Biology subjects

Milner, T. A.

Publications and source records attributed to Milner, T. A..

8 recordsLinked to original sources

bioRxiv2026

Increased expression of developmental Nav1.3 promotes hippocampal CA3 hyperexcitability in early-stage 5xFAD mice

Neuronal hyperexcitability is an early and pervasive feature of Alzheimers disease (AD) that both predicts and accelerates subsequent cognitive decline. Persistent excitability depends on activation of voltage-gated sodium channels (Nav), yet most work has focused on the Nav subtypes expressed in the mature brain. Here, we show that Nav1.3, a subtype normally confined to early development, shows aberrantly increased expression in the dentate gyrus (DG)-CA3 circuit in early-stage 5xFAD mice. Using in vivo fiber photometry, three-month-old 5xFAD mice exhibited greater CA3 neuron population activity than seven-month-old 5xFAD mice or wildtype littermates. Oligomeric A{beta} expression, assessed by immunolabeling, was sparse at three months and rose significantly by seven months, indicating that CA3 hyperactivity emerges before substantial oligomeric A{beta} accumulates. This early activity increase coincided with elevated Nav1.3 expression at the DG-CA3 mossy fiber synapse, localized by immuno-electron microscopy to presynaptic mossy fiber terminals, where it exceeded levels in age-matched controls. Lentiviral shRNA-mediated knockdown of Nav1.3 expression in CA3 normalized CA3 network activity in early-stage 5xFAD mice. These findings identify Nav1.3 expression at DG-CA3 mossy fiber synapses as a driver of early hippocampal network dysfunction in AD and suggest Nav1.3 modulation as a potential target for circuit-level intervention.

neuroscience↗

bioRxiv2026

A Versatile AAV-TH-SNCA Model to Study Early α-Synuclein Pathology and Intervention

Adeno-associated viral (AAV)-mediated overexpression of human wildtype -synuclein (-syn) in the substantia nigra (SN) is a widely used approach to model Parkinsons disease (PD) in rodents. However, variability in the ability of AAV-based systems to induce nigrostriatal pathology and motor deficits has limited reproducibility across studies, especially in mice. Here, we systematically optimized key vector features - AAV serotype, promoter, viral titer - to establish a highly efficient and reliable mouse model of PD. We compared the tropism and expression efficiency of mixed AAV2/1 and AAV2/rh10 serotypes combined with three promoters - CMV enhancer/chicken {beta}-actin (CBA), human Synapsin (hSYN), and rat Tyrosine Hydroxylase (TH) - to drive human -syn gene (SNCA) expression in nigral dopaminergic neurons. The AAV.TH.SNCA vector, delivered at an optimized titer, achieved selective and sustained -syn overexpression in nigral neurons, resulting in nigro-striatal neurochemical changes and progressive motor deficits preceding overt neuronal loss. Fine tuning -syn expression proved critical for detecting early disease processes: lower AAV.TH.SNCA titer induced early pathological signatures, including -syn hyperphosphorylation and neuroinflammation, whereas higher titers produced robust nigrostriatal degeneration not achieved with other promoter constructs. Notably, we demonstrate that motor and neurochemical impairments can occur prior to dopaminergic cell death, implicating microglial activation and -syn pathology as primary drivers of dysfunction. This observation is consistent with human genetic evidence showing that triplication of the wild-type SNCA gene alone can cause Parkinsonian pathology, highlighting that our model enables the use of a single experimental reagent to investigate the molecular, cellular, and behavioral consequences of controlled increases in -syn expression. This novel AAV.TH.SNCA model provides a powerful and versatile platform for investigating mechanisms of a -syn-mediated neurotoxicity and for evaluating disease modifying interventions targeting early, pre-degenerative stages of PD. HighlightsO_LITitrated -syn expression uncouples early dysfunction from dopaminergic neuron loss C_LIO_LIAAV2/rh10-TH-SNCA model captures prodromal and degenerative PD stages C_LIO_LIMotor deficits arise from -syn pathology and nigral molecular changes before neurodegeneration C_LI

neuroscience↗

bioRxiv2026

Presynaptic NMDA receptors on mouse mossy fiber terminals mediate rapid BDNF release by ketamine and hydroxynorketamine

Major depressive disorder is associated with deficits in hippocampal synaptic plasticity that depend on brain-derived neurotrophic factor (BDNF) release from both axonal and dendritic compartments. Antidepressant efficacy requires enhanced BDNF signaling, thought to be mediated by drug-induced BDNF release from postsynaptic dendritic spines. Here, we show that fast-acting antidepressants rapidly trigger BDNF secretion from presynaptic terminals in hippocampal area CA3. At antidepressant-relevant concentrations, ketamine and its metabolite (2R,6R)-hydroxynorketamine (HNK) induced BDNF release within minutes from mossy fiber terminals of dentate granule neurons in rat hippocampal cultures, with no detectable secretion from dendritic spines. This antidepressant-evoked BDNF release required presynaptic NMDA receptors (preNMDARs). Conditional genetic deletion of preNMDARs from granule neurons abolished ketamine- and HNK-induced BDNF exocytosis in acute mouse hippocampal slices, establishing a presynaptic receptor mechanism for antidepressant-induced neurotrophin release. In CA3 pyramidal neurons that receive mossy fiber input, both compounds induced rapid remodeling of dendritic spines, resulting in increased spine density. Together, these findings identify presynaptic terminals as a previously unrecognized source of antidepressant-evoked BDNF release and establish a new cellular mechanism for the rapid synaptic effects of fast-acting antidepressants.

neuroscience↗

bioRxiv2025

Dual pathway regulation of castration response and ferroptosis in the prostate epithelium

Understanding how sex hormones maintain tissue integrity--and how their disruption can promote cancer--is a central question in biology. In the prostate, androgen signaling is crucial for development, homeostasis, and cancer, yet the molecular mechanisms underlying tissue regression after androgen-deprivation have remained unknown. Here we show that castration induces epithelial regression via ferroptosis in the normal mouse and human prostate, as well as in prostate tumors. Using in vivo analyses in genetically-engineered mice, supported by validation in human organotypic cultures, we demonstrate that androgen receptor (AR) signaling controls castration response through two distinct pathways: an intrinsic luminal epithelial pathway regulated by the prostate-specific transcription factor NKX3.1, and an extrinsic stromal signal mediated by the secreted factor pleiotrophin (PTN). Together, these AR signaling pathways coordinately regulate biosynthesis of monounsaturated fatty acid (MUFA) phospholipids and GPX4 expression to suppress prostate epithelial ferroptosis. Our findings reveal a sex hormone-regulated ferroptotic program that governs tissue homeostasis and suggest that ferroptosis induction could represent a new therapeutic strategy for prostate cancer.

developmental biology↗

bioRxiv2025

Perimenopause promotes neuroinflammation in select hippocampal regions in a mouse model of Alzheimers disease

Alzheimers disease (AD) is the most common neurodegenerative disorder characterized by age-dependent amyloid beta (A{beta}) aggregation and accumulation, neuroinflammation, and cognitive deficits. Significantly, there are prominent sex differences in the risk, onset, progression, and severity of AD, as well as response to therapies, with disease burden disproportionately affecting women. Although menopause onset (i.e., perimenopause) may be a critical transition stage for AD susceptibility in women, the role of early ovarian decline in initial disease pathology, particularly key neuroinflammatory processes, is not well understood. To study this, we developed a unique mouse model of perimenopausal AD by combining an accelerated ovarian failure (AOF) model of menopause induced by 4-vinylcyclohexene diepoxide (VCD) with the 5xFAD transgenic AD mouse model. To target early stages of disease progression, 5xFAD females were studied at a young age ([~]4 months) and at the beginning stage of ovarian failure analogous to human perimenopause (termed "peri-AOF"), and compared to age-matched males. Assessment of neuropathology was performed by immunohistochemical labeling of A{beta} as well as markers of astrocyte and microglia activity in the hippocampus, a brain region involved in learning and memory that is deleteriously impacted during AD. Our results show that genotype, AOF, and sex contributed to AD-like pathology. Aggregation of A{beta} was heightened in female 5xFAD mice and further increased at peri-AOF, with hippocampal subregion specificity. Further, select increases in glial activation also paralleled A{beta} pathology in distinct hippocampal subregions. However, cognitive function was not affected by peri-AOF. These findings align with the hypothesis that perimenopause constitutes a period of susceptibility for AD pathogenesis in women.

neuroscience↗

bioRxiv2024

ScISOr-ATAC reveals convergent and divergent splicing and chromatin specificities between matched cell types across cortical regions, evolution, and in Alzheimer's disease.

Multimodal measurements have become widespread in genomics, however measuring open chromatin accessibility and splicing simultaneously in frozen brain tissues remains unconquered. Hence, we devised Single-Cell-ISOform-RNA sequencing coupled with the Assay-for-Transposase-Accessible-Chromatin (ScISOr-ATAC). We utilized ScISOr-ATAC to assess whether chromatin and splicing alterations in the brain convergently affect the same cell types or divergently different ones. We applied ScISOr-ATAC to three major conditions: comparing (i) the Rhesus macaque (Macaca mulatta) prefrontal cortex (PFC) and visual cortex (VIS), (ii) cross species divergence of Rhesus macaque versus human PFC, as well as (iii) dysregulation in Alzheimers disease in human PFC. We found that among cortical-layer biased excitatory neuron subtypes, splicing is highly brain-region specific for L3-5/L6 IT_RORB neurons, moderately specific in L2-3 IT_CUX2.RORB neurons and unspecific in L2-3 IT_CUX2 neurons. In contrast, at the chromatin level, L2-3 IT_CUX2.RORB neurons show the highest brain-region specificity compared to other subtypes. Likewise, when comparing human and macaque PFC, strong evolutionary divergence on one molecular modality does not necessarily imply strong such divergence on another molecular level in the same cell type. Finally, in Alzheimers disease, oligodendrocytes show convergently high dysregulation in both chromatin and splicing. However, chromatin and splicing dysregulation most strongly affect distinct oligodendrocyte subtypes. Overall, these results indicate that chromatin and splicing can show convergent or divergent results depending on the performed comparison, justifying the need for their concurrent measurement to investigate complex systems. Taken together, ScISOr-ATAC allows for the characterization of single-cell splicing and chromatin patterns and the comparison of sample groups in frozen brain samples.

genomics↗

bioRxiv2023

Single-cell long-read mRNA isoform regulation is pervasive across mammalian brain regions, cell types, and development

RNA isoforms influence cell identity and function. Until recently, technological limitations prevented a genome-wide appraisal of isoform influence on cell identity in various parts of the brain. Using enhanced long-read single-cell isoform sequencing, we comprehensively analyze RNA isoforms in multiple mouse brain regions, cell subtypes, and developmental timepoints from postnatal day 14 (P14) to adult (P56). For 75% of genes, full-length isoform expression varies along one or more axes of phenotypic origin, underscoring the pervasiveness of isoform regulation across multiple scales. As expected, splicing varies strongly between cell types. However, certain gene classes including neurotransmitter release and reuptake as well as synapse turnover, harbor significant variability in the same cell type across anatomical regions, suggesting differences in network activity may influence cell-type identity. Glial brain-region specificity in isoform expression includes strong poly(A)-site regulation, whereas neurons have stronger TSS regulation. Furthermore, developmental patterns of cell-type specific splicing are especially pronounced in the murine adolescent transition from P21 to P28. The same cell type traced across development shows more isoform variability than across adult anatomical regions, indicating a coordinated modulation of functional programs dictating neural development. As most cell-type specific exons in P56 mouse hippocampus behave similarly in newly generated data from human hippocampi, these principles may be extrapolated to human brain. However, human brains have evolved additional cell-type specificity in splicing, suggesting gain-of-function isoforms. Taken together, we present a detailed single-cell atlas of full-length brain isoform regulation across development and anatomical regions, providing a previously unappreciated degree of isoform variability across multiple scales of the brain.

genetics↗

bioRxiv2021

Single-nuclei isoform RNA sequencing reveals combination patterns of transcript elements across human brain cell types

Single-nuclei RNA-Seq is being widely employed to investigate cell types, especially of human brain and other frozen samples. In contrast to single-cell approaches, however, the majority of single-nuclei RNA counts originate from partially processed RNA leading to intronic cDNAs, thus hindering the investigation of complete isoforms. Here, using microfluidics, PCR-based artifact removal, target enrichment, and long-read sequencing, we developed single-nuclei isoform RNA-sequencing ( SnISOr-Seq), and applied it to the analysis of human adult frontal cortex samples. We found that exons associated with autism exhibit coordinated and more cell-type specific inclusion than exons associated with schizophrenia or ALS. We discovered two distinct modes of combination patterns: first, those distinguishing cell types in the human brain. These are enriched in combinations of TSS-exon, exon-polyA site, and distant (non-adjacent) exon pairs. Second, those with all isoform combinations found within one neural cell type, which are enriched in adjacent exon pairs. Furthermore, adjacent exon pairs are predominantly mutually associated, while distant pairs are frequently mutually exclusive. Finally, we observed that human-specific exons are as tightly coordinated as conserved exons, pointing to an efficient evolutionary mechanism underpinning coordination. SnISOr-Seq opens the door to single-nuclei long-read isoform analysis in the human brain, and in any frozen, archived or hard-to-dissociate sample.

genomics↗