Rafael Luján

Calcium signaling mediated by small-conductance calcium-activated potassium (SK) channels plays a critical role in regulating synaptic plasticity and neuronal excitability in the hippocampus. Although SK2 channels are strategically positioned to control calcium-dependent feedback mechanisms, their subcellular organization in Aβ pathology remains poorly understood. Here, we investigated the expression and nanoscale distribution of SK2 channels in the hippocampus of APP/PS1 mice. Using histoblot analysis, we found that overall SK2 protein levels, including the synaptically targeted SK2-L isoform, were not significantly altered in APP/PS1 mice compared with age-matched controls. In contrast, high-resolution SDS-digested freeze-fracture replica labeling revealed a marked and compartment-specific redistribution of SK2 channels. Quantitative analyses demonstrated a significant increase in extrasynaptic SK2 immunoparticles along dendritic membranes, whereas synaptic SK2 localization at postsynaptic densities remained largely preserved. These findings indicate a clear dissociation between SK2 expression and subcellular organization in Aβ pathology. Rather than altering total protein levels, AD selectively remodels the spatial distribution of SK2 channels, preferentially affecting dynamic extrasynaptic pools. This redistribution may have important consequences for the regulation of intrinsic membrane excitability and synaptic integration in hippocampal circuits. More broadly, our results support the concept that disruption of membrane protein organization at the nanoscale level represents a key mechanism underlying early neuronal dysfunction in Aβ pathology.

Background: Normal body mass index (BMI) does not necessarily indicate normal adiposity, and individuals with normal-weight obesity (NWO) may exhibit cardiometabolic abnormalities that remain undetected by BMI-based classification. We examined the cardiometabolic phenotype associated with NWO among adults with normal BMI, with particular attention to sex-related heterogeneity and the discriminatory ability of simple anthropometric measures. Methods: This cross-sectional study included 44,939 Spanish healthcare workers. NWO was defined as BMI 18.5–24.9 kg/m² combined with high or very high body-fat percentage according to sex- and age-specific classification. The primary analytical sample comprised 19,824 participants with normal BMI and either normal adiposity (n=19,040) or NWO (n=784). Cardiometabolic characteristics included glycaemic, lipid, blood-pressure, insulin-resistance-related, and metabolic-syndrome measures. Multivariable logistic regression was used to examine associations between NWO and cardiometabolic abnormalities, including NWO × sex interactions. Receiver operating characteristic analyses evaluated the ability of BMI, waist circumference, and waist-to-height ratio (WHtR) to discriminate NWO. Results: NWO was present in 3.10% (95% confidence interval [CI], 2.89–3.32%) of participants with normal BMI and was more prevalent in men than women (5.43% vs. 2.35%). Compared with normal adiposity, NWO was characterized by greater central adiposity, higher fasting glucose and insulin-resistance-related indices, lower HDL-C, and a more atherogenic lipid profile. At least one abnormal cardiometabolic domain was present in 53.1% of participants with NWO versus 29.2% with normal adiposity. After multivariable adjustment, NWO was associated with ≥1 abnormal domain (adjusted odds ratio [aOR] 2.24; 95% CI, 1.93–2.60), ≥2 abnormal domains (aOR 1.76; 95% CI, 1.36–2.29), elevated triglyceride-glucose index (aOR 3.28; 95% CI, 2.61–4.11), elevated low-density/high-density lipoprotein cholesterol ratio (aOR 3.45; 95% CI, 2.87–4.15), and elevated triglyceride/high-density lipoprotein cholesterol ratio (aOR 2.72; 95% CI, 2.18–3.38). Several associations differed by sex. In receiver operating characteristic (ROC) analyses, waist circumference showed the highest discrimination overall (area under the curve [AUC] 0.776; 95% CI, 0.756–0.795) and among men (AUC 0.836; 95% CI, 0.812–0.858), whereas BMI showed the highest discrimination among women (AUC 0.757; 95% CI, 0.733–0.780). Conclusions: Among Spanish healthcare workers with normal BMI, NWO identified a subgroup with greater concurrent cardiometabolic abnormality burden, particularly involving glycaemic, insulin-resistance-related, and atherogenic characteristics. Associations and anthropometric discrimination differed by sex, highlighting heterogeneity within the conventional normal-BMI category. Prospective studies are needed to determine the prognostic relevance of these phenotypes.

Summary: Carotenoids and apocarotenoids are widespread specialized metabolites, yet animals, including sponges, lack the ability to synthesize carotenoids de novo and must obtain them from dietary or microbial sources. The roles of carotenoid cleavage dioxygenases (CCDs) in marine animals remain largely unexplored. A CCD from the marine sponge Suberites domuncula (SdCDO) was evaluated for retinoid formation using a synthetic biology approach involving heterologous expression in Escherichia coli and tomato ( Solanum lycopersicum ), combined with metabolic, ultrastructural, and transcriptomic analyses. Unexpectedly, SdCDO did not produce retinoids. Instead, it cleaved lycopene and β‐carotene to generate crocetin dialdehyde, the direct precursor of crocins. In tomato fruits, SdCDO expression triggered crocin accumulation, particularly in locular tissues, while reducing lycopene, lutein, and β‐carotene levels. It also promoted the accumulation of upstream carotenoid intermediates, such as phytoene, phytofluene, and ζ‐carotene. These metabolic changes resulted in orange fruit pigmentation and enhanced antioxidant capacity, along with altered chromoplast ultrastructure and broad downregulation of endogenous carotenoid biosynthetic genes. Overall, these findings reveal unexpected enzymatic plasticity in a sponge‐derived CCD and demonstrate its ability to redirect plant carotenoid metabolism toward crocin biosynthesis. This work highlights the functional diversity of CCDs across kingdoms and their potential for synthetic biology and crop biofortification.

SUMMARY: NMDA receptors (NMDARs) are well-established as coincidence detectors in Hebbian learning, but this requires postsynaptic localization. Presynaptic NMDARs, however, remain controversial due to limitations of pharmacology and calcium imaging. We therefore dissected the function of distinct NMDAR pools using more direct techniques, including immunogold EM, sparse genetic deletion, and paired recordings from layer-5 (L5) pyramidal cell (PC) synapses in mouse primary visual cortex (V1). We found that pre- but not postsynaptic NMDARs regulate both spontaneous and evoked neurotransmitter release. In spike-timing-dependent plasticity, we uncovered a double dissociation: timing-dependent long-term depression (tLTD) requires pre- but not postsynaptic NMDARs, whereas timing-dependent long-term potentiation (tLTP) needs post- but not presynaptic NMDARs. Postsynaptic NMDAR loss also caused developmentally delayed dendritic spine loss and altered axonal and dendritic architecture. In summary, NMDARs do not act as a unified plasticity signal but rather confer location-specific control over diverse forms of synaptic signaling, plasticity, and circuit structure.

Background: Ca _V 2.3 (R-type) channels mediate calcium signals involved in neuronal excitability, synaptic plasticity, and neurotransmitter release in the hippocampus. However, there is currently no information about their potential changes in Alzheimer's disease (AD). Objective: This study aims to analyze the protein levels, expression, and subcellular localization of Ca _V 2.3 channels in the hippocampus of 5xFAD mice. Methods: We employed histoblot, western blot, qRT-PCR, and immunoelectron microscopy techniques. Results: Using the histoblot technique, we observed that the protein content of Ca _V 2.3 in female 5xFAD mice was downregulated in the hippocampus and caudate putamen at 5 months, with more pronounced downregulation at 10 months that also affected the septum. The hippocampus was the most affected region, where Ca _V 2.3 immunodetection was significantly decreased in a few dendritic layers at 5 months, but this reduction extended to all layers and subregions by 10 months, demonstrating age- and laminar-dependent changes. The pattern in females differed from that seen in males and remained unchanged in APP/PS1 and P301S mice. Immunoelectron microscopy of the hippocampus showed Ca _V 2.3 in apical and oblique dendrites of CA1 pyramidal cells, with a significantly higher presence in dendritic spines. Quantification revealed a significant decrease in the density of Ca _V 2.3 in oblique dendrites and dendritic spines of CA1 pyramidal cells, whereas apical dendrites and presynaptic compartments were unaffected in 5xFAD mice. Conclusions: Overall, this downregulation in postsynaptic Ca _V 2.3 channels could impact neural circuit activity and contribute to the cognitive deficits observed in 5xFAD mice.

Metabotropic GABA (GABA_B) receptors have modulatory functions on neuronal excitability and neurotransmitter release. To fulfil these functions, GABA_B receptors form macromolecular signaling complexes with G proteins, effectors, and other associated proteins. Here we investigated the postnatal development of GABA_B receptors (GABA_B1 and GABA_B2 subunits) in mouse brain, focusing on potential similarities in the spatial and temporal expression pattern of their associated proteins Ca_V2.1, Gα_o, Gβ5, and RGS7, using histoblots, immunofluorescence, and immunoelectron microscopic techniques. At all ages analyzed, histoblot showed that the six proteins were widely expressed in the brain, with mostly an overlapping pattern throughout postnatal development. In the hippocampus, immunoelectron microscopy and quantitative analysis of immunoparticles for GABA_B1, GABA_B2, Gα_o, Gβ5, and RGS7 revealed their progressive enrichment around excitatory synapses on dendritic spines of CA1 pyramidal cells toward P15. At presynaptic sites, GABA_B receptors colocalize with Ca_V2.1, Gα_o, Gβ5, and RGS7 in the active zone and extrasynaptic membranes of axon terminals, establishing synapses on dendritic spines of CA1 pyramidal cells. In the cerebellum, double immunofluorescence at P7 and P10 revealed the colocalization of GABA_B1 and Ca_V2.1 in the whole dendritic tree of developing Purkinje cells. Immunoelectron microscopy at P15 showed that GABA_B1, GABA_B2, Ca_V2.1, Gα_o, Gβ5, and RGS7 are distributed along the dendritic surface of Purkinje cells, enriched close to excitatory synapses in spines. Altogether, these data suggest that macromolecular complexes composed of GABA_B1/GABA_B2/Ca_V2.1/Gα_o/Gβ5/RGS7 are pre-assembled during key stages of postnatal development in hippocampal and cerebellar neurons.

Kufs disease/CLN4 is an autosomal dominant neurodegenerative disorder caused by unknown mechanisms through Leu ¹¹⁵ Arg and Leu ¹¹⁶ Δ mutations in the DNAJC5 gene that encodes the synaptic vesicle co-chaperone cysteine string protein α (CSPα/DNAJC5). To investigate the disease mechanisms in vivo, we generated three independent mouse lines overexpressing different versions of CSPα/DNAJC5 under the neuron-specific Thy1 promoter: wild-type (WT), Leu ¹¹⁵ Arg, and Leu ¹¹⁶ Δ. Mice expressing mutant Leu ¹¹⁵ Arg CSPα/DNAJC5 are viable but develop motor deficits. As described in patients with Kufs disease, we observed the pathological lipofuscinosis and intracellular structures resembling granular osmiophilic deposits (GRODs) in the mutant but not in the WT transgenic lines. Microglia engulf lipofuscin and lipofuscin-containing neurons. Notably, conventional or conditional knockout mice lacking CSPα/DNAJC5 did not exhibit any signs of increased lipofuscinosis or GRODs. Our novel mouse models provide a valuable tool to investigate the molecular mechanisms underlying Kufs disease/CLN4. DNAJC5 mutations cause neuronal lipofuscinosis through a cell-autonomous gain of a pathological function of CSPα/DNAJC5.

Opioids trigger structural and functional neural adaptations of the reward circuit that lead to dependence. Synaptic cell adhesion molecules (CAMs) play a pivotal role in circuit organization and present prime candidates for orchestrating remodeling of neural connections in response to drug exposure. However, the contribution of CAMs to opioid-induced rewiring of the reward circuit has not been explored. Here, we used unbiased molecular profiling to identify CAMs in the nucleus accumbens (NAc) modulated by morphine administration. We found that opioid exposure induces the expression of ELFN1, a CAM selectively expressed in cholinergic interneurons in the NAc. We determined that ELFN1 acts trans-synaptically to modulate the strength and plasticity of the glutamatergic inputs onto cholinergic neurons via the recruitment of presynaptic metabotropic glutamate receptor 4 (mGlu4). Disruption of Elfn1 diminished morphine reward and intake in self-administering mice. Together, our findings identify a key molecular factor responsible for adjusting the strength of opioid effects by modulating the configuration of striatal circuitry in an experience-dependent fashion and unveil potential therapeutic target for combating opioid abuse.

We are in the midst of a revolution in the fields of neuroanatomy and electron microscopy. The monumental advancements in the neuroscience field during the last decade have led to unprecedented scientific discoveries about our brain and to the development of new technologies and applications that have significantly contributed to such advances. Conventional applications of transmission electron microscopy have revolutionized neurosciences and are critical for determining the fine morpho-functional characterization of brain cells and their connections. Electron microscopy has progressively evolved toward the development of both more sensitive approaches to unravel the bidimensional subcellular localization of proteins and tools that allow for the three-dimensional characterization of different nerve cells and their connections. The development of new technological advances in two- and three-dimensional electron microscopy to study and map the brain has led to the development of essential tools to decipher the complexity of the brain. For two-dimensional, the sodium dodecyl sulfate-digested freeze-fracture replica labeling technique is a technique with the main goal of chemically identifying the structural components viewed in freeze-fracture replicas and has significant advantages over conventional immunoelectron microscopic techniques for revealing the subcellular organization of proteins along the neuronal surface in the brain. For three-dimensional, volume electron microscopy methods can be applied to structural studies of cell components and organelles, just as conventional transmission electron microscopy has been traditionally applied, but with advantages derived from the possibility of three-dimensional visualization and analysis. The development of volume electron microscopy has greatly facilitated the study of brain structure and connectivity at the synaptic level. Dedicated software tools for the analysis of highly complex connectivity patterns in three dimension are evolving in parallel, allowing the extraction of relevant information from large datasets. Moreover, by applying these new methodologies, the field of pathology is expected to advance, potentially with the identification of the pathogenesis generating these diseases. This review aims to present the possibilities and fundamentals of two- and three-dimensional electron microscopy for high-resolution ultrastructural analyses of neurons and their connections. These technological tools have improved the ability to study the brain, thus providing new insights into brain structure and function.