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Journal of Neurochemistry

Published by Wiley and International Society for Neurochemistry

Online ISSN: 1471-4159

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Print ISSN: 0022-3042

Disciplines: Neuroscience

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Glutamate links central metabolic pathways in the brain. Glutamate serves as a metabolic hub linking amino acid, neurotransmitter, and energy metabolism through the TCA cycle intermediate α‐ketoglutarate. Glutamate synthesis and metabolism is primarily mediated by aspartate aminotransferase (AAT) and glutamate dehydrogenase (GDH), which are essential cerebral enzymes. However, three other transamination reactions also catalyze the conversion between glutamate and α‐ketoglutarate: alanine aminotransferase (ALAT), branched‐chain amino acid aminotransferase (BCAT), and GABA transaminase (GABA‐T). GABA‐T transfers the nitrogen of GABA to α‐ketoglutarate forming glutamate, whereas the carbon skeleton of GABA is converted into succinic semialdehyde (SSA). Succinic semialdehyde dehydrogenase (SSADH) subsequently converts SSA to the TCA cycle intermediate succinate for oxidation of the GABA carbon skeleton. Glutamate also serves as the precursor of GABA by glutamate decarboxylase (GAD) activity. Finally, glutamate is also the precursor of glutamine, the synthesis of which is catalyzed by the astrocytic enzyme glutamine synthetase (GS) under the fixation of ammonia. Conversely, glutamine can be converted back into glutamate by phosphate‐activated glutaminase (PAG). Glutamine synthesis and metabolism are particularly important metabolic pathways for neurotransmitter recycling (see Figure 2). Abbreviations not explained above: BCAA, branched‐chain amino acid; BCKA, branched‐chain α‐keto acid; OAA, oxaloacetate; Pyr, pyruvate.
The glutamate/GABA‐glutamine cycle connects neurotransmitter homeostasis and cellular energy metabolism. Glutamate, GABA, and glutamine are extensively recycled between neurons and astrocytes, a process which is coordinated and regulated by cell‐specific expression of transporters and enzymes. The majority of glutamate, and a substantial fraction of GABA, released from neurons is recovered from the synapse by uptake into astrocytes. Astrocytes express the glutamate transporters GLT‐1 and GLAST and the GABA transporters GAT1 and GAT3, whereas glutamatergic and GABAergic neurons express GLT‐1 and GAT1, respectively. In the astrocyte, glutamate and GABA are metabolized, which supports the synthesis of the non‐neuroactive amino acid glutamine, which is subsequently released. The astrocyte‐derived glutamine is taken up by neurons and is converted into glutamate by phosphate‐activated glutaminase (PAG) to replenish the neurotransmitter pools. Glutamine transfer is primarily mediated by the sodium‐coupled neutral amino acid transporters (SNATs). Astrocytes express SNAT3 and SNAT5, whereas SNAT1, SNAT2, SNAT7, and SNAT8 are neuronal glutamine transporters. The recycling of glutamate, GABA and glutamine is known as the GABA‐glutamine cycle (left) and the glutamate‐glutamine cycle (right), which is also collectively called the glutamate/GABA‐glutamine cycle. Neurotransmitter recycling is closely connected to energy metabolism, as glutamate, GABA, and glutamine all can undergo oxidation in the TCA cycle and hereby support energy production in both neurons and astrocytes. Furthermore, the TCA cycle intermediate α‐ketoglutarate is the obligatory precursor of glutamate synthesis (Figure 1). Abbreviations not explained above: AAT: aspartate aminotransferase; GAD: glutamate decarboxylase; GDH: glutamate dehydrogenase; GABA‐T: GABA transaminase; SSADH: succinic semialdehyde dehydrogenase.
Application of nuclear magnetic resonance (NMR) spectroscopy to functionally explore astrocytic and neuronal metabolism. NMR spectroscopy and stable isotope tracing, that is, mapping metabolism of substrates enriched with stable isotopes, were applied to explore metabolism of cultured neurons and astrocytes. (a) Uptake and subsequent metabolism of exogenous [U‐¹³C]glutamate (0.5 mM) leads to extensive glutamine synthesis and release in cultured astrocytes (Sonnewald, Westergaard, Petersen, et al., 1993). Furthermore, the ¹³C‐enriched carbon skeleton of glutamate was recovered as lactate in the media. This occurs through partial pyruvate recycling catalyzed by malic enzyme (ME) or the concerted actions of phosphoenolpyruvate carboxykinase (PEPCK) and pyruvate kinase (PK). The physiological roles of partial pyruvate recycling are not completely clear but may serve as a pathway to sustain glutamate uptake in astrocytes. (b) By analyzing the media of cultured neurons and astrocytes following metabolism of [U‐¹³C]glucose, a preferential astrocytic release of alanine, citrate, and glutamine was demonstrated (Sonnewald et al., 1991), whereas both neurons and astrocyte in culture released large quantities of lactate. This study prompted the notion that astrocyte‐derived metabolites could play an active role for neuronal biosynthesis and function. (c) Incubating co‐cultures of astrocytes and GABAergic neurons with [2‐¹³C]acetate, a substrate preferentially metabolized in astrocytes, showed extensive ¹³C enrichment of neuronal GABA (Sonnewald, Westergaard, Schousboe, et al., 1993). By applying L‐methionine sulfoximine (MSO), an inhibitor of the astrocytic enzyme glutamine synthetase (GS), a drastic reduction of the ¹³C enrichment of GABA was observed, hereby functionally demonstrating that astrocyte‐derived glutamine serves as direct precursor of neuronal GABA synthesis. This observation is now a well‐established feature of the GABA‐glutamine cycle (Figure 2). Abbreviations not explained above: ALAT: alanine aminotransferase; LDH: lactate dehydrogenase; PAG: phosphate‐activated glutaminase.
Distinct metabolic features of astrocyte are essential for brain function. Astrocyte metabolism is primed to sustain neurotransmitter recycling. (1) Astrocytes exhibit a large capacity for uptake of neurotransmitter glutamate and GABA. (2) GABA taken up by astrocytes is extensively metabolized as astrocytes display high activity of GABA transaminase (GABA‐T) and succinic semialdehyde dehydrogenase (SSADH). (3) Astrocytes are the primary compartment of glutamate dehydrogenase (GDH) expression and activity, facilitating efficient glutamate oxidation in the TCA cycle. (4) Crucially, astrocytes synthesize large amounts of glutamine via glutamine synthetase (GS) activity. Glutamine is released from astrocytes and serves essential roles in restoring neuronal neurotransmitter pools as part of the glutamate/GABA‐glutamine cycle (Figure 2). Furthermore, glutamine synthesis is the primary route of cerebral ammonia fixation. (5) Astrocytes release the TCA cycle intermediate citrate which is able to regulate NMDA receptor activity. (6) To sustain the extensive biosynthesis and release of metabolites, astrocytes express pyruvate carboxylase (PC) being the primary anaplerotic enzyme of the brain. PC converts pyruvate to oxaloacetate and hereby replenish the pools of TCA cycle intermediates, which is essential to counteract the drain of metabolites. (7) Astrocytes are the primary compartment of fatty acid metabolism in the brain. Notably, acetate can be used as a selectively marker of astrocyte energy metabolism. (8) Astrocytes release lactate, which may support neuronal energy metabolism. (9) Astrocytes hold significant stores of the polysaccharide glycogen, which serves as an emergency fuel, but also facilitates multiple other functions. The functionality of the specialized features of astrocyte metabolism, including glutamate metabolism, glutamine synthesis, anaplerosis, and glycogen mobilization are all crucial to sustain synaptic function. Abbreviations not explained above: AAT, aspartate aminotransferase; AceCS, acetyl CoA synthetase; G‐1‐P, glucose‐1‐phosphate; G‐6‐P, glucose‐6‐phosphate; LDH, lactate dehydrogenase; PDH, pyruvate dehydrogenase.
Modulation of astrocyte and neuron energy metabolism via lipid and ketone supplementation. Pathological dysfunction of the energy metabolism of neurons and astrocytes may mediate or aggravate synaptic dysfunction and neurodegeneration. (1) Excessive release or impaired clearance of synaptic glutamate may lead to overstimulation, excitotoxicity, and neuronal death. (2) Diminished metabolic function of astrocytes may deprive neurons of metabolic support, including transfer of serine, lactate, ketones, and glutamine. (3) Reduced oxidative astrocyte metabolism and deficient glutamine synthesis, may lead to an intracellular accumulation of glutamate, which ultimately may impair glutamate uptake and hence contribute to excitotoxicity. (4) Ketones, for example, β‐hydroxybutyrate, are a commonly applied supplementary fuel, which can either be given as dietary supplement or be generated from hepatic fatty acid metabolism. Ketones are the primary alternative fuel of the brain and are excellent substrates to support neuronal energy metabolism. (5) Astrocytes are the primary compartment of fatty acid metabolism in the brain and exogenous fatty acids support the metabolic function of astrocytes. Particularly, the medium‐chain fatty acids octanoic acid (C8) and decanoic acid (C10) have been found to boost lactate, ketone and glutamine synthesis is astrocytes. (6) Targeting astrocyte metabolism, by improving TCA cycle function, glycolytic activity, and glutamine synthesis, may aid to restore activity of the glutamate/GABA‐glutamine cycle, increase synaptic function, and ultimately halt pathological progression. Dietary supplementation with a combination of ketones and fatty acids, for example, via medium‐chain triglycerides, will thus provide auxiliary fuels for both neurons and astrocytes. Abbreviations not explained above: AAT: aspartate aminotransferase; GDH: glutamate dehydrogenase; GS: glutamine synthetase; PDH: pyruvate dehydrogenase.
Milestone Review: Metabolic dynamics of glutamate and GABA mediated neurotransmission — The essential roles of astrocytes

March 2023

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The Journal of Neurochemistry is dedicated to disseminating research covering all aspects of neurochemistry including molecular, cellular, biochemical and behavioural aspects of the nervous system, with a focus on pathogenesis, biomarkers and treatment of neurological and psychiatric disorders. We prioritize original research that demonstrates a mechanistic advance as well as critical reviews that highlight progression of knowledge in the field. We are owned by the International Society for Neurochemistry.

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mTORC2 Regulates Actin Polymerization in Auditory Cells
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February 2025

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2 Reads

Michael Lanz

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Maurizio Cortada

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Yu Lu

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Daniel Bodmer

Mammalian target of rapamycin complex 2 (mTORC2) is essential for hearing by regulating auditory hair cell structure and function. However, mechanistic details of how mTORC2 regulates intracellular processes in sensory hair cells have not yet been clarified. To further elucidate the role of mTORC2 in auditory cells, we generated a Rictor knockout cell line from HEI‐OC1 auditory cells. mTORC2‐deficient auditory cells exhibited significant alterations in actin cytoskeleton morphology and decreased proliferation rates. Additionally, we observed a reduction in phosphorylation of protein kinase C alpha (PKCα) and disrupted actin polymerization in mTORC2‐deficient cells. Using proteomics, we found that mTORC2 disruption altered expression of cytoskeleton‐related proteins in auditory cells. These findings provide valuable mechanistic insights into the functional role of mTORC2 in auditory cells, potentially opening new perspectives to address sensorineural hearing loss. image


PPARβ/δ activation reduces corticosterone‐induced astrocytic ROS levels and cell death. (a) After treatment with corticosterone, GW0742 can elevate cellular viability in a concentration‐dependent manner. n = 6 cell culture preparations, F(4,25) = 18.04; p < 0.0001. ***p < 0.001 versus Ctrl group; ##p < 0.01, ###p < 0.001 versus Cort group. The Ctrl group means vehicle for Cort/GW/GSK. (b) Pretreatment with GSK3787 reverses the effect of GW0742. n = 6 cell culture preparations, F(3,20) = 65.68; p < 0.0001. ***p < 0.001 versus Ctrl group; ###p < 0.001 versus Cort group; &&&p < 0.001 versus Cort + GW group. The Ctrl group means vehicle for Cort/GW/GSK. (c) The representative photos of the treated astrocytes. (d) The representative photos of intracellular ROS in astrocytes. Scale bar is 200 μm. (e) Quantification of intracellular ROS in astrocytes. n = 4 cell culture preparations, F(3,11) = 97.79; p < 0.0001. ***p < 0.001 versus Ctrl group; ###p < 0.001 versus Cort group, &&&p < 0.001 versus Cort + GW group. The Ctrl group means vehicle for Cort/GW/ GSK. (f) The representative photos of cell apoptosis determined by flow cytometry. (g) Quantification of cell apoptosis. n = 4 cell culture preparations, F(3,12) = 130.2; p < 0.0001. ***p < 0.001 versus Ctrl group; #p < 0.05 versus Cort group; &&&p < 0.001 versus Cort + GW group. The Ctrl group means vehicle for Cort/GW/ GSK. Data were presented as mean ± SD and analyzed by a one‐way ANOVA followed by Turkey's post hoc tests.
PPARβ/δ activation partially reverses corticosterone‐induced decrease in pexophagy in astrocytes. (a) The representative photographs of peroxisome‐tracing combined with LC3 immunofluorescence. Scale bar is 20 μm. (b) Quantitative analysis of the Manders' Colocalization Coefficients between LC3 and mScarlet‐peroxisome. n = 4 cell culture preparations, F(3,19) = 18.62; p < 0.0001. ***p < 0.001 versus Con group; ###p < 0.001 versus Cort group; ⁺⁺⁺p < 0.001 versus Cort+GW group. Each data point represents the mean ± SEM. (c) The representative photographs of peroxisome‐tracing combined with LAMP1 immunofluorescence. Scale bar is 20 μm. (d) Quantitative analysis of the Manders' Colocalization Coefficients between LAMP1 and mScarlet‐peroxisome. n = 4 cell culture preparations, F(3,19) = 19.61; p < 0.0001. ***p < 0.001 versus Con group; ###p < 0.001 versus Cort group; ⁺⁺⁺p < 0.001 versus Cort+GW group. (e) The representative photographs of peroxisome‐tracing combined with SQSTM1/p62 immunofluorescence. Scale bar is 20 μm. (f) Quantitative analysis of the Manders' Colocalization Coefficients between P62/SQSTM1 and mScarlet‐peroxisome. n = 4 cell culture preparations, F(3,18) = 21.92; p < 0.0001. ***p < 0.001 versus Con group; ###p < 0.001 versus Cort group; ⁺⁺⁺p < 0.001 versus Cort+GW group. Each data point represents the mean ± SEM. (g) The representative blots of autophagy associated proteins. (h) Quantitative analysis LC3. n = 6 cell culture preparations, F(3,20) = 23.48; p < 0.0001. ***p < 0.001 versus Ctrl group; ###p < 0.001 versus Cort group; &&&p < 0.001 versus Cort + GW group. The Ctrl group means vehicle for Cort/GW/ GSK. (i) Quantitative analysis Atg5. n = 6 cell culture preparations, F(3,20) = 17.45; p < 0.0001. ***p < 0.001 versus Ctrl group; ###p < 0.001 versus Cort group; &&p < 0.01 versus Cort + GW group. The Ctrl group means vehicle for Cort/GW/ GSK. (j) Quantitative analysis Beclin1. n = 6 cell culture preparations, F(3,8) = 8.212; p < 0.0001. *p < 0.05 versus Ctrl group; #p < 0.05 versus Cort group; &p < 0.05 versus Cort + GW group. The Ctrl group means vehicle for Cort/GW/ GSK. (k) Quantitative analysis SQSTM1/p62. n = 6 cell culture preparations, F(3,8) = 14.84; p < 0.0001. **p < 0.01 versus Ctrl group; #p < 0.05 versus Cort group; &&p < 0.01 versus Cort + GW group. The Ctrl group means vehicle for Cort/GW/ GSK. (l) Quantitative analysis ULlK1. n = 6 cell culture preparations, F(3,19) = 37.72; p < 0.0001. ***p < 0.001 versus Ctrl group; ###p < 0.001 versus Cort group; &&&p < 0.001 versus Cort + GW group. The Ctrl group means vehicle for Cort/GW/ GSK. Data were presented as mean ± SD and analyzed by a one‐way ANOVA followed by Turkey's post hoc tests. (m) The representative photographs of pexophagy flux traced by mCherry‐EGFP‐LC3B in astrocytes. n = 4. Scale bar is 20 μm. (n) The quantification of autolysosomes in astrocytes in each group. n = 6 cell culture preparations, F(3,20) = 83.24; p < 0.0001. ***p < 0.01 versus Con group; ###p < 0.01 versus Cort group; &&&p < 0.01 versus Cort + GW group. (o) The quantification of autophagosomes in astrocytes in each group. n = 6 cell culture preparations, F(3,20) = 36.14; p < 0.0001. **p < 0.01 versus Con group; ###p < 0.01 versus Cort group; &&&p < 0.01 versus Cort + GW group.
PPARβ/δ activation promotes ATM phosphorylation and interaction with PEX5 in astrocytes under corticosterone treatment conditions. (a) The representative photographs of immunofluorescent co‐labeled phosphorylated ATM and PMP70 in astrocytes. n = 4. Scale bar is 50 μm. (b) The representative blots (the top) and quantification (the bottom) of phosphorylated ATM and ATM in cytoplasm. n = 5 cell culture preparations, F(3,19) = 19.31; p < 0.0001. ***p < 0.001 versus Ctrl group; ##p < 0.01 versus Cort group; &&&p < 0.001 versus Cort + GW group. The Ctrl group means vehicle for Cort/GW/ GSK. (c) The representative blots (the top) and quantification (the bottom) of phosphorylated ATM and ATM in nucleus. n = 6 cell culture preparations, F(3,20) = 2.115; p = 0.1304. (d) The representative photographs of immunofluorescent co‐labeled phosphorylated ATM and PEX5 in astrocytes. n = 4 cell culture preparations. Scale bar is 50 μm. (e) The representative blots of interaction between phosphorylated ATM and PEX5 determined by co‐immunoprecipitation. n ≥ 3.
PPARβ/δ activation facilitates pexophagy and alleviates oxidative injury by enhancing UBR5‐ATM signaling pathway. (a) The representative blots of UBR5 and ATMIN. (b) Quantitative analysis ATMIN in cytoplasm. n = 6 cell culture preparations, F(3,20) = 39.38; p < 0.0001. *p < 0.05 versus Ctrl group; #p < 0.05 versus Cort group; &p < 0.05 versus Cort + GW group. The Ctrl group means vehicle for Cort/GW/ GSK. (c) Quantitative analysis ATMIN in nucleus. n = 6 cell culture preparations, F(3,20) = 11.43; p = 0.0001. **p < 0.01 versus Ctrl group; ##p < 0.01 versus Cort group; &&p < 0.01 versus Cort + GW group. The Ctrl group means vehicle for Cort/GW/ GSK. (d) Quantitative analysis UBR5 in cytoplasm. n = 6 cell culture preparations, F(3,20) = 39.78; p < 0.0001. ***p < 0.001, **p < 0.01 versus Ctrl group; ###p < 0.001 versus Cort group; &p < 0.001 versus Cort + GW group. The Ctrl group means vehicle for Cort/GW/ GSK. (e) Quantitative analysis UBR5 in nucleus. n = 6 cell culture preparations, F(3,19) = 22.67; p < 0.0001. ***p < 0.001 versus Ctrl group; ###p < 0.001 versus Cort group; &&&p < 0.001 versus Cort + GW group. The Ctrl group means vehicle for Cort/GW/ GSK. Data were presented as mean ± SD and analyzed by a one‐way ANOVA followed by Turkey's post hoc tests. (f) The diagram of interaction between UBR5 and PPARβ/δ determined by ChIP assay. (g) Quantification of interaction between UBR5 and PPARβ/δ. n = 3 cell culture preparations, F(2,12) = 11.32; p = 0.0017. F(2,12) = 187.8; p < 0.0001. ****p < 0.001 versus Ctrl group; ###p < 0.001 versus Cort group. The Ctrl group means vehicle for Cort/GW/ GSK. (h) The representative blots of autophagy associated proteins. (i) Quantitative analysis LC3. n = 3 cell culture preparations, F(1,20) = 51.43; p < 0.0001. ***p < 0.001 versus NC Cort group. (j) Quantitative analysis Beclin1. n = 3 cell culture preparations, F(1,20) = 25.59; p < 0.0001. ***p < 0.001 versus NC Cort group. (k) Quantitative analysis Atg5. n = 3 cell culture preparations, F(1,20) = 14.07; p = 0.0013. ***p < 0.001 versus NC Cort group. (l) Quantitative analysis SQSTM1/p62. n = 3 cell culture preparations, F(1,20) = 6.191; p = 0.0218. ***p < 0.001 versus NC Cort group. (m) GW0742 reverses corticosterone‐induced decreased cellular viability depending on UBR5. n = 3 cell culture preparations, F(2–12) = 57.52; p < 0.0001. ***p < 0.001 versus NC Ctrl group; ###p < 0.001 versus NC Cort group. The Ctrl group means untransfected cells. (n) The representative photographs of intracellular ROS in astrocytes. n = 3 cell culture preparations. Scale bar is 50 μm. (o) Quantification of intracellular ROS in astrocytes. n = 3 cell culture preparations, F(1–8) = 120.5 p < 0.0001. ***p < 0.001 versus NC Cort group. Data were presented as mean ± SD and analyzed by two‐way ANOVA followed by Turkey's post hoc tests.
Schematic diagram of PPARβ/δ activation improving corticosterone‐induced astrocytic damage. PPARβ/δ directly interacts with UBR5 and its activation facilitates PEX5‐mediated peroxisomal autophagy by promoting ATM phosphorylation, so relieves intracellular ROS levels and cell death induced by corticosterone in astrocytes.
PPARβ/δ Activation Improves Corticosterone‐Induced Oxidative Stress Damage in Astrocytes by Targeting UBR5/ATM Signaling

Juan Ji

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Ye‐Fan Chen

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Chen Hong

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[...]

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Xiu‐Lan Sun

Oxidative stress‐mediated astrocytic damage contributes to nerve injury and the development of depression, especially under stress conditions. Peroxisomes and pexophagy are essential for balancing oxidative stress and protein degradation products. Our previous findings suggest that peroxisome proliferators‐activated receptor β/δ (PPARβ/δ) activation significantly alleviates depressive behaviors by preventing astrocytic injury. However, the underlying mechanisms remain unclear. In the present study, we established oxidative injury by treating astrocytes with corticosterone. Subsequently, PPARβ/δ agonists and antagonists were applied to determine the effects of PPARβ/δ on balancing peroxisomes and pexophagy in astrocytes. The PPARβ/δ agonist (GW0742) significantly improved cell viability and decreased intracellular reactive oxygen species (ROS) production induced by corticosterone, while pretreatment with the PPARβ/δ, antagonist GSK3787 reversed the effects of GW0742. Moreover, activating PPARβ/δ promoted peroxisomal biogenesis factor 5 (PEX5)‐mediated pexophagy by enhancing the phosphorylation of ataxia‐telangiectasia mutated (ATM) kinase. Conversely, blocking PPARβ/δ with GSK3787 partially abolished the effects of GW0742. Further investigations demonstrated that activation of PPARβ/δ not only induced transcription of the ubiquitin protein ligase E3 component n‐recognin 5 (UBR5) but also enhanced the interaction between PPARβ/δ and UBR5, contributing to ATM interactor (ATMIN) degradation, and increased phosphorylated ATM kinase levels. Therefore, this study revealed that activating PPARβ/δ improves corticosterone‐induced oxidative damage in astrocytes by enhancing pexophagy. PPARβ/δ directly interacts with UBR5 to facilitate ATMIN degradation and promotes ATM phosphorylation, thereby maintaining the balance between peroxisomes and pexophagy. These findings suggest that PPARβ/δ is a potential target for promoting pexophagy in astrocytes upon stress. image


Overview of discovery study design.
Exemplar image annotating brain regions analyzed in MAP (top panel) and UKB (bottom panel). Amygdala and substantia nigra (UKB) are not annotated.
Genetic Markers of Postmortem Brain Iron

February 2025

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10 Reads

Marilyn C. Cornelis

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Amir Fazlollahi

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David A. Bennett

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Scott Ayton

Brain iron (Fe) dyshomeostasis is implicated in neurodegenerative diseases. Genome‐wide association studies (GWAS) have identified plausible loci correlated with peripheral levels of Fe. Systemic organs and the brain share several Fe regulatory proteins but there likely exist different homeostatic pathways. We performed the first GWAS of inductively coupled plasma mass spectrometry measures of postmortem brain Fe from 635 Rush Memory and Aging Project (MAP) participants. Sixteen single nucleotide polymorphisms (SNPs) associated with Fe in at least one of four brain regions were measured (p < 5 × 10⁻⁸). Promising SNPs (p < 5 × 10⁻⁶) were followed up for replication in published GWAS of blood, spleen, and brain imaging Fe traits and mapped to candidate genes for targeted cortical transcriptomic and epigenetic analysis of postmortem Fe in MAP. Results for SNPs previously associated with other Fe traits were also examined. Ninety‐eight SNPs associated with postmortem brain Fe were at least nominally (p < 0.05) associated with one or more related Fe traits. Most novel loci identified had no direct links to Fe regulatory pathways but rather endoplasmic reticulum‐Golgi trafficking (SORL1, SORCS2, MARCH1, CLTC), heparan sulfate (HS3ST4, HS3ST1), and coenzyme A (SLC5A6, PANK3); supported by nearest gene function and omic analyses. We replicated (p < 0.05) several previously published Fe loci mapping to candidate genes in cellular and systemic Fe regulation. Finally, novel loci (BMAL, COQ5, SLC25A11) and replication of prior loci (PINK1, PPIF, LONP1) lend support to the role of circadian rhythms and mitochondria function in Fe regulation more generally. In summary, we provide support for novel loci linked to pathways that may have greater relevance to brain Fe accumulation; some of which are implicated in neurodegeneration. However, replication of a subset of prior loci for blood Fe suggests that genetic determinants or biological pathways underlying Fe accumulation in the brain are not completely distinct from those of Fe circulating in the periphery. image


Maternal alcohol consumption frequency and newborn clinical presentation. Identification of the age range of interviewed pregnant women (A). Prevalence of alcohol consumption during gestation (B). Identification of gestational trimester of alcohol consumption (C). Age range of women reporting alcohol consumption during gestation (C′). Raw values of weight (g), length (cm), APGAR (minute 1/5), and sex of newborns of Control and Ethanol‐exposed group newborns (D). Mean weight (E–G), body length (F) and APGAR score (G) of newborns from Control and Ethanol‐exposed mothers. ni, not informed [(A–C) n = 77; (D–G) control group n = 5, ethanol group n = 5].
UCBS from ethanol‐exposed newborns decreases the levels of BBB‐related endothelial proteins. Phase contrast photomicrographs of HBMEC cultivated in medium containing 10% FBS, S‐Control or S‐Ethanol (A–C). Photomicrographs of cells cultured under the same conditions and stained with the nuclear marker DAPI (D–F). Quantification of DAPI‐positive nuclei showing no statistical differences among FBS, S‐Control and S‐Ethanol conditions [(G) Mann–Whitney test, U value 3908; p = 0.4438]. Immunofluorescence photomicrographs of HBMEC cells treated with S‐Control showing the typical distribution of ZO‐1 (H, I) and GLUT1 (N, O) proteins. Treatment with S‐Ethanol reduces labeling levels of ZO‐1 (J, K) and GLUT‐1 (P, Q) on the cell surface. Patient‐specific integrated density of immunofluorescence of ZO‐1 (L) and GLUT‐1 (R) in cells exposed to S‐Control (five patients) and S‐Ethanol (four patients) . Integrated density analysis provided by grouped S‐Control and S‐Ethanol immunofluorescence of ZO‐1 [(M) Mann–Whitney test, ***p = 0.0008] and GLUT‐1 [(S) Mann–Whitney test, *p = 0.0166] proteins revealed decreases by 68% and 38% labeling intensity levels, respectively. n = 3–4 cultures treated with S‐Control (n = 5) and S‐Ethanol (n = 4).
UCBS from ethanol‐exposed newborns impairs BBB‐related endothelial angiogenic functions. Schematic representation the Evans blue dye permeability assay in HBMEC cultures grown in transwell inserts (A). Analysis of permeability index after 1, 5, and 24 h, demonstrating increased permeability in cells treated with S‐Ethanol after 5 h of dye addition when compared to the control condition [(B) Mann–Whitney test, U value = 0, *p = 0.0159]. Phase contrast photomicrographs showing kinetics of endothelial migration on scratch demonstrating that cells treated with S‐Ethanol showed lower migration index after 5 h, compared to initial (0 h) time of analysis [(C–F, I) t‐test, **p = 0.003; df = 44; t value = 3.139] and, after 24 h, no difference was observed when compared to S‐Control‐treated cells [(G–I) t‐test, p = 0.3601; df = 22; t value = 0.9348]. Angiogenesis array of conditioned medium (CM) from HBMECs treated with control serum (CM‐S‐Control) or ethanol‐exposed serum (CM‐S‐Ethanol) revealed the presence of 15 proteins (J), some of which were specifically modulated by S‐Ethanol treatment (K). Analysis of protein–protein interactions revealed functional correlations among them (L). n = 3 cultures treated with S‐Control (n = 5) and S‐Ethanol (n = 4).
Proteomic analysis of UCBS reveals specific profile enrichment. Venn diagram showing the number of identified proteins in S‐Control and S‐Ethanol samples (A). Heatmap showing differential expression (Log2 fold change) of proteins significantly modulated in S‐Ethanol (n = 3) samples in relation to S‐Control (n = 3) (B). Analysis of protein–protein interaction networks showing differentially regulated proteins (up and down, Log2 fold change) in S‐Ethanol in comparison with S‐Control. Overrepresented proteins in alcohol binding molecular function and immune system pathway are indicated. Red, represent overregulated proteins in S‐Ethanol, and blue downregulated (C). Analysis of gene ontology terms and pathways enriched from proteins found exclusively in S‐Control (D) and in S‐Ethanol (E) samples. Hyper geometric p values < 0.05.
Prenatal Alcohol Consumption Alters Protein Fingerprint in Umbilical Cord Blood Serum and Induces Brain Microvascular Endothelial Cell Dysfunction

Paula Silva Lacerda Almeida

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Dayana Araújo

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Juliana Minardi Nascimento

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Joice Stipursky

Consumption of alcoholic beverages during pregnancy is directly related to the establishment of fetal alcohol spectrum disorders (FASD), which includes craniofacial changes, body growth restriction, and neurodevelopment impairments. Proper functioning of the central nervous system (CNS) depends on blood–brain barrier (BBB) development, which is formed by interactions of vascular endothelial cells, pericytes, astrocytes, and basal lamina. Gestational exposure to ethanol has been demonstrated to impair CNS development; however, little is known about ethanol modulation of blood circulating factors and impacts on human developing BBB. Here we investigated the prevalence of alcohol consumption during pregnancy and found that 27% of pregnant women reported alcohol consumption, mainly in the first trimester. Control and alcohol‐exposed newborns showed no differences in weight, length, and appearance, pulse, grimace, activity, respiration (APGAR) score at birth. In vitro, we cultivated human brain microcapillary endothelial cells (HBMEC) and treated with umbilical cord blood serum (UCBS) from control (S‐Control) newborns or ethanol‐exposed ones (S‐Ethanol). S‐Ethanol treatment induced 68% and 38% decreases in protein levels of ZO‐1 (tight junction) and GLUT‐1 (glucose transporter type‐1), respectively, increased endothelial monolayer permeability, migratory potential impairment, and changes in angiogenesis‐related secreted proteins profile, compared to S‐Control treatments. UCBS proteomics revealed a total of 392 proteins, 10 exclusively found in S‐Ethanol, mostly related to innate and adaptive immunity and tissue injury response. These results suggest that gestational exposure to ethanol contributes to blood altered protein profiles triggering BBB endothelial. image


Deletion analysis of the human SORL1 gene promoter. (A) Schematic illustration of human SORL1 promoter deletion constructs in the pGL3‐basic vector. The arrow indicates the direction of transcription, and the numbers indicate the start and end points of each construct insertion relative to the transcription start site. The size of the pGL3‐basic vector is 4.8 kb, and the insert sizes range from 519 to 3019 bp. A series of deletion constructs were cotransfected with pCMV‐RLuc into SH‐SY5Y cells. Cell lysates were collected 24 h post‐transfection, and luciferase activity was measured using a luminometer. SORL1 promoter luciferase activity was normalized to pCMV‐Luc luciferase activity to assess transfection efficiency and compared to the luciferase activity of the pGL3‐basic vector, expressed as fold change. Statistical analysis: one‐way ANOVA with Sidak's post hoc test, F(4,20) = 38.32, p < 0.001; pGL3‐basic versus pSORL1‐A: p = 0.0290; pGL3‐basic versus pSORL1‐B: p =  0.0003; pGL3‐basic versus pSORL1‐C: p =  0.0004; pGL3‐basic versus pSORL1‐D: p < 0.0001. (B–D) A series of deletion constructs were co‐transfected with pCMV‐Luc into SH‐SY5Y cells and measured using a luminometer. Statistical analysis: One‐way ANOVA with Sidak's post hoc test, (B) F(5,24) = 100.5, p < 0.0001; pGL3‐basic versus pSORL1‐D: p =  0.0001; pGL3‐basic versus pSORL1‐E: p =  0.0006; pGL3‐basic versus pSORL1‐F: p < 0.0001; pGL3‐basic versus pSORL1‐G: p < 0.0001. pGL3‐basic versus pSORL1‐H: p = 0.0019. (C) F(4,20) = 26.71, p < 0.0001; pGL3‐basic versus pSORL1‐I: p < 0.0001; pGL3‐basic versus pSORL1‐J: p = 0.1768; pGL3‐basic versus pSORL1‐K: p < 0.0759; pGL3‐basic versus pSORL1‐L: p < 0.0001. (D) F(4,20) = 153.3, p < 0.0001; pGL3‐basic versus pSORL1‐M: p < 0.3598; pGL3‐basic versus pSORL1‐N: p = 0.1375; pGL3‐basic versus pSORL1‐O: p < 0.0001; pGL3‐basic versus pSORL1‐P: p < 0.1163. Values represent means ± SD, n = 5 independent cell culture preparations per group, *p < 0.05; **p < 0.01; ***p < 0.001.
The binding of SORL1 gene promoter with transcription factor FOXP1. (A) The indicated fragments from SORL1 promoter region with biotin labeling were incubated with the nuclear extracts of SH‐S5Y5 cells, and the reactions were subjected to EMSA assay. (B) The fragment −200~−161 was used to pull‐down binding proteins from SH‐SY5Y nuclear extracts, and the −160~−131 fragment without promoter activity was used as a control. The precipitated proteins were resolved on SDS‐PAGE and stained with Coomassie blue. (C) After pull‐down as in (B), the precipitated proteins were blotted for the indicated proteins identified by mass spectrometry. (D) The fragments −200~−161 and −160~−131 as a negative control were incubated with SH‐SY5Y nuclear extracts. After separation on the native gel for EMSA, proteins were transferred onto nitrocellulose membrane for the western blot of FOXP. (E) The EMSA reaction as before was supplemented with or without unlabeled −200~−161 at the indicated amounts, and the upshifted −200~−161 with biotin labeling was examined by EMSA. 1× and 5×: labeled to unlabeled probe = 1:1 and 1:5, respectively. (F) The EMSA reaction as before was supplemented with anti‐FOXP1 antibody or a nonspecific normal IgG, and the upshifted −200~−161 with biotin labeling were examined by EMSA.
FOXP1 regulates the human SORL1 protein expression. (A, B) SH‐SY5Y cells were transfected with the siRNA targeting FOXP1, and the cell lysates were blotted for SORL1 and FOXP1. SORL1 and FOXP1 were quantified for comparison. Statistical analysis: unpaired t test, FOXP1: T(4) = 4.570, p = 0.0103; SORL1: T(4) = 2.849, p = 0.0464. (C, D) SH‐SY5Y cells were transfected with the siRNA targeting Jun, and the cell lysates were blotted for SORL1 and Jun. SORL1 and Jun were quantified for comparison. Statistical analysis: unpaired t test, JUN: T(4) = 4.737, p = 0.0091; SORL1: T(4) = 0.5940, p = 0.5844. (E, F) SH‐SY5Y cells were transfected with the siRNA targeting TFAP2A, and the cell lysates were blotted for SORL1 and TFAP2A. SORL1 and TFAP2A were quantified for comparison. Statistical analysis: unpaired t test, TFAP2A: T(4) = 8.844, p = 0.0009; SORL1: T(4) = 0.9626, p = 0.3902. (G, H) SH‐SY5Y cells were transfected with the siRNA targeting SMAD4, and the cell lysates were blotted for SORL1 and SMAD4. SORL1 and SMAD4 were quantified for comparison. Statistical analysis: unpaired t test, SMAD4: T(4) = 5.990, p = 0.0039; SORL1: T(4) = 0.2438, p = 0.8194. (I) After FOXP1 suppression in SH‐SY5Y cells, mRNA levels of FOXP1 and SORL1 were determined by quantitative PCR (qPCR). Statistical analysis: unpaired t test, FOXP1: T(4) = 3.011, p = 0.0395; SORL1: T(4) = 4.427, p = 0.0115. The values represent means ± SD, n = 3 independent cell culture preparations per group, *p < 0.05.
Both FOXP1 and SORL1 respond to BafA1. (A) SH‐SY5Y cells were treated overnight with 1 nmol/mL BafA1. The protein expression of FOXP1 and SORL1 was detected using immunoblotting. (B) The protein levels of FOXP1 and SORL1 were compared with the control group. FOXP1: T(4) = 5.150, p = 0.0067; SORL1: T(4) = 2.849, p = 0.0464. (C) The FOXP1 siRNAs were transfected into SH‐SY5Y cells 48 h before the luciferase reporter plasmids transfection. BafA1 was added 24 h after plasmid transfection. The luciferase activity was measured by a luminometer and expressed as fold change in comparison with pGL3‐Basic vector. Statistical analysis: two‐way ANOVA with Tukey's test, BafA1: F(3,32) = 21.00, p < 0.0001; different treatments: F(1,32) = 0.596, p = 0.4455; BafA1 × different treatments: F(3,32) = 2.172, p = 0.1107. BafA1 versus control: p = 0.0369, FOXP1 siRNA versus Ctrl siRNA: p < 0.0001. The values represent means ± SD, n = 3–5 independent cell culture preparations per group, *p < 0.05, ***p < 0.001.
Quantification of FOXP1 and SORL1 in the prefrontal cortex of 5xFAD. (A) Representative images of co‐immunofluorescence staining of FOXP1 (red) with neurons (NeuN, green) and nuclei DAPI (blue), (B) comparison levels of FOXP1 and NeuN in the prefrontal cortex of WT (10 months) and 5xFAD (10 months) mice. Magnification 40×, scale bar = 50 μm. (C) Representative images of co‐immunofluorescence staining of SORL1 (red) with neurons (NeuN, green) and nuclei DAPI (blue), (D) comparison levels of SORL1 and NeuN in the prefrontal cortex of WT (10 months) and 5xFAD (10 months) mice. Statistical analysis: unpaired t test, total FOXP1: t(10) = 2.403, p = 0.0371; total NeuN: t(10) = 2.342, p = 0.0412; NeuN‐positive cells expressing FOXP1: t(10) = 2.294, p = 0.0447. Magnification 40×, scale bar = 50 μm. The values represent means ± SD, n = 6 mice per group. *p < 0.05.
FOXP1 is a Transcription Factor for the Alzheimer's Disease Risk Gene SORL1

Sortilin‐related receptor 1 (SORL1) is a risk gene of Alzheimer's disease (AD), and some protein‐truncating (PTV) and rare missense variants causing the loss of function of SORL1 contribute to AD pathogenesis. SORL1 is an endosomal receptor that interacts with multiple protein sorting complexes to facilitate the transport of various cargoes through the endolysosomal network (ELN). However, the regulatory mechanisms governing SORL1 expression remain unknown. Through biochemical methods, we identified Forkhead Box P1 (FOXP1) as a binding protein to the minimal promoter region of SORL1 gene. Silencing FOXP1 using siRNA significantly decreased the activity of the SORL1 minimal promoter and reduced SORL1 protein and mRNA levels in the neuroblastoma cell line SH‐SY5Y. Additionally, using 5xFAD mouse models of AD, we observed significantly decreased FOXP1 and SORL1 expression in neurons within the prefrontal cortex. Disruption of ELN and the autophagy degradation system by bafilomycin A1 (BafA1) appeared to be a specific condition to suppress FOXP1 and hence SORL1 in SH‐SY5Y cells. These findings highlight the critical role of FOXP1 in regulating SORL1 expression and suggest that FOXP1 could be a potential target to maintain SORL1 expression for AD prevention and therapy. image


Transcript and Lipid Profile Alterations in Astrocyte‐Neuron Mitochondrial Transfer Under Lipopolysaccharide Exposure: An In Vitro Study

February 2025

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4 Reads

Sepsis‐associated encephalopathy (SAE) is a brain dysfunction for which no effective therapy currently exists. Recent studies suggest that transferring mitochondria from astrocytes to neurons may benefit SAE patients, though the underlying mechanism remains unclear. We cultured astrocytes and neurons from mice in vitro. Astrocytes were stimulated with lipopolysaccharide (LPS) for 24 h, and the astrocyte‐conditioned medium (ACM) was collected. Neuronal cultures were then treated with ACM or mitochondria‐depleted ACM (mdACM) for further analysis. Mitochondrial transfer was examined under a fluorescence microscope. Western blotting analyzed the protein expression of genes related to apoptosis and mitochondrial metabolism. RNA sequencing and mass spectrometry were employed to investigate the mechanisms underlying mitochondrial transfer. Astrocyte‐derived mitochondria migrated toward and connected with LPS‐exposed neurons. The addition of ACM significantly attenuated LPS‐induced alterations in the proteins linked to apoptosis and mitochondrial dynamics. RNA sequencing revealed notable alterations in the transcript profile of neurons upon ACM treatment, highlighting the involvement of mitochondria metabolism, inflammation, and apoptosis‐related factors. Additionally, mitochondrial transfer modified the lipid composition of neurons, increasing phosphatidylserine levels, which correlated with neuroinflammation and enriched pathways related to cytokine and MAPK signaling. Our findings suggest that astrocyte‐neuron mitochondrial transfer holds therapeutic potential for alleviating SAE, possibly through the anti‐inflammatory effects of lipids, particularly phosphatidylserine. image


Focused Ultrasound Modulates Dopamine in a Mesolimbic Reward Circuit

February 2025

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5 Reads

Dopamine is a neurotransmitter that plays a significant role in reward and motivation. Dysfunction in the mesolimbic dopamine pathway has been linked to a variety of psychiatric disorders, including addiction. Low‐intensity focused ultrasound (LIFU) has demonstrated effects on brain activity, but how LIFU affects dopamine neurotransmission is not known. Here, we applied three different intensities (6.5, 13, and 26 W/cm² ISPPA) of 2‐min LIFU to the prelimbic cortex (PLC) and measured dopamine in the nucleus accumbens (NAc) core using fast‐scan cyclic voltammetry. Two minutes of LIFU sonication at 13 W/cm² to the PLC significantly reduced dopamine release by ~50% for up to 2 h. However, double the intensity (26 W/cm²) resulted in less inhibition (~30%), and half the intensity (6.5 W/cm²) did not result in any inhibition of dopamine. Anatomical controls applying LIFU to the primary somatosensory cortex did not change NAc core dopamine, and applying LIFU to the PLC did not affect dopamine release in the caudate or NAc shell. Histological evaluations showed no evidence of cell damage or death. Modeling temperature rise demonstrates a maximum temperature change of 0.5°C with 13 W/cm², suggesting that modulation is not due to thermal mechanisms. These studies show that LIFU at a moderate intensity provides a noninvasive, high spatial resolution means to modulate specific mesolimbic circuits that could be used in future studies to target and repair pathways that are dysfunctional in addiction and other psychiatric diseases. image


Systematic flowchart and publication year of articles included in this meta‐analysis review. (a) PRISMA flow diagram illustrates the process of article selection, detailing the steps from identification and screening to inclusion in the review. (b) Histogram distribution of included articles by publication year, highlighting trends in research focus over time.
An overview of 6‐OHDA‐based methodologies for modeling Parkinson's disease: Considerations for subject selection and neurotoxin infusion parameters. (a) Total number of articles reviewed (purple), number of 6‐OHDA approaches identified (gray), distribution of rodent choice (blue) and the breakdown by sex (last bar). (b) Proportion of studies using rat (r) and mouse (m) as well as preferred strain. (c) Distribution of selected 6‐OHDA injection sites. (d) Number of 6‐OHDA injections per hemisphere in unilateral and bilateral approaches. (e) Schematic representation of 6‐OHDA injection sites based on reported stereotaxic coordinates. Scale bar, distance between bregma and lambda reference points, as defined by Franklin and Paxinos (2019) and Paxinos and Watson (2007). (f) Profile of injection site variability for rats. Heatmap depicts 6‐OHDA concentration (μg/μL), and injection site stereotaxic position (top panel; mediolateral, ML. bottom panel; dorsoventral, DV) by anteroposterior (AP) axis. (g) Average amount of 6‐OHDA mass and volume infused per brain region. Data are presented as mean ± SEM.
Most common motor tests used in 6‐OHDA lesion articles. (a) Tests used for motor phenotype analysis. (b) Self‐report of motor phenotype as a confounder to non‐motor symptoms evaluation.
Diversity in 6‐OHDA animal models of Parkinson's Disease: Limited replication and prevalence of cognitive and affective symptom evaluation. (a) Grouping of experimental approaches based on parameters used to generate the lesion. Each model is characterized by specific choices of species/strain, 6‐OHDA injection coordinates, number of injections, and 6‐OHDA concentration. A total of 125 unique models were identified, with only 12 having been replicated. (b) Distribution of model usage frequency across studies. Each circle represents a unique model, with the color coding indicating the number of replications. (c) Number of models tested (count, n) for each class of non‐motor symptom, including both positive and negative findings. (d) Venn diagram depicting the overlap between the two most commonly evaluated classes of non‐motor symptoms. Models presenting cognitive deficits (red circles), affective deficits (green), or both (brown) are shown inside the diagram. Models in which these deficits were absent or not assessed are displayed outside the diagram (gray). (e) Venn diagram showing the subcategories of cognitive and affective symptoms, with areas proportional to the number of models presenting each phenotype. (f) Bar graphs displaying the distribution of cognitive and affective symptoms evaluated per infused nigrostriatal region (MFB, SN, STR) and lesion laterality (bilateral vs. unilateral). (g) Heatmap illustrating the conditional probabilities for all pairs of behavioral features reported across models.
Low similarity of references and lack of cross‐citation in studies using 6‐OHDA models of Parkinson's Disease. (a) Shared references between article pairs. The index (x‐axis) measures how similar two articles are based on the references they share relative to the total set of references they cite. The similarity score of each article pair is a value between 0 and 1, where 0 indicates no similarity (no shared references) and 1 indicates complete similarity (all references are shared). The y‐axis represents the percentage of article pairs with shared bibliographies. (b) Number of cross‐citations between reviewed articles (at least one cross‐citation required). Node color indicates if an article cites (dark blue) or is cited (light blue). Only three articles cite and are cited by other articles in the review list. Right panel, citation node graph. Arrows help depict citation direction, and article ID was used to identify each node. Article ID and respective DOI‐reference can be found in Table S1.
Challenges and Opportunities in Exploring Non‐Motor Symptoms in 6‐Hydroxydopamine Models of Parkinson's Disease: A Systematic Review

Parkinson's disease (PD) is a neurodegenerative disorder characterized by the progressive loss of midbrain dopaminergic neurons, leading to motor symptoms such as tremors, rigidity, and bradykinesia. Non‐motor symptoms, including depression, hyposmia, and sleep disturbances, often emerge in the early stages of PD, but their mechanisms remain poorly understood. The 6‐hydroxydopamine (6‐OHDA) rodent model is a well‐established tool for preclinical research, replicating key motor and non‐motor symptoms of PD. In this review, we systematically analyzed 135 studies that used 6‐OHDA rodent models of PD to investigate non‐motor symptoms. The review process adhered to the Preferred Reporting Items for Systematic Reviews and Meta‐Analyses (PRISMA) guidelines. Our analysis highlights the growing use of 6‐OHDA PD models for experimental research of non‐motor symptoms. It also reveals significant variability in methodologies, including choices of brain target, toxin dosage, lesion verification strategies, and behavioral assessment reporting. Factors that hinder reproducibility and comparability of findings across studies. We highlight the need for standardization in 6‐OHDA‐based models with particular emphasis on consistent evaluation of lesion extent and reporting of the co‐occurrence of non‐motor symptoms. By fostering methodological coherence, this framework aims to enhance the reproducibility, reliability, and translational value of 6‐OHDA models in PD non‐motor symptom research. image


Modular elements of human, bovine, and murine MFG‐E8. Each form has an N‐terminal signal peptide (S) that guides its secretion. An EGF‐like domain (EGF2) contains an arginine–glycine–aspartic acid (RGD) motif, and two F5/8 type C domains (C1 and C2). The C2 domain is mainly responsible for phospholipid binding. Murine and bovine MFG‐E8 contain two EGF‐like domains (EGF1 and EGF2), although only EGF2 possesses the RGD domain required for integrin binding. Both a long and a short form of MFG‐E8 are expressed in mice; the long form contains an additional proline/threonine‐rich domain (P/T).
In the immune system, MFG‐E8 acts as a bridging ligand between exposed phosphatidylserine (PS) on apoptotic cell membranes and integrin αVβ3/5 on the surface of phagocytes. MFG‐E8 recognizes PS via its C2 domain which possesses three membrane‐interactive loops or spikes. It can also act as ligand for the membrane receptor integrin αVβ3/5, which recognizes its arginine–glycine–aspartic acid (RGD) motif. Upon MFG‐E8 binding, integrin αVβ3/5 triggers a signaling cascade which leads to Rac1 activation and subsequent engulfment of the apoptotic cell by the phagocyte.
Activation of integrin αVβ5 and MERTK via their ligands MFG‐E8 and Gas6/ProS, respectively, leads to downstream Rac1 activation and subsequent preparation for phagocytic activity via redundant cascades. Binding of MFG‐E8 to integrin αVβ5 induces autophosphorylation of FAK at Tyr397, which can direct it to MERTK for further phosphorylation at Tyr861. Alternatively, binding of Gas6/ProS to MERTK facilitates Src‐dependent phosphorylation of FAK at Tyr861, which translocates to integrin αVβ5 for further phosphorylation at Tyr397 to provide a binding site for Src. The activated FAK‐Src complex is required to phosphorylate p130Cas, which in turn recruits the CrkII/DOCK180/ELMO module for activation of Rac1. Some evidence suggests that ligation of integrin αVβ5 with MFG‐E8 may also activate Rac1 in a FAK‐independent manner, potentially via activation of RhoG and its GEF TRIO.
A summary of established and putative roles for MFG‐E8 in synapse elimination. MFG‐E8's opsonization capabilities have been shown to have both a positive and negative effect on synapse elimination through opsonization of synapses and protein aggregates. MFG‐E8 may also have direct and indirect effects on synapse elimination via opsonization‐independent signaling related to the regulation of the cytoskeleton and inflammation. Last, MFG‐E8 can participate in and increase formation of toxic protein aggregates via cleavage into medin.
Emerging Roles for MFG‐E8 in Synapse Elimination

Synapse elimination is an essential process in the healthy nervous system and is dysregulated in many neuropathologies. Yet, the underlying molecular mechanisms and under what conditions they occur remain unclear. MFG‐E8 is a secreted glycoprotein well known to act as an opsonin, tagging stressed and dying cells for engulfment by phagocytes. Opsonization of cells and debris by MFG‐E8 for microglial phagocytosis in the CNS is well established, and its role in astrocytic phagocytosis, and trogocytosis‐like engulfment of synapses is beginning to be explored. However, MFG‐E8's function in other tissues is highly diverse, and evidence suggests that its role in the nervous system and on synapse elimination in particular may be more complex and varied than opsonization. In this review, we outline the documented direct and indirect effects of MFG‐E8 on synapse elimination, while also proposing potential roles to be explored further, in particular, cytoskeletal reorganization of neurites and glia leading to synapse elimination by various mechanisms. Finally, we demonstrate the need for several open questions to be answered—chiefly, under what conditions might MFG‐E8‐mediated synapse elimination occur in favor of other mechanisms, and when might its activity be dysregulated, increasing unwanted synapse elimination and neurotoxicity? image


Schematic of PrPC structure. Key structural features of PrPC are highlighted, including the charged cluster, hexarepeats and tandem octarepeats of the N‐terminal domain, the hydrophobic linker and the C‐terminal domain with its three α‐helices and two β‐strands. A disulphide bond (SS) stabilizes the fold of the globular C‐terminal domain. The signal peptides (SIG) are not present in the mature protein that is expressed on the cell surface.
α‐Cleavage sites and putative α‐PrPases. Alignment of PrPC sequences from various mammalian species (using NCBI blastp) showing that the region containing the α‐cleavage sites is well‐conserved at the amino acid level, with dots used to denote sequence identity. Arrows indicate the cleavage sites identified by N‐terminal radiosequencing of fragments purified from post‐mortem human brain tissue (Chen et al. 1995) or by mass spectrometry after immunoprecipitation from mouse brain (Gomez‐Cardona et al. 2023). Potentially responsible α‐PrPases are indicated based on data from in vitro cleavage assays (McDonald et al. 2014; Praus et al. 2003).
β‐Cleavage sites, mechanisms and potential C2Sc‐generating pathways. (A) Alignment of PrPC sequences from various mammalian species (using NCBI blastp) covering the OR domain and flanking sequences. ORs 2–4 are almost identical among all the species, with a little more sequence variability found on either side. (B) Visualization of cleavage sites in the indicated regions (cleavages within the imperfect OR1 have not been reported). Sequence variation versus the human sequence is shown (for the species listed in panel A). Solid red arrows correspond to cleavages detected in prion‐infected brain tissue (Hope et al. 1988), solid blue arrows to cleavages in normal brain tissue (Gomez‐Cardona et al. 2023) and dashed blue arrows to cleavages in uninfected cells or in vitro assays (Castle et al. 2023; Mangé et al. 2004; McDonald et al. 2014). Potentially responsible β‐PrPases are indicated based on data from in vitro cleavage assays (Castle et al. 2023; McDonald et al. 2014; Praus et al. 2003). DPP4‐mediated cleavage at G/SQG↓GGT/S is the only proteolytic event conclusively detected both in brain tissue and in vitro (hence the solid outline for the protease). (C) The alternative pathways potentially leading to C2Sc generation: (1) attempted lysosomal degradation of FL PrPSc that progressively trims the protease‐sensitive N‐terminus, leaving the protease‐resistant core intact and (2) conversion of C2 generated by β‐endoproteolysis of normally folded PrPC, possibly also followed by N‐terminal trimming of the longer fragments in the lysosome.
Alignment of PrPC N‐terminal sequences from various mammalian species. The alignment (obtained using NCBI blastp) demonstrates the complete sequence identity of the PrPC N‐terminal region between humans and rodents, with only minimal changes found in other mammalian species. The position of DPP4‐mediated cleavage demonstrated in vitro is indicated.
Prion Protein Endoproteolysis: Cleavage Sites, Mechanisms and Connections to Prion Disease

Highly abundant in neurons, the cellular prion protein (PrPC) is an obligatory precursor to the disease‐associated misfolded isoform denoted PrPSc that accumulates in the rare neurodegenerative disorders referred to either as transmissible spongiform encephalopathies (TSEs) or as prion diseases. The ability of PrPC to serve as a substrate for this template‐mediated conversion process depends on several criteria but importantly includes the presence or absence of certain endoproteolytic events performed at the cell surface or in acidic endolysosomal compartments. The major endoproteolytic events affecting PrPC are referred to as α‐ and β‐cleavages, and in this review we outline the sites within PrPC at which the cleavages occur, the mechanisms potentially responsible and their relevance to pathology. Although the association of α‐cleavage with neuroprotection is well‐supported, we identify open questions regarding the importance of β‐cleavage in TSEs and suggest experimental approaches that could provide clarification. We also combine findings from in vitro cleavage assays and mass spectrometry‐based studies of prion protein fragments in the brain to present an updated view in which α‐ and β‐cleavages may represent two distinct clusters of proteolytic events that occur at multiple neighbouring sites rather than at single positions. Furthermore, we highlight the candidate proteolytic mechanisms best supported by the literature; currently, despite several proteases identified as capable of processing PrPC in vitro, in cell‐based models and in some cases, in vivo, none have been shown conclusively to cleave PrPC in the brain. Addressing this knowledge gap will be crucial for developing therapeutic interventions to drive PrPC endoproteolysis in a neuroprotective direction. Finally, we end this review by briefly addressing other cleavage events, specifically ectodomain shedding, γ‐cleavage, the generation of atypical pathological fragments in the familial prion disorder Gerstmann–Sträussler–Scheinker syndrome and the possibility of an additional form of endoproteolysis close to the PrPC N‐terminus. image


TRIM9 Controls Growth Cone Responses to Netrin Through DCC and UNC5C

The guidance cue netrin‐1 promotes both growth cone attraction and growth cone repulsion. How netrin‐1 elicits diverse axonal responses, beyond engaging the netrin receptor DCC and UNC5 family members, remains elusive. Here, we demonstrate that murine netrin‐1 induces biphasic axonal responses in cortical neurons: Attraction at lower concentrations and repulsion at higher concentrations using both a microfluidic‐based netrin‐1 gradient and bath application of netrin‐1. We find that repulsive turning in a netrin gradient is blocked by knockdown of UNC5C, whereas attractive turning is impaired by knockdown of DCC. TRIM9 is a brain‐enriched E3 ubiquitin ligase previously shown to bind and cluster the attractive receptor DCC at the plasma membrane and regulate netrin‐dependent attractive responses. However, whether TRIM9 also regulated repulsive responses to netrin‐1 remained to be seen. In this study, we show that TRIM9 localizes and interacts with both the attractive netrin receptor DCC and the repulsive netrin receptor, UNC5C. We find that deletion of murine Trim9 alters both attractive and repulsive axon turning and changes in growth cones size in response to murine netrin‐1. TRIM9 was required for netrin‐1‐dependent changes in the surface levels of DCC and UNC5C in the growth cone during morphogenesis. We demonstrate that DCC at the membrane regulates the growth cone area and show that TRIM9 negatively regulates FAK activity in the absence of both repulsive and attractive concentrations of netrin‐1. Together, our work demonstrates that TRIM9 interacts with and regulates both DCC and UNC5C during attractive and repulsive axonal responses to netrin‐1. image


GABAB Receptor Modulation of Membrane Excitability in Human Pluripotent Stem Cell‐Derived Sensory Neurons by Baclofen and α‐Conotoxin Vc1.1

January 2025

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8 Reads

GABAB receptor (GABABR) activation is known to alleviate pain by reducing neuronal excitability, primarily through inhibition of high voltage‐activated (HVA) calcium (CaV2.2) channels and potentiating G protein–coupled inwardly rectifying potassium (GIRK) channels. Although the analgesic properties of small molecules and peptides have been primarily tested on isolated murine dorsal root ganglion (DRG) neurons, emerging strategies to develop, study, and characterise human pluripotent stem cell (hPSC)‐derived sensory neurons present a promising alternative. In this study, hPSCs were efficiently differentiated into peripheral DRG‐induced sensory neurons (iSNs) using a combined chemical and transcription factor‐driven approach via a neural crest cell intermediate. Molecular characterisation and transcriptomic analysis confirmed the expression of key DRG markers such as BRN3A, ISLET1, and PRPH, in addition to GABABR and ion channels including CaV2.2 and GIRK1 in iSNs. Functional characterisation of GABABR was conducted using whole‐cell patch clamp electrophysiology, assessing neuronal excitability under current‐clamp conditions in the absence and presence of GABABR agonists baclofen and α‐conotoxin Vc1.1. Both baclofen (100 μM) and Vc1.1 (1 μM) significantly reduced membrane excitability by hyperpolarising the resting membrane potential and increasing the rheobase for action potential firing. In voltage‐clamp mode, baclofen and Vc1.1 inhibited HVA Ca²⁺ channel currents, which were attenuated by the selective GABABR antagonist CGP 55845. However, modulation of GIRK channels by GABABRs was not observed in the presence of baclofen or Vc1.1, suggesting that functional GIRK1/2 channels were not coupled to GABABRs in hPSC‐derived iSNs. This study is the first to report GABABR modulation of membrane excitability in iSNs by baclofen and Vc1.1, highlighting their potential as a future model for studying analgesic compounds. image


Ionic Mechanisms Involved in β3‐Adrenoceptor‐Mediated Augmentation of GABAergic Transmission Onto Pyramidal Neurons in Prefrontal Cortex

Activation of the brain‐penetrant beta3‐adrenergic receptor (Adrb3) is implicated in the treatment of depressive disorders. Enhancing GABAergic inputs from interneurons onto pyramidal cells of prefrontal cortex (PFC) represents a strategy for antidepressant therapies. Here, we probed the effects of the activation of Adrb3 on GABAergic transmission onto pyramidal neurons in the PFC using in vitro electrophysiology. We found that Adrb3 agonist SR58611A increased both the frequency and the amplitude of miniature IPSCs (mIPSCs). Ca²⁺ influx through T‐type voltage‐gated Ca²⁺ channel (T‐type VGCC) contributed to SR58611A‐enhanced mIPSC frequency. We also found that SR58611A facilitated GABA release probability and the number of releasable vesicles through interaction with T‐type VGCC. SR58611A depolarized somatostatin (Sst) interneurons with no effects on the firing rate of action potential of Sst interneurons. SR58611A‐induced depolarization of Sst interneurons and enhancement of mIPSC frequency required inward rectifier K⁺ channel (Kir). Our results suggest that Kir and T‐type VGCC in Sst interneurons participate in SR58611A‐induced increase in GABA release in PFC. image


Interactions of Oligodendrocyte Precursor Cells and Dopaminergic Neurons in the Mouse Substantia Nigra

January 2025

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19 Reads

Parkinson's disease (PD) is a prevalent neurodegenerative disease caused by the death of dopaminergic neurons within the substantia nigra pars compacta (SNpc) region of the midbrain. Recent genomic and single cell sequencing data identified oligodendrocytes and oligodendrocyte precursor cells (OPCs) to confer genetic risk in PD, but their biological role is unknown. Although SNpc dopaminergic neurons are scarcely or thinly myelinated, there is a gap in the knowledge concerning the physiological interactions between dopaminergic neurons and oligodendroglia. We sought to investigate the distribution of OPCs with regard to the myelination state in the mouse substantia nigra (SN) by high‐resolution imaging to provide a morphological assessment of OPC‐dopaminergic neuron interactions and quantification of cell numbers across different age groups. OPCs are evenly distributed in the midbrain throughout the lifespan and they physically interact with both the soma and axons of dopaminergic neurons. The presence of OPCs and their interaction with dopaminergic neurons does not correlate with the distribution of myelin. Myelination is sparse in the SNpc, including dopaminergic fibers originating from the SNpc and projecting through the substantia nigra pars reticulata (SNpr). We report that OPCs and dopaminergic neurons exist in a 1:1 ratio in the SNpc, with OPCs accounting for 15%–16% of all cells in the region across all age groups. This description of OPC‐dopaminergic neuron interaction in the midbrain provides a first look at their longitudinal distribution in mice, suggesting additional functions of OPCs beyond their differentiation into myelinating oligodendrocytes. image


Key amino acid residues of physiological relevance within synaptic vesicle glycoprotein protein 2A (SV2A). Highlighted sites involved in the phosphorylation‐dependent binding of SV2A to Syt1 are displayed, accompanied with mutation (T84A) that perturbs the interaction (purple). Y46 is a residue critical for SV2A retrieval during SV endocytosis, with a mutant version (Y46A) incompetent for retrieval (yellow). Important glycosylated residues of SV2A are also highlighted (N498, N548 and N573: green) thought to be required for botulinum neurotoxin binding. Tryptophan residues proposed to be important for maintenance of neurotransmission (W300 and W666) are indicated in blue. Pathogenic mutations observed in humans with intractable epilepsy (R289X, R383Q, R570C and G660R) are highlighted in red. Finally, a spontaneous mutation that results in epilepsy in a rodent model is indicated (L174Q, orange).
Proposed mechanism of synaptic vesicle glycoprotein protein 2A (SV2A)‐dependent Syt1 retrieval during synaptic vesicle (SV) endocytosis. (a) After SV fusion and neurotransmitter release, all SV cargoes are present on the presynaptic plasma membrane, including the cis‐SNARE complex with Syt1 attached. (b) The cis‐SNARE complex is then broken apart freeing Syt1. Simultaneous to this, the N‐terminus of SV2A is phosphorylated by casein kinase I family members, facilitating an interaction with the C2B domain of Syt1 (Initial SV2A‐Syt1 retrieval complex). (c) Stonin‐2 interacts with the C2 domains of Syt1 to create the final SV2A‐Syt1 retrieval complex. The complex is then clustered for retrieval via the μ‐homology domain of either SGIP1α (c(i)) which shares interactions with Syt1 and possibly SV2A, or AP2 (c(ii)), which has interactions with all three molecules within the final retrieval complex. (d) On formation of the nascent SV, SV2A is dephosphorylated, freeing Syt1 to perform its essential role in triggering synchronous neurotransmitter release. Disruption of this retrieval process may result in disrupted neurotransmitter release, short‐term plasticity and potentially culminates in epilepsy.
Key amino acid residues implicated in racetam binding to synaptic vesicle glycoprotein protein 2A (SV2A). Residues demonstrated to be involved in racetam binding via either in silico modelling, binding assays or a combination of both that are conserved between rat and human models are highlighted. Contributing studies for this figure were: (Shi et al. 2011; Correa‐Basurto et al. 2015; Lee et al. 2015; Mittal et al. 2024; Yamagata et al. 2024; Wood et al. 2018).
Control of Synaptotagmin‐1 Trafficking by SV2A—Mechanism and Consequences for Presynaptic Function and Dysfunction

Synaptic vesicle protein 2A (SV2A) is an abundant synaptic vesicle cargo with an as yet unconfirmed role in presynaptic function. It is also heavily implicated in epilepsy, firstly being the target of the leading anti‐seizure medication levetiracetam and secondly with loss of function mutations culminating in human disease. A range of potential presynaptic functions have been proposed for SV2A; however its interaction with the calcium sensor for synchronous neurotransmitter release, synaptotagmin‐1 (Syt1), has received particular attention over the past decade. In this review we will assess the evidence that the primary role of SV2A is to control the expression and localisation of Syt1 at the presynapse. This will integrate biochemical, cell biological and physiological studies where the interaction, trafficking and functional output of Syt1 is altered by SV2A. The potential for SV2A‐dependent epilepsy to be a result of dysfunctional Syt1 expression and localisation is also discussed. Finally, a series of key open questions will be posed that require resolution before a definitive role for SV2A in Syt1 function in health and disease can be confirmed. image


(A) Schematic figure of the experimental design related to cuprizone model characterization. (B) Representative Luxol fast blue–crystal violet staining in the corpus callosum of the control and cuprizone‐treated groups. A notable reduction in myelin content was evident in the fifth week among cuprizone‐treated animals (demyelination) compared to the control group; a noticeable recovery was observed after the subsequent recovery phase (remyelination). (C) Representative immunofluorescence in the corpus callosum of the control and cuprizone‐treated groups during the fifth week of cuprizone treatment (demyelination) and the 10th week of the experiment, corresponding to the recovery phase (remyelination). (D) mRNA expression levels of Mbp, Plp, and Cnp were assessed in the corpus callosum, prefrontal cortex, and hippocampus of animals exposed to cuprizone for 5 weeks (demyelination), followed by a 5‐week recovery phase (remyelination). The mRNA expression levels of each gene were normalized with the endogenous controls Actb and 18S. Data are represented as mean ± SD, n = 3–6 animals per group. Statistical significance was determined by one‐way ANOVA followed by Tukey's post test, *p < 0.05, **p < 0.005, and ***p < 0.0005 compared to the control group. (E) Effects of cuprizone exposure on locomotion of mice as evaluated by the open‐field test (OFT) and on short‐ and long‐term memory as evaluated by the novel object recognition (NOR) behavioral test. In the OFT, there was no statistically significant difference between the groups after 5 weeks of cuprizone exposure in relation to: (a) distance moved; (b) periphery time; (c) time spent in the center; (d) mobility; and (e) immobile state below 0.5%. However, the demyelination group had a lower velocity parameter than the control group (f; p = 0.0085). Cognitive impairment was observed in animals treated with cuprizone (demyelination) compared to the vehicle group (control) after the demyelination process (h, j). The discrimination index was calculated by the time spent exploring the novel object/total exploring time. No differences were found in the overall interaction time. Data represent the mean ± SEM (n = 24–26). Statistical significance was determined by Student's t‐test, ****p < 0.0001 compared to the control group. Shapiro–Wilk and Kolmogorov–Smirnov normality tests were applied to ensure that the data met the criteria for performing parametric tests. Once the criteria were met, the results were expressed as a mean ± 95% confidence interval (CI).
(A) Schematic representation of the experimental design for cuprizone and VPC‐80051 treatment. (B) Effects of cuprizone and VPC‐80051 exposure on mice locomotion (open‐field test) and memory (novel object recognition test). After 10 weeks (demyelination period + remyelination phase), no significant difference was seen due to VPC‐80051 exposure for (a) distance moved or (c) time spent in the center. Two‐way ANOVA identified impacts on (b) time spent in the periphery (p = 0.0397; F(1,30) = 4.623), (d) mobility (p = 0.0182; F(1,30) = 6.244), and (e) immobility below 0.5% (p = 0.0162; F(1,30) = 6.491). Two‐way ANOVA also indicated that cuprizone affected (f) velocity (p = 0.0383; F(1,30) = 4.694); (d) mobility (p = 0.0046; F(1,30) = 9.383); and (e) immobility below 0.5% (p = 0.0076; F(1,30) = 8.202). After applying Tukey's multiple comparison test, differences between VPC and CUP + VPC were observed for (f) velocity (p = 0.0114) and (e) immobility below 5% (p = 0.0143). Regarding the novel object recognition test, no significant differences were observed between the groups after the 5‐week remyelination period, regardless of the presence or absence of the inhibitor VPC‐80051 (h, j). Data represent the mean ± SEM (n = 6–8). Shapiro–Wilk and Kolmogorov–Smirnov normality tests were applied to ensure that the data met the criteria for performing parametric tests. Once the criteria were accepted, the results were expressed as a mean ± 95% CI.
Upset plot (A) illustrating the proteins identified as differentially expressed in the corpus callosum (CC) and prefrontal cortex (PFC) of animals exposed to cuprizone for 5 weeks followed by a remyelination period (CUP), compared to those not receiving cuprizone (CTL). The lower bar plot shows the number of entities associated with each group, while the upper bar plot displays the intersection size for each comparison. This is visually represented by colored and linked circles within the frame. (B) The cellular compartment analysis demonstrates the enrichment of various cellular compartments by differentially expressed proteins from CC and PFC. Circle size represents the number of proteins found in a specific cellular compartment, whereas colors indicate the enrichment‐adjusted p value. (C) The Circos plot visually represents differentially expressed myelin sheath proteins in the PFC and CC. Edge colors depict the log2 fold‐change, comparing animals exposed to cuprizone followed by remyelination (CUP) against those not receiving cuprizone (CTL).
Upset plot (A) showing the differentially expressed proteins in each brain region in response to VPC exposure (CUP + VPC) during the remyelination period compared to controls (CUP). The lower bar plot shows the number of entities associated with each group, whereas the upper bar plot displays the intersection size for each comparison. This is visually represented by colored and linked circles within the frame. (B) The cellular compartment analysis demonstrates the enrichment of differentially expressed proteins in the CC, PFC, and HPC for various cellular compartments. Bubble size represents the number of proteins found in a specific cellular compartment, whereas color indicates the enrichment‐adjusted p value. (C) The Circos plot shows differentially expressed myelin sheath proteins in the PFC, CC, and HPC. Edge colors depict the log2 fold‐change, comparing animals exposed to CUP followed by a period of remyelination against those treated with VPC‐80051 during the remyelination period. (D) The abundance of PLP protein was found to be differentially expressed across the proteomic data obtained from the PFC, CC, and HPC regions. (E) The mRNA expression of Plp in the PFC, CC, and HPC is illustrated. mRNA expression levels for each gene were normalized with the endogenous controls Actb and 18S. Data are represented as mean ± SD, n = 3–4 animals per group. Statistics: Two‐way ANOVA followed by Tukey's post test.
The Top 20 dysregulated KEGG pathways identified through the analysis of differentially expressed proteins in the PFC, CC, and HPC between cuprizone‐exposed animals treated with the inhibitor VPC‐80051 during the remyelination period (CUP + VPC) and those that received the vehicle during the remyelination period (CUP) (A). The Circos plot showcases the differentially expressed proteins in the PFC, CC, and HPC that are related to the sphingolipid signaling pathway, retrograde endocannabinoid signaling pathway (B), and synaptic vesicle, GABAergic, and dopaminergic synapses (C). The Circos plot edge color illustrates the log2 fold‐change, comparing animals exposed to cuprizone followed by remyelination against cuprizone‐exposed animals treated with VPC‐80051 during the remyelination period. N = 3 animals per group.
Impacts of hnRNP A1 Splicing Inhibition on the Brain Remyelination Proteome

January 2025

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31 Reads

Oligodendrocytes, the myelinating cells in the central nervous system, are implicated in several neurological disorders marked by dysfunctional RNA–binding proteins (RBPs). The present study aimed at investigating the role of hnRNP A1 in the proteome of the corpus callosum, prefrontal cortex, and hippocampus of a murine cuprizone–induced demyelination model. Right after the cuprizone insult, we administered an hnRNP A1 splicing activity inhibitor and analyzed its impact on brain remyelination by nanoESI‐LC‐MS/MS label‐free proteomic analysis to assess the biological processes affected in these brain regions. Significant alterations in essential myelination proteins highlighted the involvement of hnRNP A1 in maintaining myelin integrity. Pathways related to sphingolipid and endocannabinoid signaling were affected, as well as the synaptic vesicle cycle and GABAergic synapses. Although behavioral impairments were not observed, molecular changes suggest potential links to memory, synaptic function, and neurotransmission processes. These findings enhance our understanding of the multifaceted roles of hnRNP A1 in the central nervous system, providing valuable insights for future investigations and therapeutic interventions in neurodegenerative and demyelinating diseases. image


Evidence of senescent cells in the demyelinated CNS. An overview of how chronic demyelination and senescence affect various CNS cells, contributing to the disease pathology of MS. Senescent markers, such as SA‐β‐gal, p16, p21, and SASP, have been found in various cell types, including astrocytes, microglia, oligodendrocytes, endothelial cells of the blood brain barrier, and progenitor cells. Markers are denoted by if they have been observed in rodent models of MS (*), post‐mortem MS tissue (†), or both (bolded text). The expected consequences of senescence on each cell type are listed. NPC = neural progenitor cells. OPC = oligodendrocyte progenitor cells. SASP = senescence associated secretory phenotype. Created in Biorender. Maupin, E (2024). https://BioRender.com/f10l606.
Cellular Senescence in Glial Cells: Implications for Multiple Sclerosis

Aging is the most common risk factor for Multiple Sclerosis (MS) disease progression. Cellular senescence, the irreversible state of cell cycle arrest, is the main driver of aging and has been found to accumulate prematurely in neurodegenerative diseases, including Alzheimer's and Parkinson's disease. Cellular senescence in the central nervous system of MS patients has recently gained attention, with several studies providing evidence that demyelination induces cellular senescence, with common hallmarks of p16INK4A and p21 expression, oxidative stress, and senescence‐associated secreted factors. Here we discuss the current evidence of cellular senescence in animal models of MS and different glial populations in the central nervous system, highlighting the major gaps in the field that still remain. As premature senescence in MS may exacerbate demyelination and inflammation, resulting in inhibition of myelin repair, it is critical to increase understanding of cellular senescence in vivo, the functional effects of senescence on glial cells, and the impact of removing senescent cells on remyelination and MS. This emerging field holds promise for opening new avenues of treatment for MS patients. image


Diet‐Induced Obesity in the Rat Impairs Sphingolipid Metabolism in the Brain and This Metabolic Dysfunction Is Transmitted to the Offspring via Both the Maternal and the Paternal Lineage

Obesity leads to a number of health problems, including learning and memory deficits that can be passed on to the offspring via a developmental programming process. However, the mechanisms involved in the deleterious effects of obesity on cognition remain largely unknown. This study aimed to assess the impact of obesity on the production of sphingolipids (ceramides and sphingomyelins) in the brain and its relationship with the learning deficits displayed by obese individuals. We also sought to determine whether the effects of obesity on brain sphingolipid synthesis could be passed on to the offspring. Learning abilities and brain concentration of sphingolipids in male and female control and obese founder rats (F0) and their offspring (F1) were evaluated, respectively, by the novel object recognition test and by ultra‐performance liquid chromatography tandem mass spectrometry. In addition, a global lipidome profiling of the cerebral cortex and hippocampus was performed. Both male and female F0 rats showed impaired learning and increased concentrations of ceramides and sphingomyelins in the hippocampus and frontal cortex compared to their control counterparts. However, the overall lipidome profile of these brain regions did not change with obesity. Remarkably, the alterations in brain sphingolipid synthesis, as well as the cognitive impairment induced by obesity, were also present in adult F1 male rats born to obese mothers or sired by obese fathers and were associated with enhanced expression of mRNAs coding for enzymes involved in the de novo synthesis of ceramides. These results show that the cognitive deficits and impaired sphingolipid metabolism induced by obesity can be transmitted to the offspring through both the maternal and paternal lineages and suggest that an increase in the brain concentration of sphingolipids could play a causal role in the cognitive deficits associated with obesity. image


Nanostructural Modulation of G‐Quadruplex DNA in Neurodegeneration: Orotate Interaction Revealed Through Experimental and Computational Approaches

January 2025

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31 Reads

The natural compound orotic acid and its anionic form, orotate, play a pivotal role in various biological processes, serving as essential intermediates in pyrimidine de novo synthesis, with demonstrated connections to dietary, supplement, and neurodrug applications. A novel perspective on biomolecular aggregation at the nanoscale, particularly pertinent to neurodegeneration, challenges the established paradigm positing that peptide (amyloid beta) and protein (tau) aggregation mainly govern the molecular events underlying prevalent neuropathologies. Emerging biological evidence indicates a notable role for G‐quadruplex (G4) DNA aggregation in neurodegenerative processes affecting neuronal cells, particularly in the presence of extended (G4C2)n repeats in nuclear DNA sequences. Our study concerns d[(GGGGCC)3GGGG], a G4‐forming DNA model featuring G4C2 repeats that is in correlation with neurodegeneration. Through different investigations utilizing spectroscopic techniques (CD, UV, and thermal denaturations), PAGE electrophoresis, and molecular docking, the study explores the influence of orotate on the aggregation of this neurodegeneration‐associated DNA. A computational approach was employed to construct an in silico model of the DNA aggregate, which involved the docking of multiple G4 units and subsequent integration of the ligand into both the DNA monomer and its in silico aggregated model. The convergence of computational analyses and empirical data collectively supports the hypothesis that orotate possesses the capability to modulate the aggregation of neurodegeneration‐related DNA. Notably, the findings suggest the potential utility of orotate as a neurodrug, especially for the therapy of amyotrophic lateral sclerosis (ALS) and Frontotemporal Dementia (FTD), with its current status as a dietary supplement indicating minimal safety concerns. Additionally, orotate demonstrated a slight increase in mitochondrial dehydrogenase activity as assessed by the MTT assay, which is beneficial for a neurodrug as it suggests a potential role in enhancing mitochondrial function and supporting neuronal health. image


Sub‐Microliter H Magnetic Resonance Spectroscopy for In Vivo High‐Spatial Resolution Metabolite Quantification in the Mouse Brain

January 2025

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22 Reads

Proton magnetic resonance spectroscopy (MRS) offers a non‐invasive, repeatable, and reproducible method for in vivo metabolite profiling of the brain and other tissues. However, metabolite fingerprinting by MRS requires high signal‐to‐noise ratios for accurate metabolite quantification, which has traditionally been limited to large volumes of interest, compromising spatial fidelity. In this study, we introduce a new optimized pipeline that combines LASER MRS acquisition at 11.7 T with a cryogenic coil and advanced offline pre‐ and post‐processing. This approach achieves a signal‐to‐noise ratio sufficient to reliably quantify 19 distinct metabolites in a volume as small as 0.7 μL within the mouse brain. The resulting high spatial resolution and spectral quality enable the identification of distinct metabolite fingerprints in small, specific regions, as demonstrated by characteristic differences in N‐acetylaspartate, glutamate, taurine, and myo‐inositol between the motor and somatosensory cortices. We demonstrated a decline in taurine and glutamate in the primary motor cortex between 5 and 11 months of age, against the stability of other metabolites. Further exploitation to cortical layer‐specific metabolite fingerprinting of layer I–III to layer VI–V in the primary motor cortex, with the latter showing reduced taurine and phosphoethanolamine levels, demonstrates the potential of this pipeline for detailed in vivo metabolite fingerprinting of cortical areas and subareas. image


RNA methylation system of m6A, including “writers” (methyltransferases), “erasers” (demethylases), and “readers” (proteins that recognize RNA methylation, such as YTHDC1 in nucleus, and YTHDF1 in cytoplasm, also see Table 1 for details).
Using Zebrafish Models to Study Epitranscriptomic Regulation of CNS Functions

Epitranscriptomic regulation of cell functions involves multiple post‐transcriptional chemical modifications of coding and non‐coding RNA that are increasingly recognized in studying human brain disorders. Although rodent models are presently widely used in neuroepitranscriptomic research, the zebrafish (Danio rerio) has emerged as a useful and promising alternative model species. Mounting evidence supports the importance of RNA modifications in zebrafish CNS function, providing additional insights into epitranscriptomic mechanisms underlying a wide range of brain disorders. Here, we discuss recent data on the role of RNA modifications in CNS regulation, with a particular focus on zebrafish models, as well as evaluate current problems, challenges, and future directions of research in this field of molecular neurochemistry. image


Workflows for our label‐free approaches. (A) Workflow for immunofluorescence (IF) guided Fourier transform infrared (FTIR) imaging. Unstained brain tissue sections are first imaged with FTIR subsequently stained against Aβ and scanned. Both modalities are registered, and region‐specific FTIR spectra are analyzed. (B) Unstained brain tissue sections are first imaged with QCL‐IR, and plaques are detected using a neural network (Müller et al. 2024). Plaques and surrounding tissue areas are extracted via laser microdissection (LMD) and analyzed using flow injection analysis mass spectrometry (FIA‐MS).
Lipid unsaturation in plaques in human brain tissue derived by immunofluorescence (IF) and Fourier‐transform infrared (FTIR) microspectroscopy. (A1) Tissue sections are stained against Aβ using the fluorophore‐labeled antibody 4G8 (blue) and the fluorescent probe Thioflavin T (ThT) in red. (A2) The gray matter region of an advanced AD TL (TL) is typically abundant in Aβ plaques. (A3) A mature plaque is readily identified in IF images by their characteristic core and corona structure. (B) The combination of FTIR and IF images of the same sample produce clearly separated mean FTIR spectra of a plaque core (red), corona (blue), and its surrounding tissue (black). The inset displays the marked decrease of the =C‐H stretching band of alkenes at 3012 cm⁻¹ in the plaque. (C) The ratio between alkenes and ester groups (1738 cm⁻¹) indicates the degree of lipid unsaturation and was calculated for 208 gray matter regions from three control cases and 121 plaques from three AD cases. Unsaturated lipids are significantly decreased in plaques, compared to their surrounding and gray matter of HC cases. The full statistical reports, including p‐values, t‐values, and degrees of freedom, are provided in Supporting Information S2. ** < 0.01, **** < 0.0001.
Lipid unsaturation derived by flow injection analysis mass spectrometry (FIA‐MS) of label‐free extracted Aβ plaques, surrounding tissue, and gray matter from healthy control (HC) cases. (A) The distribution of lipid unsaturation (number of C=C double bonds per lipid) across all samples (n = 24) from all cases (n = 16) and tissue groups as percentage of the total lipid content. Figure S4B illustrates the distribution of data on a patient‐wise basis. In some instances, the degree of lipid unsaturation differs significantly between tissue groups. (B) Subsequently, the relative content of unsaturated lipids differs significantly between the tissue groups and is lowest in plaques. (C) The distribution difference of lipid unsaturation between plaques and their surrounding tissue reveals that unsaturated lipids are significantly increased in plaques, whereas unsaturated lipids are decreased. The complete statistical reports, including p‐values, t‐values, and degrees of freedom, can be found in Supporting Information S2. ns > 0.05, ** < 0.01, **** < 0.0001.
Sum of acyl chain composition derived by label‐free flow injection analysis mass spectrometry (FIA‐MS) of label‐free extracted Aβ plaques, surrounding tissue, and gray matter from healthy control (HC) cases. (A) The distribution of FA length (number of C atoms in FAs per lipid molecule) across all samples (n = 24) from all cases (n = 16) and tissue groups as percentage of the total lipid content. Figure S3D illustrates the distribution of data on a patient‐wise basis. The FA length distribution differs significantly between the tissue groups. (B) Subsequently, the average FA length differs significantly between the tissue groups and is lowest in plaques. (C) The distribution difference between plaques and their surrounding tissue reveals that short FAs (< 35 C atoms per lipid) are significantly increased in plaques, whereas long FAs (> 35 C atoms per lipid) are mostly decreased. The full statistical reports, including p‐values, t‐values, and degrees of freedom, are provided in Supporting Information S2. ns > 0.05, * < 0.05, ** < 0.01, *** < 0.001, **** < 0.0001.
The lipid changes in plaques. (A) The mean difference of the total molar contributions of the most increased lipid species in plaques (n = 8), compared to their surroundings. Phosphatidylcholine (PC) in blue, PE in orange and Ceramides (Cer) in green. (B) Principal component analysis (PCA) of the entire lipidomes, comparing plaques (blue) and their surrounding (gray) from the eight AD cases included in this study. The black line indicates a linear separation of the cluster. (C) Correlation matrix between saturated and all PC species. Red indicates correlation, whereas blue indicates anti‐correlation. Strong correlations (R < 0.6) are labeled, and significant correlations (p > 0.05) are marked with an asterisk, excluding self‐correlations.
Unsaturated Fatty Acids Are Decreased in Aβ Plaques in Alzheimer's Disease

January 2025

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37 Reads

Alzheimer's disease (AD) is characterized by the accumulation of amyloid‐beta (Aβ) plaques in the brain, contributing to neurodegeneration. This study investigates lipid alterations within these plaques using a novel, label‐free, multimodal approach. Combining infrared (IR) imaging, machine learning, laser microdissection (LMD), and flow injection analysis mass spectrometry (FIA‐MS), we provide the first comprehensive lipidomic analysis of chemically unaltered Aβ plaques in post‐mortem human AD brain tissue. IR imaging revealed decreased lipid unsaturation within plaques, evidenced by a reduction in the alkene (=C‐H) stretching vibration band. The high spatial resolution of IR imaging, coupled with machine learning‐based plaque detection, enabled precise and label‐free extraction of plaques via LMD. Subsequent FIA‐MS analysis confirmed a significant increase in short‐chain saturated lipids and a concomitant decrease in long‐chain unsaturated lipids within plaques compared to the surrounding tissue. These findings highlight a substantial depletion of unsaturated fatty acids (UFAs) in Aβ plaques, suggesting a pivotal role for lipid dysregulation and oxidative stress in AD pathology. This study advances our understanding of the molecular landscape of Aβ plaques and underscores the potential of lipid‐based therapeutic strategies in AD. image


Brain Transcriptome Changes Associated With an Acute Increase of Protein O‐GlcNAcylation and Implications for Neurodegenerative Disease

January 2025

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15 Reads

Enhancing protein O‐GlcNAcylation by pharmacological inhibition of the enzyme O‐GlcNAcase (OGA) has been considered as a strategy to decrease tau and amyloid‐beta phosphorylation, aggregation, and pathology in Alzheimer's disease (AD). There is still more to be learned about the impact of enhancing global protein O‐GlcNAcylation, which is important for understanding the potential of using OGA inhibition to treat neurodegenerative diseases. In this study, we investigated the acute effect of pharmacologically increasing O‐GlcNAc levels, using the OGA inhibitor Thiamet G (TG), in normal mouse brains. We hypothesized that the transcriptome signature in response to a 3 h TG treatment (50 mg/kg) provides a comprehensive view of the effect of OGA inhibition. We then performed mRNA sequencing of the brain using NovaSeq PE 150 (n = 5 each group). We identified 1234 significant differentially expressed genes with TG versus saline treatment. Functional enrichment analysis of the upregulated genes identified several upregulated pathways, including genes normally down in AD. Among the downregulated pathways were the cell adhesion pathway as well as genes normally up in AD and aging. When comparing acute to chronic TG treatment, protein autophosphorylation and kinase activity pathways were upregulated, whereas cell adhesion and astrocyte markers were downregulated in both datasets. AMPK subunit Prkab2 was one gene in the kinase activity pathway, and the increase after acute and chronic treatment was confirmed using qPCR. Interestingly, mitochondrial genes and genes normally down in AD were up in acute treatment and down in chronic treatment. Data from this analysis will enable the evaluation of the mechanisms underlying the impact of OGA inhibition in the treatment of AD. In particular, OGA inhibitors appear to have downstream effects related to bioenergetics which may limit their therapeutic benefits. image


Prenatal Valproic Acid Induces Autistic‐Like Behaviors in Rats via Dopaminergic Modulation in Nigrostriatal and Mesocorticolimbic Pathways

Autism spectrum disorder (ASD) is a complex developmental disorder characterized by several behavioral impairments, especially in socialization, communication, and the occurrence of stereotyped behaviors. In rats, prenatal exposure to valproic acid (VPA) induces autistic‐like behaviors. Previous studies by our group have suggested that the autistic‐like phenotype is possibly related to dopaminergic system modulation because tyrosine hydroxylase (TH) expression was affected. The objective of the present study was to understand the dopaminergic role in autism. Wistar rats on gestational day 12.5 received VPA (400 mg/kg) and behaviors related to rat models of ASD were evaluated in juvenile offspring. Neurochemical and genetic dopaminergic components were studied in different brain areas of both juvenile and adult rats. Prenatal VPA‐induced autistic‐like behaviors in comparison to a control group: decreased maternal solicitations by ultrasonic vocalizations, cognitive inflexibility and stereotyped behavior in the T‐maze test, decreased social interaction and play behavior, as well as motor hyperactivity. Prenatal VPA also decreased dopamine synthesis and activity in the striatum and prefrontal cortex, as well as dopamine transporter, D1 and D2 receptors, and TH expressions. Moreover, prenatal VPA increased TH+ immunoreactive neurons of the ventral tegmental area–substantia nigra complex. In conclusion, the dopaminergic hypoactivity associated with the behavioral impairments exhibited by the rats that received prenatal VPA suggests the important role of this system in the establishment of the characteristic symptoms of ASD in juvenile and adult males. Dopamine was demonstrated to be an important biomarker and a potential pharmacological target for ASD. image


Regulation of Dendrite and Dendritic Spine Formation by TCF20

January 2025

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30 Reads

Mutations in the Transcription Factor 20 (TCF20) have been identified in patients with autism spectrum disorders (ASDs), intellectual disabilities (IDs), and other neurological issues. Recently, a new syndrome called TCF20‐associated neurodevelopmental disorders (TAND) has been described, with specific clinical features. While TCF20's role in the neurogenesis of mouse embryos has been reported, little is known about its molecular function in neurons. In this study, we demonstrate that TCF20 is expressed in all analyzed brain regions in mice, and its expression increases during brain development but decreases in muscle tissue. Our findings suggest that TCF20 plays a central role in dendritic arborization and dendritic spine formation processes. RNA sequencing analysis revealed a downregulation of pre‐ and postsynaptic pathways in TCF20 knockdown neurons. We also found decreased levels of GABRA1, BDNF, PSD‐95, and c‐Fos in total homogenates and in synaptosomal preparations of knockdown TCF20 rat cortical cultures. Furthermore, synaptosomal preparations of knockdown TCF20 rat cortical cultures showed significant downregulation of GluN2B and GABRA5, while GluA2 was significantly upregulated. Overall, our data suggest that TCF20 plays an essential role in neuronal development and function by modulating the expression of proteins involved in dendrite and synapse formation and function. image


Journal metrics


4.2 (2023)

Journal Impact Factor™


37%

Acceptance rate


9.3 (2023)

CiteScore™


18 days

Submission to first decision


$5,080 / £3,400 / €4,270

Article processing charge

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