Biology subjects

Harkany, T.

Publications and source records attributed to Harkany, T..

12 recordsLinked to original sources

bioRxiv2026

A uniform tissue-clearing framework and mesoSPIM-ultra enable cm-scale single-neuron tracing

Tissue-clearing and light-sheet microscopy have transformed volumetric imaging of intact organs, yet limited mechanistic understanding of dehydration-based clearing continues to constrain rational protocol design and broader applicability. Here, we define the cardinal chemical and physical principles underlying dehydration-based tissue-clearing and establish a new pipeline for large-volume imaging. To maximize imaging performance, we developed the "mesoSPIM-ultra", an upgraded mesoSPIM platform with a temperature-controlled sample chamber, a large field-of-view (FoV) camera and specialized optics to achieve long-working-distance, high-resolution imaging of cleared samples. We applied this approach to investigate the projectome of Chx10+ neurons, a cell population with complex axonal morphologies along the entire mouse spinal-cord and brain, and implicated in ipsilateral orienting behaviors. By combining behavioral analysis with post-hoc single-neuron reconstructions, we revealed previously inaccessible branching architectures and long-range projections extending from the brainstem to the spinal cord. Together, our work establishes a mechanistic foundation for tissue-clearing and scalable imaging.

neuroscience↗

bioRxiv2026

Methamphetamine-induced disruption of neuropeptide expression in mice

Psychoactive and psychotoxic drugs are particularly harmful, if their use coincides with critical developmental windows of brain maturation. Methamphetamine is one such stimulant with developmental exposure increasing seizure susceptibility and long-term neuronal maladaptation in children. Nevertheless, the extent at which infant and adult vulnerability to methamphetamine could differ in time-course and severity remains incompletely understood. Here, we developed a method to monitor methamphetamine-induced hyperactivity in infant mice at high temporal resolution, differentiate it from a biphasic response in adults, and link it to activity changes in cortical areas executing goal-directed (escape) behaviors in infant subjects when using Fos expression as a molecular surrogate. Subsequently, we hypothesized that methamphetamine could alter the expression and cellular distribution of inhibitory neuropeptides, which, when co-released with fast neurotransmitters, could protect circuit plasticity by counteracting methamphetamine-induced hyperexcitability. Methamphetamine differentially altered somatostatin, cholecystokinin, and galanin expression in corticolimbic areas. These data suggest that methamphetamine can evoke age-specific neurocircuit modifications, at least in mice.

neuroscience↗

bioRxiv2026

Stratifying cellular injury in Alzheimer's disease by chaperonin containing TCP1 subunits 2 and 3

Chaperonins complex into double-ringed octamers to aid peptide folding. Recent evidence implicates dysfunctional chaperonin subunits in cancer and neurodegenerative diseases because their deregulation exacerbates cellular injury. Nevertheless, a gap of knowledge exists regarding the expression and localization of chaperonin subunits in relation to amyloidogenic processes in Alzheimers disease (AD). Here, we show that reduced levels of chaperonin-containing TCP-1 subunits 2 (CCT2) and 3 (CCT3) stratify AD, with the subcellular distribution of their residua being mutually exclusive with both {beta}-amyloid and hyperphosphorylated tau in neurons. We find CCT3 localized to a subset of glial fibrillary acidic protein-positive astrocytes in AD. Increased oxidative stress in vitro upregulated CCT3 expression in astrocyte-like U251 cells. Conversely, CCT3, but not CCT2, loss-of-function in neuron-like SH-SY5Y cells increased intracellular {beta}-amyloid load. These data suggest that CCT2/CCT3 are faithful disease-state indicators and implicate CCT3 in oxidative stress-dependent cellular damage pathways.

neuroscience↗

bioRxiv2026

Astrocytes modulate mitochondrial proteostasis and membrane potential in neurons through S100A6 - calcyclin-binding protein signaling

Calcyclin binding protein (CaCyBp) is an evolutionarily conserved protein that converts S100 protein family-derived signals to protein turnover through an ubiquitin ligase interaction domain within its C-terminus. Despite its predicted roles in cellular differentiation, CaCyBp expression alone and in relation to S100A6, its prototypic ligand, during fetal and postnatal organ development remains incompletely understood. Here, we combined cell-resolved neuroanatomy and biochemistry to reveal not only organ system-level CaCyBp expression but also its region-, circuit-, and neuron-specific distribution in the mouse brain from embryonic day 18.5. We extended these studies to human brains past gestational week 27, corroborating that neurons, but not astrocytes, microglia or oligodendrocytes, harbored CaCyBp. Conversely, S100A6 was detected only in astrocytes and ependymoglia in both species. Ultrastructural studies localized CaCyBp to mitochondria in neurons, with genetic loss-of-function and recombinant S100A6-induced signaling implicating CaCyBp in the regulation of the mitochondrial electrochemical gradient controlling ATP production. Overall, pleiotropic CaCyBp expression in neurons suggests a maintenance receptor-like role impacting cellular respiration, thus affecting proteostasis upon glia-derived S100A6 signals.

cell biology↗

bioRxiv2025

(S)-ketamine augments behavioral and physiological responses to Δ9-tetrahydrocannabinol

(S)-ketamine is a fast-acting therapeutic to manage treatment-resistant depression as well as psychiatric emergencies. As the non-prescribed use of ketamine has rapidly increased over the past decade, its possible interactions with other recreational drugs are of increasing interest. Here, we addressed if {Delta}9-tetrahydrocannabinol (THC), the main psychoactive constituent of Cannabis sp., and (S)-ketamine could affect each others psychoactive profile in a pharmacological triad. In healthy male mice, the sequential use of (S)-ketamine followed by THC at human-relevant doses did not result in either synergistic or antagonistic effects acutely. In contrast, THC-induced hypothermia was nearly doubled by subsequent (S)-ketamine administration. THC-induced hypolocomotion was not only prolonged by (S)-ketamine, but axial rotation and stumbling during ambulation in an open field were increased in subjects exposed sequentially to both drugs. Likewise, the performance of subjects exposed to THC and then (S)-ketamine worsened on a rotarod. Combined drug treatment led to unusual epinephrine and corticosterone profiles in blood plasma. We suggest that (S)-ketamine augments adverse THC effects, which at the endocrine level invoke the acute deregulation of the hypothalamus-pituitary-adrenal axis. Our findings support the clinical notion that combining cannabis and (S)-ketamine should be avoided.

pharmacology and toxicology↗

bioRxiv2025

Alk-Fam150b (augmentor α) expression in the paraventricular nucleus of the mouse hypothalamus at molecular resolution, and its sensitivity to acute stress

Augmentor (Fam150b) action on the ALK receptor (Alk) has gained significance as a hypothalamic signaling pathway with relevance to the control of food intake and energy homeostasis. In contrast, much less is known about the sensitivity of Fam150b-Alk expression and signaling upon noxious challenges. In this regard, acute stress is of particular interest because augmentor , released from afferents of the food intake circuit of the arcuate hypothalamus in the paraventricular hypothalamus (PVN), could link stress-induced changes in food consumption. Nevertheless, conflicting data exist on whether Fam150b mRNA is expressed in the PVN. Here, we combined single-cell RNA-seq and multiplexed in situ hybridization to demonstrate that both Fam150b and Alk are expressed in the PVN of adult mice, including corticotropin-releasing hormone (CRH)-containing neurons. As such, a dichotomy of CRH neurons is present through their mutually exclusive expression of either Fam150b or Scgn (secretagogin). Fam150b and Alk were not co-expressed. When inducing inflammation-associated stress, Fam150b but not Alk mRNA expression increased in a mifepristone-sensitive manner, implying regulation by peripheral glucocorticoid feedback. We suggest that augmentor -ALK signaling could underpin, at least partly, stress-induced changes in feeding and the control of body weight.

neuroscience↗

bioRxiv2023

Ependymocytes control cerebrospinal fluid flow to the peripheral organs through periaxonal pathway

Mechanisms controlling movement of the CSF through the central canal towards the peripheral nerves are poorly characterized. We found that fluorescent dyes injected into cisterna magna are carried with cerebrospinal fluid (CSF) through the central canal and peripheral nerves to the peripheral organs such as liver, and pancreas. We also found close connection between spinal axons and ependymocytes, suggesting synaptic interactions. Serotonin, acting through the 5-HT2B receptors abundantly expressed in ependymal cells, trigger Ca2+ signal that induces polymerization of cytoskeleton protein F-actin, consequently reducing the volume of ependymocytes. Shrinkage of the latter opens one-way route to facilitate CSF outflow from the central canal into the spinal cord parenchyma and peripheral nerves. In liver, CSF is received by stellate cells. Ependymal control over transfer of the CSF from central canal to peripheral organs by the periaxonal space (PAS) represents a novel mechanism dynamically connecting the CNS with the periphery. In briefEpendymocytes control CSF flow from CNS to peripheral organs by periaxonal pathway, and serotonin evokes the ependymal shrinkage by the aggregation of F-actin. HighlightsEpendymocytes control CSF flow from central canal to peripheral organs; CSF flows through the peripheral periaxonal space to reach the peripheral organs; Serotonin makes ependymocytes shrunk by 5-HT2B receptor-mediated Ca2+ signaling and F-actin polymerization; The hepatic stellate cells are potential collectors of CSF in the liver.

neuroscience↗

bioRxiv2023

Astrocytes modulate neuronal development by S100A6 signaling

Neuronal morphogenesis relies on intercellular signaling. Astrocytes release metabolites, trophic, and guidance factors to promote neuronal maturation. In contrast, the mechanisms by which astrocytes could limit and stabilize neuronal connectivity remain less explored. Here, we show cortical astrocytes to express and release S100A6, a Ca2+-binding protein ( calcyclin). Simultaneously, the majority of cortical neurons express calcyclin-binding protein (CaCyBp), a bona fide binding partner for S100A6. In neurons, CaCyBp maintains the unfolded protein response pathway, thereby controlling proteostasis. When released, S100A6 inhibits CaCyBp-mediated signaling, thus slowing protein turnover, and, consequently, neuritogenesis. S100A6-CaCyBp signaling during gestation and lactation is sensitive to the mothers nutritional status, particularly eicosapentaenoic acid intake. Thus, a member of the S100 protein family acts as an astroglia-derived morphogen, whose action on neurons is modulated by environmental factors.

neuroscience↗

bioRxiv2023

Crucial neuroprotective roles of the metabolite BH4 in dopaminergic neurons

Dopa-responsive dystonia (DRD) and Parkinsons disease (PD) are movement disorders caused by the dysfunction of nigrostriatal dopaminergic neurons. Identifying druggable pathways and biomarkers for guiding therapies is crucial due to the debilitating nature of these disorders. Recent genetic studies have identified variants of GTP cyclohydrolase-1 (GCH1), the rate-limiting enzyme in tetrahydrobiopterin (BH4) synthesis, as causative for these movement disorders. Here, we show that genetic and pharmacological inhibition of BH4 synthesis in mice and human midbrain-like organoids accurately recapitulates motor, behavioral and biochemical characteristics of these human diseases, with severity of the phenotype correlating with extent of BH4 deficiency. We also show that BH4 deficiency increases sensitivities to several PD-related stressors in mice and PD human cells, resulting in worse behavioral and physiological outcomes. Conversely, genetic and pharmacological augmentation of BH4 protects mice from genetically- and chemically induced PD-related stressors. Importantly, increasing BH4 levels also protects primary cells from PD-affected individuals and human midbrain-like organoids (hMLOs) from these stressors. Mechanistically, BH4 not only serves as an essential cofactor for dopamine synthesis, but also independently regulates tyrosine hydroxylase levels, protects against ferroptosis, scavenges mitochondrial ROS, maintains neuronal excitability and promotes mitochondrial ATP production, thereby enhancing mitochondrial fitness and cellular respiration in multiple preclinical PD animal models, human dopaminergic midbrain-like organoids and primary cells from PD-affected individuals. Our findings pinpoint the BH4 pathway as a key metabolic program at the intersection of multiple protective mechanisms for the health and function of midbrain dopaminergic neurons, identifying it as a potential therapeutic target for PD.

neuroscience↗

bioRxiv2022

Preclinical establishment of a divalent vaccine against SARS-CoV-2

First-generation vaccines against SARS-CoV-2 have been administered to more than 60% of the population in developed countries. However, the monovalent vaccines currently available in Europe do not confer adequate and durable immune protection. To satisfy the need for a novel vaccine, we engineered a divalent gene construct consisting of the receptor binding domain (RBD, 300-685 aa) of the spike protein and the immunodominant region of the nucleocapsid (100-300 aa). This fusion protein was cloned into a pET-30a plasmid and expressed either in Escherichia coli or in a recombinant baculovirus in insect cells. Following purification via its His-tag, the fusion protein was mixed with adjuvant, and administered to mice in a prime-booster-mode. Upon testing for IgG antibody response against nucleocapsid and RBD, a titer of 10-4 - 10-5 was demonstrated 14 days after the first booster injection in 72% of the animals, which could be increased to 100% by a second booster. Notably, comparable IgG responses were detected against the delta, gamma and omicron variants of the RBD region. Durability testing revealed the presence of IgG beyond 90 days. In addition, granzyme A and perforin mRNA expression (cytolytic effector cell molecules) was increased in cytotoxic lymphocytes isolated from peripheral blood. Ex vivo stimulation of T-cells by nucleocapsid and RBD peptides showed antigen-specific upregulation of CD44 in vaccinated mice among their CD4+ and CD8+ T-cells. No side-effect was documented in the central nervous system, be it either endothelial inflammation or neuronal damage. Cumulatively, the combined induction of B-cell and T-cell response by a bivalent protein-based vaccine directed against two structural SARS-CoV-2 proteins represents a proof-of-principle approach alternative to existing mRNA vaccination strategies, which could confer long-lasting immunity against all known viral strains.

bioengineering↗

bioRxiv2020

POMC neurons functional heterogeneity relies on mTORC1 signaling

Hypothalamic Pro-opiomelanocortin (POMC) neurons are classically known to trigger satiety. However, they encompass heterogeneous subpopulations whose functions are unknown. Here we show that POMC neurons releasing GABA, glutamate or both neurotransmitters possess distinct spatial distribution, molecular signatures and functions. Functional specificity of these subpopulations relies on the energy sensor mechanistic Target of Rapamycin Complex 1 (mTORC1), since pharmacological blockade of mTORC1, by mimicking a cellular negative energy state, simultaneously inhibited POMC/glutamatergic and activated POMC/GABAergic neurons. Chemogenetics and conditional deletion of mTORC1 then demonstrated that mTORC1 blockade in POMC neurons causes hyperphagia. This is due to decreased POMC-derived anorexigenic -melanocyte-stimulating hormone and the recruitment of POMC/GABAergic neurotransmission, which is restrained by cannabinoid type 1 receptor signaling. Genetic inhibition of glutamate release from POMC neurons also produced hyperphagia, recapitulating the phenotype caused by mTORC1 blockade. Altogether, these findings pinpoint the molecular mechanisms engaged by POMC neurons to oppositely control feeding, thereby challenging conventional views about their functions.

neuroscience↗

bioRxiv2019

Brain-wide genetic mapping identifies the indusium griseum as a prenatal and shared target of pharmacologically-unrelated psychostimulants

Psychostimulant use is an ever-increasing socioeconomic burden, including a dramatic rise during pregnancy. Nevertheless, brain-wide effects of psychostimulant exposure are incompletely understood. Here, we performed Fos-CreERT2-based activity mapping, correlated for pregnant mouse dams and their fetuses with amphetamine, nicotine and caffeine applied acutely during mid-gestation. While light-sheet microscopy-assisted intact tissue imaging revealed drug- and age-specific neuronal activation, the indusium griseum (IG) appeared indiscriminately affected. By using GAD67gfp/+ mice we subdivided the IG into a dorsolateral domain populated by GABA interneurons and a ventromedial segment containing glutamatergic neurons, many showing drug-induced activation and sequentially expressing Pou3f3/Brn1 and secretagogin (Scgn) during differentiation. We then combined Patch-seq and circuit mapping to show that the ventromedial IG is a quasi-continuum of glutamatergic neurons (IG-Vglut1+) reminiscent to dentate granule cells in both rodents and humans, whose dendrites emanate perpendicularly towards, while their axons course parallel with the superior longitudinal fissure. IG-Vglut1+ neurons receive Vglut1+ and Vglut2+ excitatory afferents that topologically segregate along their somatodendritic axis. In turn, their efferents terminate in the olfactory bulb, thus being integral to a multi-synaptic circuit that could feed information antiparallel to the olfactory-cortical pathway. In IG-Vglut1+ neurons, prenatal psychostimulant exposure delayed the onset of Scgn expression. Genetic ablation of Scgn was then found to sensitize adult mice towards methamphetamine-induced epilepsy, suggesting a role for this Ca2+-binding protein in scaling IG-Vglut1+ neuronal excitability. Overall, our study identifies brain-wide targets of the most common psychostimulants, among which Scgn+/Vglut1+ neurons of the IG link limbic and olfactory circuits.\n\nSignificance statementDrug abuse during pregnancy is a significant socioeconomic problem. The use of psychostimulants is particularly common during pregnancy even though a risk to the developing fetus is significant. Here, we show that short-lived exposure to amphetamine, nicotine and caffeine during pregnancy induces neuronal activation in the fetal brain with the indusium griseum (IG), a brain area situated parallel to the central surface of the cortical hemispheres, becoming indiscriminately activated. By using mouse genetics, we find that psychostimulants preferentially target glutamatergic IG neurons, and delay their differentiation postnatally. Notably, the expressional onset of secretagogin, a Ca2+-sensor amenable for synaptic integration, is deregulated. This is significant because these neurons are integral to a multi-synaptic neuronal pathway that links limbic and olfactory circuits. As such, genetic deletion of secretagogin brings about heightened sensitivity to psychostimulants, manifesting as epileptiform discharges. Cumulatively, we describe a novel psychostimulant-sensitive neuronal subtype and its circuit arrangement whose developmental delay seems critical for behavioral abnormalities in offspring prenatally exposed to the most common psychostimulants.

neuroscience↗