Molecular and Cellular Neuroscience

Calcium functions as an important second messenger in a wide variety of intracellular processes. In photoreceptor cells, calcium is involved in activation, deactivation, and adaptation in response to light stimuli. Calcium-binding protein 53E (Cbp53E, also known as calbindin-32 or cbn), a protein with 6 EF-Hand domains thought to act as a calcium buffer, was previously identified to have elevated expression levels in the eye of drosophila. While a recent study showed that transgenic flies lacking Cbp53E have aberrant axonal arborization at the neuromuscular junction, nothing is known about the role of Cbp53E in the visual system. We performed electroretinogram (ERG) recordings on Cbp53E mutant flies to test whether eye function was affected. Here, we report that Cbp53E null mutants exhibit a prolonged repolarization (or slow termination) phenotype which can be rescued by expressing Cbp53E in photoreceptor cells. The human homologs Calbindin 2, Calbindin 1, and S100G also rescue the Drosophila ERG phenotype. This supports a role for Cbp53E in regulating intracellular calcium levels of photoreceptor cells and contributing to normal sensory neuron response dynamics in vivo in Drosophila and suggests a similar function in human photoreceptor cells as well.

Voltage-gated sodium channels (VGSCs) are conventionally described as heterotrimers composed of one alpha and two beta subunits. However, the patterns of co-expression of alpha- and beta-subunits in neurons remain unclear. In the present study, we report that alpha- (Nav1.1, Nav1.2, and Nav1.6) and beta- (beta-1 and beta-2) subunits are densely expressed in axon initial segments (AISs) of neurons in the neocortex, hippocampus and cerebellum at postnatal days 14-15 (P14-15) and 8-9 weeks (8-9W). These distributions are largely unique and partially overlapping among brain regions. Notably, in the neocortex and hippocampus, AISs of presumptive parvalbumin-positive inhibitory neurons are positive for Nav1.1 and beta-1, whereas those of excitatory ones are positive for Nav1.2 and beta-2. Similarly, AISs of cerebellar basket cells, which are inhibitory neurons, are positive for Nav1.1 and beta-1, whereas those of granule cells, which are excitatory neurons, are positive for Nav1.2 and beta-2. Nav1.6 is expressed in many of these neurons. Some subunits exhibited distinct distribution patterns at the two postnatal stages analyzed, possibly because of their developmental changes of subcellular localizations. Taken together, these results indicate that combinations of VGSC subunits are largely unique among different neuronal subpopulations. These findings provide a useful reference for understanding the distribution and interactions of VGSC subunits in the brain.

Insulin-like growth factor-1 (IGF-1) plays a critical role in neuronal signaling. Disrupted insulin/IGF-1 signaling is implicated in Alzheimers disease, among other conditions, yet its specific influence on glutamate receptor-mediated calcium responses remains unclear. We examined the impacts of IGF-1 on glutamate receptor function in primary rat neurons monitored for intraneuronal calcium following stimulation with glutamate, AMPA, or NMDA/glycine. Pharmacological blockers (CNQX for AMPA receptors, APV for NMDA receptors, and nimodipine for L-type calcium channels) were applied to define receptor-specific contributions. In hippocampal neurons, IGF-1 and insulin altered responses to glutamate in different directions, with IGF-1 tending to evoke and enhanced response. In neocortical neurons, by contrast, IGF-1 consistently reduced glutamate- and AMPA-evoked calcium peaks, suggesting an inhibitory effect on AMPA receptors. To rule out effects on voltage-gated calcium channels downstream of AMPA receptors, we tested effects of IGF-1 on depolarization with potassium chloride; calcium elevation in this case was unaffected by IGF-1. Likewise, IGF-1 did not inhibit responses to NMDA/glycine; and IGF-1 did not affect glutamate responses in the presence of CNQX, a selective AMPA receptor blocker. These findings, combined with the observation that IGF-1 effects persisted in the presence of APV (an NMDA receptor antagonist), indicate that the inhibition of glutamate responses by IGF-1 is mediated by suppression of AMPA receptor activity. IGF-1 may thus contribute to normal neurophysiology, and given the role that glutamate receptors play in excitotoxicity, IGF-1 may confer neuroprotection in the neocortex. Disruption of IGF-1 signaling, as seen in states resembling insulin resistance, may therefore worsen glutamate-driven excitotoxicity and contribute to adverse outcomes.

Excitatory glutamatergic synapses in the brain are remarkably plastic. Two forms of plasticity have received the most attention: long-term potentiation (LTP) and synaptic homeostasis. While LTP requires the activation of NMDA receptors, synaptic homeostasis does not. However, both phenomena are mediated by the recruitment of postsynaptic AMPA receptors to the synapses. Recently a new form of plasticity has been described referred to as presynaptic homeostatic plasticity (PHP) (Chipman et al., 2022; Chipman et al., 2025). Pharmacological inhibition of AMPA synaptic responses in CA1 hippocampal pyramidal cells initiates a rapid homeostatic response that results in the recovery of the AMPA responses to normal values in the continued presence of the inhibitor. Accompanying this recovery is a doubling of the NMDA response which is interpreted as an increase in the release of glutamate. This is provocative since it is the first report claiming that a reduction in AMPA responses triggers an enhancement in NMDA responses. Using three different protocols to monitor synaptic responses we fail to observe any recovery of synaptic responses in the presence of an AMPA inhibitor. Furthermore, there was no enhancement in NMDA responses. Thus, we find no evidence for the presence of PHP at CA1 hippocampal synapses.

Tau protein, the primary component in neurofibrillary tangles characteristic of Alzheimers Disease and related dementia disorders, normally regulates microtubule growth and stability. While tau dysfunction contributes to the progression of tauopathies, the role of microtubules in disease has remained unclear. Through forward genetic screening in Caenorhabditis elegans tauopathy models, we found multiple tubulin gene mutations that rescue tau-mediated neurodegeneration. Whole animal behavioral and in vitro biochemical assays were employed to characterize mutation-driven effects on neuron function, neurodegeneration, and effects on tubulin and tau proteins as well as microtubule function. Mutant tubulin genes were found to confer different levels of suppression correlating with the level of mutant gene expression. Mutant tubulins did not drastically alter total tau protein levels, tau phosphorylation or aggregation, however tau-induced neurodegeneration was rescued. The suppression of tau toxicity by tubulin gene mutations cannot be explained by changes in tau or tubulin expression, tau phosphorylation, or tau aggregation state. Rather the tubulin mutations appear to act by influencing global microtubule properties. In vitro experiments using C. elegans tubulin in semi-isolated and isolated contexts have indicated changes to microtubule properties without observable changes to tau-tubulin affinity. This work suggests that manipulation of microtubules can rescue tauopathy even when pathological tau species persist, supporting the importance of understanding microtubule contributions to disease progression and investigation into microtubule targeted gene therapy or small molecule approaches for tauopathy intervention.

Traumatic brain injury (TBI) induces a series of neuropathological changes in the brain including neurogenesis, an important cellular response involved in brain repair and regeneration. TBI-enhanced neurogenesis in the dentate gyrus (DG) of the hippocampus is of particular importance due its contribution to learning and memory functions. In the neurogenic process, proliferation and differentiation of neural stem cells (NSCs) follow a well-characterized sequence controlled by many factors including Notch1, which plays essential roles in regulating NSC fate determination under physiological conditions in both developing and adult brains. Following TBI, the dynamic changes of NSCs and the involvement of Notch1 on their development at different stages post-injury are not fully characterized. In the current study, we examined the impact of TBI and Notch1 on NSCs proliferation, survival and neuronal differentiation. Utilizing transgenic mice with tamoxifen-induced GFP expression and Notch1 knock-out in nestin+ NSCs, we examined DG neurogenic response at acute, subacute and chronic stages following a moderate lateral fluid percussion injury. We found that TBI enhanced a proliferative response in the DG at the acute stage following injury; however, this injury response was abolished when Notch1 was conditionally deleted from nestin+ NSCs. We also found that injury and Notch1 deletion drove NSCs committing fate choice towards neuronal differentiation. The results of this study provides further knowledge regarding TBI-induced neurogenic response and Notch1 as the key regulating mechanism.

A single allele of any of the [~]1100 functional mouse odorant receptor (OR) genes is expressed by mature olfactory sensory neurons (mOSNs) through a poorly understood mechanism of singular gene choice. We were interested in developing an expression system to study OR selection in a mouse embryonic stem cell (mESC) carrying an Olfr151-IRES-CRE knock-in and a CRE-recombination-dependent ROSA26-MTMG reporter. Recombination of the ROSA reporter could not be observed after exposing this mESC to thousands of chemical compounds, including 175 compounds known to interfere with epigenetic regulatory processes nor by transfection with high probability Olfr151 promoter minigenes coexpressing CRE. The only two known regulatory elements that control OR promoters are not olfactory specific. Still, our inability to elicit even leaky CRE recombination in mESCs suggests that a specific transcriptional machinery within the OSN lineage is needed to drive OR promoter activation. Author SummaryThe Olfr151 promoter is inactive in mESCs.

Nociception is an essential response for organisms to avoid potential harm and promote survival. Its molecular determinants are largely conserved across Eumetazoa. TRPV receptors are polymodal ion channels exhibiting selective peripheral expression and functional coupling that underpins nociception and pain modulation in complex organisms. However, the execution of protective behaviours triggered by TRPVs is also found in species with a simpler nervous organisation, thus encouraging their investigation in invertebrate model organisms to increase understanding of animal nociception. Cephalopods represent an interesting invertebrate phylum with respect to the evolution of the nervous system, whose complexity suggests it might support pain-like states that exist in vertebrates. This possibility is reflected by the inclusion of cephalopods in the UK and EU animal welfare legislations. Despite this, there is poor characterisation of cephalopod molecular nociceptors. For this reason, we used in silico analysis to identify two TRPV channels in Octopus vulgaris genome (Ovtrpv1 and Ovtrpv2). We validated the putative transcript sequences and highlighted prevalent expression in sensory tissues. We investigated the functional competence of these TRPVs by heterologously expressing Ovtrpv1 and Ovtrpv2 cDNA into Caenorhabditis elegans null mutants of the orthologous genes, ocr-2 and osm-9 respectively. Ovtrpvs successfully rescued the aversive response to chemical and mechanical noxious stimuli in the C. elegans mutants, suggesting these receptors are polymodal nociceptors. Additionally, complementary investigation using Xenopus laevis oocytes showed Ovtrpv1 and Ovtrpv2 form an active heteromeric channel gated by nicotinamide. This study highlights Ovtrpvs as an important route to better understand nociceptive detection in cephalopods.

Leber hereditary optic neuropathy (LHON) is primarily caused by pathogenic mitochondrial DNA (mtDNA) variants, most commonly the m.11778G>A variant in the MT-ND4 gene. The presence of this variant alone is insufficient to trigger disease symptoms, of which vision loss is the hallmark. Given the incomplete penetrance and inter-population variability in modifying factors, this study aimed to investigate two previously proposed genetic risk factors for LHON in the Polish population. Using quantitative PCR, we measured the mtDNA copy number in peripheral blood of affected and unaffected carriers of the m.11778G>A variant. In addition, we assessed the frequency of the PRICKLE3 c.157C>T variant in symptomatic, asymptomatic and control individuals using PCR-RFLP. Our results indicate that neither mtDNA copy number nor the presence of the PRICKLE3 variant is associated with LHON symptom manifestation in the Polish cohort under conditions tested, in contrast to previously reported associations in other populations. These findings suggest that the incomplete penetrance of LHON in the Polish population may involve other modifying factors, such as yet unidentified nuclear DNA variants. Research highlightsO_LIMitochondrial DNA (mtDNA) copy number and the presence of the c.157C>T variant in the PRICKLE3 gene do not influence the manifestation of Leber hereditary optic neuropathy (LHON) symptoms in the Polish population. C_LIO_LIThe results support a geographic dependence of genetic risk factors affecting the penetrance of LHON-associated mtDNA variants. C_LI

Mutations in human LIS1 cause lissencephaly, a severe developmental brain malformation. Although most stud-ies focus on development, LIS1 is also expressed in adult mouse tissues. We previously induced LIS1 knockout (iKO) in adult mice using a Cre-Lox approach with an actin promoter driving CreERT2 expression. This proved to be rapidly lethal, with evidence pointing toward nervous system dysfunction. CreERT2 activity was observed in astrocytes, brainstem and spinal motor neurons, and axons and Schwann cells in the sciatic and phrenic nerves, suggesting dysfunctional cardiorespiratory and motor circuits. However, it is unclear how LIS1 knockout in these different cell types contributes to the lethal phenotype. We now report that LIS1 depletion from astro-cytes is not lethal to mice (male or female), although glial fibrillary protein (GFAP) expression is increased in all LIS1-depleted astrocytes. In contrast, LIS1 depletion from projection neurons causes motor deficits and rapid lethality in both males and females. This is accompanied by progressive, widespread axonal degeneration along the entire length of both motor and sensory axons. Interestingly, sensory neurons harvested from iKO mice ini-tially extend axons in culture but soon develop axonal swellings and fragmentation, indicating axonal degenera-tion. LIS1 is a prominent regulator of cytoplasmic dynein 1 (dynein, hereafter), a microtubule motor whose dis-ruption can cause both cortical malformations and later-onset neurodegenerative diseases, such as Charcot-Marie-Tooth disease. Our results raise the possibility that LIS1 depletion, through disruption of dynein function in mature axons, may lead to Wallerian-like axon degeneration without traumatic nerve injury.

Voltage-gated CaV2.2 channels are essential for neurotransmitter release throughout the nervous system including areas related to learning and memory like the hippocampus. Previous results have shown that CaV2.2 channels are involved in cognitive processes. However, a link between alternative splicing of the Cacna1b (gene that encodes for CaV2.2) pre-mRNA and cognitive processes has not been described. The Cacna1b pre-mRNA undergoes extensive cell-specific alternative splicing. In this body of work, we focus on the cassette exon 18a. Alternative splicing of exon 18a generates two splice variants, +18a-Cacna1b and {Delta}18a-Cacna1b. Exon 18a encodes a 21-amino acid sequence within the SYNaptic PRotein INTeraction (synprint) site. Splice variants containing exon 18a (+18a-CaV2.2) show reduced cumulative inactivation and increased Ca{superscript 2} current density compared to splice variants lacking exon 18a ({Delta}18a-CaV2.2), suggesting functional specialization. We previously showed that +18a-Cacna1b splice variants are enriched in cholecystokinin-expressing interneurons (CCK+INs). This neuronal type is strongly implicated in associative learning. Therefore, we tested whether alternative splicing of exon 18a contributes to associative learning. To test this hypothesis, we used genetically engineered mice that constitutively express either +18a-Cacna1b (+18a) or {Delta}18a-Cacna1b ({Delta}18a). We first validated that restricted splicing of exon 18a did not alter downstream alternative or constitutive spliced exons in the Cacna1b pre-mRNA, nor total CaV2.2 protein levels. We then performed a comprehensive behavioral analysis that included assessment of associate learning. We found that in the trace fear conditioning task, +18a mice exhibited less freezing during the trace interval in both the acquisition and memory phases compared to WT mice. Whereas {Delta}18a mice showed enhanced freezing during the same intervals relative to WT mice. These bidirectional phenotypes reveal that exon 18a shapes aversive associative learning. Furthermore, exon 18a splicing did not influence spatial working memory, spatial navigation under stress, nociceptive responses in basal and inflammatory conditions, overall locomotion or exploratory behavior. These results suggest that the behavioral impact of exon 18a splicing is highly selective. Together, our findings identify alternative splicing of exon 18a as a molecular contributor to associative learning.

Mechanosensing involves proteins detecting mechanical changes in the cytoskeleton or at cell adhesion sites. These interactions initiate signaling cascades that produce biochemical effects such as post-translational modifications or cytoskeletal rearrangements. Filamin is a ubiquitous mechanosensing protein that binds actin filaments and senses pulling forces within the cytoskeleton. Drosophila Filamin (Cheerio) is structurally similar to mammalian Filamin, with roles in egg chamber development, embryo cellularization, and integrity of muscle attachment sites and Z discs in Drosophila indirect flight muscles (IFMs). Here we report a potential novel binding partner of Drosophila Filamins: the death-associated protein kinase Drak that functions as a myosin light chain kinase. We found that Drak biochemically bound to an open mutant of Filamin that resembles the mechanically activated form partially bound to wild type Filamin and did not bind to closed mutant of Filamin. The interaction site was mapped to the intrinsically unfolded C-terminal region of Drak. To study the functional role of Drak-Filamin interaction, we studied two developmental events where Drak has been earlier shown to be expressed and where Filamin also functions: early embryonic cellularization and indirect flight muscle development at pupal stages. We found partial colocalization between Drak-GFP and Filamin-mCherry during the initiation of cellularization furrow, and at the time of myotube attachment site maturation in tendon cells. However, functionally we could not show direct correlation between Filamin and Drak. Our studies reveal interesting new expression patterns of Drak during Drosophila development and provide detailed information about Filamin localization during IFM development.

AO_SCPLOWBSTRACTC_SCPLOWThe laminins are a family of extracellular matrix proteins that regulate numerous cellular processes, including adhesion, neurite outgrowth, and axon guidance. However, it remains unclear whether laminin regulates axon guidance through local translation. Here, we show that laminin is necessary for local translation in axonal growth cones. Local translation is significantly increased in growth cones of embryonic day 17 mouse cortical neurons, either cultured on or acutely stimulated with soluble laminin 111, in the presence of BDNF. When cultured on laminin isoforms 211 or 221 in the presence of BDNF, there was a remarkable decrease in local translation in growth cones. Using a puromycin-proximity ligation assay to examine newly synthesized {beta}-actin specifically, we find a significant increase in growth cones of neurons cultured on laminin 111 in the presence of BDNF. However, soluble laminin 111 alone results in a significant reduction in nascent {beta}-actin protein synthesis. These results indicate that laminin isoforms can act in multiple ways, including synergistically with guidance cues and independently, to modulate local mRNA translation, thereby differentially influencing axon growth and guidance during development. SO_SCPLOWUMMARYC_SCPLOW SO_SCPLOWTATEMENTC_SCPLOWLocal translation in axons is critical for axon guidance. Laminin, a key component of the extracellular matrix, is necessary to induce local translation and thus mediate axon growth and guidance.

Acute spinal cord injury is a condition with a poor prognosis, leading to reduced quality of life and high economic costs for both patients and health systems, and only a minority of patients with this injury can achieve a meaningful recovery. Given the high frequency of traumatic events such as vehicle collisions and falls, research aimed at limiting injury extension, promoting neuronal recovery, and improving prognosis is essential. It has been shown that ketone bodies have anti-inflammatory properties and also mediate the Nrf2 pathway, exerting antioxidant effects. The aim was to identify any alternative to mitigate the extension and progression of secondary injury by using 1,3 butanediol as a {beta}-hydroxybutyrate source. An experimental model (n= 60) of clinically healthy male Wistar rats were used, divided into five groups (n=12) as a control group, while the remaining rats were subjected to extradural spinal cord clipping according to the following groups (n=12): Spinal Cord Injury (SCI); endogenous ketosis + methylprednisolone (Endo-K+MP); exogenous ketosis + methylprednisolone (Exo-K+MP), and methylprednisolone (MP). After 8 hours of spinal cord injury, tissue was collected, immunohistochemistry and PCR analyses were carried out. Nrf2 and 3NT for the antioxidant pathway, and HIF-1, NFkB, NLRP3, TNF-[a] and IL-1{beta} for inflammation were analyzed. Results demonstrated that the groups thrown into a ketosis state had better outcomes, and, according to the Exo-k+MP and Endo-k+MP groups, increased Nrf2 and decreased 3NT (p < 0.05), which resulted in an upregulation of antioxidant pathways. According to HIF-1 and NF{kappa}B, Endo-K+MP showed better outcomes p<0.05 and proinflammatory cytokines showed the same pattern as the standard treatment (MP) p<0.05). Overall, our results also demonstrated a downregulation of inflammatory pathways. Author SummarySpinal cord injury is a devastating condition that frequently results in permanent neurological damage, reduced quality of life, and high social and healthcare costs. Current treatments are limited and mainly focus on reducing inflammation after injury, with methylprednisolone being one of the most commonly used therapies despite its associated adverse effects. Previous studies have shown that ketosis, a metabolic state characterized by increased ketone bodies, has anti-inflammatory and antioxidant properties in several neurological conditions. In this study, we investigated whether inducing ketosis could improve early outcomes after acute spinal cord injury. Using a rat model, we compared the effects of endogenous ketosis (induced by fasting) and exogenous ketosis (induced by ketone precursors), alone or in combination with methylprednisolone. We analyzed inflammatory and oxidative stress pathways using immunohistochemistry and molecular techniques. Our findings show that ketosis enhances the anti-inflammatory and antioxidant effects of methylprednisolone, leading to reduced activation of inflammatory pathways and increased antioxidant responses during the first hours after injury. These results suggest that metabolic interventions such as ketosis may represent a promising complementary strategy to improve early management of spinal cord injury.

The rat vestibular system plays a critical role in anti-gravity responses such as the tail-lift reflex and the air-righting reflex. In a previous study in male rats, we obtained evidence that these two reflexes depend on the function of non-identical populations of vestibular sensory hair cells (HC). Here, we caused graded lesions in the vestibular system of female rats by exposing the animals to several different doses of an ototoxic chemical, 3,3-iminodipropionitrile (IDPN). After exposure, we assessed the anti-gravity responses of the rats and then assessed the loss of type I HC (HCI) and type II HC (HCII) in the central and peripheral regions of the crista, utricle and saccule. As expected, we recorded a dose-dependent loss of vestibular function and loss of HCs. The relationship between hair cell loss and functional loss was examined using non-linear models fitted by orthogonal distance regression. The results indicated that both the tail-lift reflex and the air-righting reflexes mostly depend on HCI function. However, a different dependency was found on the epithelium triggering the reflex: while the tail-lift response is sensitive to loss of crista and/or utricle HCIs, the air-righting response rather depends on utricular and/or saccular integrity.

PLA2G6-associated neurodegeneration (PLAN) is a rare, progressive neurological disorder caused by mutations in the PLA2G6 gene, which encodes the calcium-independent phospholipase A2 enzyme essential for phospholipid remodeling and membrane lipid homeostasis through the Lands cycle. Although mitochondrial dysfunction has been implicated in PLAN, the mechanisms linking PLA2G6 loss to mitochondrial degeneration across tissues, age, and sex remain poorly defined. Drosophila melanogaster (fruit flies) contains the human ortholog of the PLA2G6 gene, called iPLA2-VIA, homozygous mutation of which shows neurodegenerative phenotypes, including severely reduced lifespan, loss of locomotory ability, reduced fecundity, and mitochondrial structural and functional impairment at an early age. Thus, we use the Drosophila melanogaster iPLA2-VIA homozygous mutant flies to systematically examine mitochondrial structure, abundance, function, and the altered gene expression of the genes associated with the mitochondrial biogenesis cycle. Transmission electron microscopy revealed mitochondrial ultrastructural abnormalities in the brain, thorax, and ovary of iPLA2-VIA mutant flies, including disrupted cristae, abnormal mitochondrial morphology, and abnormal membrane integrity. Quantitative analysis demonstrated a significant, age-dependent reduction in mitochondrial number across multiple tissues in both sexes. Consistent with these structural defects, mutant flies exhibited reduced ATP production and altered reactive oxygen species (ROS) levels in a tissue-, age-, and sex-specific manner, indicating impaired mitochondrial bioenergetic capacity. At the transcriptional level, loss of function of iPLA2-VIA significantly altered the expression of genes governing mitochondrial biogenesis and dynamics. Key biogenesis regulators, including mTOR and PGC-1, were downregulated in young mutants, while genes involved in mitochondrial fusion and fission (Opa1, Mfn2, Drp1, and Fis1) showed selective, age- and sex-dependent dysregulation. Collectively, our findings demonstrate that iPLA2-VIA is essential for maintaining mitochondrial integrity, abundance, and bioenergetic function. This work establishes a mechanistic framework linking disrupted phospholipid remodeling to mitochondrial degeneration in PLAN. It highlights Drosophila as a powerful model for dissecting age- and sex-dependent mitochondrial pathology in neurodegenerative disease.

The nasal epithelium is a complex tissue composed of both respiratory and olfactory tissue, and is constantly exposed to environmental insults, including toxins and pathogens. The main olfactory epithelium (MOE) serves as the critical site for olfaction, or sense of smell. Dysfunction at this critical barrier tissue can result in partial or total loss of olfactory function, resulting in significant impact to quality of life. The MOE is heterogeneous, comprised of many cell types including olfactory sensory neurons, support cells, and immune cells. It is not well understood how these diverse cell types in the MOE interact to regulate this tissue during homeostasis, and during times of injury and inflammation. We investigated how environmental olfactory exposures impact cell type specific transcriptional responses in the mouse MOE. We performed single-cell RNA sequencing (scRNA-seq) of the MOE following controlled environmental exposure to both well-known odorants and allergens. We identified major cell types and subtypes within the MOE, and identified transcriptional changes in response to the olfactory exposures. We identified transcriptional changes in OSNs, sustentacular cells, and resident immune cells to each condition. This indicated that environmental olfactory exposures drive changes to multiple cell types in the MOE. To our knowledge, this is the first study to identify effects of environmental olfactory exposures on cell-type specific transcription at homeostasis. These findings highlight the potential importance of multi-cellular interactions and communication in regulation of the olfactory epithelium.

In adult superficial dorsal horn, 90% of Reelin (Reln+) and 70% of Disabled-1 (Dab1+) neurons co-express the transcription factor LIM-homeobox 1-beta (Lmx1b+) and therefore are glutamatergic neurons. Here we asked if embryonic Reln+Lmx1b+ and Dab1+Lmx1b+ dorsal horn neurons are derived from Lmx1b-expressing early-born dI5 or late-born dILB dorsal neurons. On Embryonic day (E)11.5, Reln+ and Dab1+ neurons appear to be part of the migration of early-born dI5 Lmx1b-expressing neurons. Between E12.5-E15.5, the lateral Reln+Lmx1b+ and Dab1+Lmx1b+ neurons migrate circumferentially along the rim of what will become the superficial dorsal horn, whereas medial Reln+Lmx1b+ and Dab1+Lmx1b+ neurons move into the dorsal midline and then migrate into lamina V. The small, late-born dILB Reln+Lmx1b+ and Dab1-Lmx1b+ neurons fill the superficial dorsal horn. In Reln mutants, large Dab1+Lmx1b+ neurons were mispositioned in lamina I and at the border between the superficial and deep dorsal horn. To confirm the identity of the circumferential and midline Reln+Lmx1b+ and Dab1+Lmx1b+ neurons, we asked if they expressed the transcription factor Zfhx3, a marker of dI5 projection neurons. We detected examples of Reln+Lmx1b+Zfhx3+ and Dab1+Lmx1b+Zfhx3+ projection neurons that migrated along the outer rim of the superficial dorsal horn and others that migrated from the midline into lamina V. Taken together, our study demonstrates that the larger Reln+Lmx1b+Zfhx3+ and Dab+Lmx1b+Zfhx3+ neurons represent two subsets of dI5 projections neurons, whereas smaller Reln+Lmx1b+ and Dab1+Lmx1b+ neurons concentrated in lamina II are likely dILB interneurons.

Alzheimers disease (AD) is a devastating neurodegenerative disorder characterized by memory loss and a decline in cognitive function. Hallmarks of AD include an age-dependent accumulation of toxic amyloid beta (A{beta}) 42 in the brain, energy dyshomeostasis caused by mitochondrial dysfunction, and iron overload. However, the role of iron overload and mitochondrial dysfunction in AD pathology is unknown and their precise relationship with A{beta} 42 toxicity in AD pathology is unclear. C. elegans provide a powerful model system to untangle and clarify these relationships. In this study, we quantify the temperature-dependence of iron toxicity (16, 20 and 25C) in neurons and muscle of C. elegans that overexpress A{beta} 42. We found that A{beta} 42, regardless of the cell-type expression, caused accelerated paralysis compared to age-matched WT worms with the greatest degree of paralysis observed at an elevated temperature (25C). Moreover, the combination of iron toxicity and A{beta} 42 results in an enhanced paralytic phenotype at 16C. Thus, iron exposure potentiates A{beta} toxicity observed at low temperatures. Iron toxicity stimulated both maximum (State 3) and leak (State 4) respiration in WT and A{beta} 42 worms. A{beta} 42 worms also exhibited increased leak respiration at baseline that was further exacerbated by iron toxicity. Iron burden and sensitivity increased A{beta} 42 peptide toxicity. A{beta} 42 worms exhibited reduced levels of Ca, Zn, Mn, and K. Overall, our results suggest that iron potentiates A{beta} toxicity at low temperature and enhances A{beta} peptide mediated mitochondrial bioenergetic dysfunction in C. elegans. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=140 SRC="FIGDIR/small/714217v1_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@9eaf46org.highwire.dtl.DTLVardef@542eforg.highwire.dtl.DTLVardef@16d9678org.highwire.dtl.DTLVardef@1b1b16d_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LITemperature stress modulates the synergetic interactions of iron toxicity and A{beta} 42 pathology C_LIO_LIIron sensitivity drives increased cell-type specific A{beta} 42 pathology C_LIO_LIEnergy dyshomeostasis via impaired mitochondrial function and increased proton leak contributes to iron- and A{beta}-induced pathology C_LI

Hypoxic-ischemic injury is a major cause of olfactory dysfunction, yet the cellular and morphological mechanisms underlying this sensory loss remain poorly understood. Here, we investigated the structural, cellular, and functional effects of acute hypoxic exposure on the olfactory system of adult zebrafish (Danio rerio) of both sexes, a model organism with remarkable neuroregenerative capacity. Fish were subjected to 15 minutes of acute severe hypoxia (0.8 mg/L dissolved oxygen) and assessed at 1 and 5 days post-hypoxia (dph). We evaluated olfactory function by means of cadaverine-evoked aversive behavioral assays. Structural and morphological integrity and inflammation of the olfactory epithelium (OE) and olfactory bulb (OB) were characterized using immunohistochemistry, histological stainings, and a 2,3,5-triphenyltetrazolium chloride (TTC) colorimetric assay. Acute hypoxic exposure impaired olfactory-mediated behaviors without affecting locomotion or exploratory behavior. In the peripheral OE, hypoxia caused neurodegeneration, disruption of the nasal mucus layer, and robust leukocytic infiltration. We observed reduced mitochondrial dehydrogenase activity in the olfactory bulb (OB) along with reactive astrogliosis. Olfactory function recovered by 5 days, coinciding with full restoration of OE morphology, and supported by a strong proliferative response. These findings reveal a coordinated degenerative and regenerative response to hypoxia across the olfactory axis, with implications for understanding hypoxia-induced sensory loss and neural repair. SIGNIFICANCEThis work addresses an important gap in knowledge regarding the mechanisms linking hypoxic insult and olfactory dysfunction. By using adult zebrafish, an extraordinarily regenerative vertebrate, it also provides insight into neuronal repair and regenerative processes supporting olfactory recovery. The novelty of our study resides in that, to our knowledge, there are no studies that provide a comprehensive characterization of the effects of hypoxia in the olfactory system across molecular, histological, and functional levels. These findings advance our understanding of hypoxia-induced sensory neurodegeneration and regeneration, and highlight the zebrafish olfactory system as a powerful model for investigating neural repair mechanisms relevant to hypoxic-ischemic brain injury.