defect in O-GalNAc glycan synthesis, while other galactose-containing glycoconjugates remained intact. O-GalNAc glycans were absent from their normal location within neuronal tracts of the corpus callosum, and truncated precursors accumulated in the cortex on critical extracellular matrix molecules. Cultured primary neurons lacking Slc35a2 showed impaired development, hyperexcitability, and impaired O-GalNAc glycosylation. Finally, human brain tissue from cases of SLC35A2 -associated intractable epilepsy displayed a strong correlation between variant burden and truncated O-GalNAc glycans. These findings provide a mechanistic link between genetic causes of SLC35A2 -associated epilepsy and protein O-glycosylation that can be targeted for biomarker and therapeutic development.

Pitt-Hopkins syndrome (PTHS) is a neurodevelopmental disorder caused by haploinsufficiency of TCF4 which encodes transcription factor 4. As PTHS therapeutics advance toward clinical trials, identifying the optimal timing for treatment is crucial. Our previous research demonstrated that restoring TCF4 during embryonic or neonatal stages, corresponding to prenatal or neonatal periods in humans, improved phenotypes in a PTHS mouse model (Kim et al., 2022). However, PTHS diagnosis generally occurs much later, when infants fail to reach developmental milestones and undergo genetic testing. This raises an essential question: can genetic therapeutics initiated at more clinically relevant time points retain effectiveness? Here, we examined whether reinstating TCF4 in juvenile PTHS model mice could reverse behavioral phenotypes, simulating a gene therapy. Our findings indicate that this delayed intervention largely fails to correct most phenotypes, except for a measure of cognitive function. These results reveal phenotype-specific plasticity and underscore a narrow, early critical window for effective treatment in PTHS. Our study also identifies the hippocampus as a potential target for PTHS therapeutics and suggests that while some cognitive functions may still retain therapeutic plasticity, reversing most core PTHS symptoms may require intervention during the very early postnatal, or potentially prenatal periods, in humans.

Angelman syndrome (AS) is a neurodevelopmental disorder caused by UBE3A loss. In humans, UBE3A generates three isoforms that localize to distinct subcellular compartments—one mainly nuclear and two cytoplasmic. The nuclear and most highly expressed cytoplasmic UBE3A isoform are highly conserved in mice, whereas the cytoplasmic human isoform accounting for just ~1% of total UBE3A has no mouse counterpart. Loss of the nuclear-enriched UBE3A isoform causes AS and behavioral deficits in mice; no specific contribution of the predominant cytoplasmic UBE3A isoform has yet been identified. Because the nuclear-enriched isoform constitutes ~80% of total UBE3A protein, it is unclear if its outsized phenotypic impact upon deletion is due to a loss of nuclear UBE3A function or simply a dramatic reduction in overall UBE3A levels. If the former, overexpression of the cytoplasmic UBE3A isoform would be unable to rescue AS phenotypes. To test this, we developed a mouse model that overexpresses the cytoplasmic UBE3A isoform (mIso2-OE) and crossed it with an AS mouse model to yield wildtype (WT), mIso2-OE, AS, and AS/mIso2-OE mice, the latter of which lacked the endogenous nuclear UBE3A isoform (mIso3) in neurons. Unexpectedly, we found that overexpression of the cytoplasmic UBE3A isoform alone rescued most tested AS-related behavioral deficits, except for kindling-induced epileptogenesis or seizure-linked perineuronal net (PNN) accumulation in AS mice. Thus, while many AS phenotypes may be caused by isoform non-selective reductions in UBE3A levels, AS-associated epilepsies appear linked to isoform-selective nuclear UBE3A loss. This information is expected to inform AS gene therapy studies.

Thetis cells (TCs) are a recently identified lineage of RORγt ⁺ antigen-presenting cells comprising four subsets, TC I to TC IV, including a tolerogenic subset (TC IV) that instructs tolerance to gut microbiota and food antigens ^1–6 . A developmental wave of TCs during early life creates a crucial window of opportunity for establishing intestinal tolerance ^1,5 . The ontogeny of TCs and the cues that shape their abundance and heterogeneity remain unknown, however, limiting efforts to harness their therapeutic potential. Here we identify a population of RORγt ⁺ progenitors, termed Thetis–lymphoid tissue inducer progenitors (TLPs), that give rise to the immediate TC progenitor (TCP) and the lymphoid tissue inducer (LTi) progenitor (LTiP), and identify PU.1 as the transcription factor that governs TC fate. Despite transcriptional similarity to myeloid-derived conventional dendritic cells, we show that TCs descend from the common lymphoid progenitor. Deletion of the plasmacytoid dendritic cell (pDC) lineage-determining transcription factor TCF4 expands TLPs and TCs, indicating a shared developmental branch with pDCs. TLPs are enriched in fetal liver, but, unlike LTi cells, TCs emerge postnatally, indicating that developmentally timed environmental cues promote TCP differentiation. We identify one such cue, RANKL provision by lymphoid tissue organizer cells, that is essential for TC I differentiation. Together, these findings define the ontogeny of TCs and the transcription factors that promote TC differentiation and heterogeneity, facilitating future investigations of these enigmatic cells and their therapeutic potential for tolerance induction in food allergy and autoimmunity.