KA-mediated excitotoxicity induces neuronal ferroptosis through activation of ferritinophagy.

OBJECTIVES: This study aims to elucidate the role of Fe2+ overload in kainic acid (KA)-induced excitotoxicity, investigate the involvement of ferritinophagy selective cargo receptor NCOA4 in the pathogenesis of excitotoxicity. METHODS: Western blotting was used to detect the expression of FTH1, NCOA4, Lamp2, TfR, FPN, and DMT1 after KA stereotaxic injection into the unilateral striatum of mice. Colocalization of Fe2+ with lysosomes in KA-treated primary cortical neurons was observed by using confocal microscopy. Desferrioxamine (DFO) was added to chelate free iron, a CCK8 kit was used to measure cell viability, and the Fe2+ levels were detected by FerroOrange. BODIPY C11 was used to determine intracellular lipid reactive oxygen species (ROS) levels, and the mRNA levels of PTGS2, a biomarker of ferroptosis, were measured by fluorescent quantitative PCR. 3-Methyladenine (3-MA) was employed to inhibit KA-induced activation of autophagy, and changes in ferritinophagy-related protein expression and the indicated biomarkers of ferroptosis were detected. Endogenous NCOA4 was knocked down by lentivirus transfection, and cell viability and intracellular Fe2+ levels were observed after KA treatment. RESULTS: Western blot results showed that the expression of NCOA4, DMT1, and Lamp2 was significantly upregulated, while FTH1 was downregulated, but there were no significant changes in TfR and FPN. The fluorescence results indicated that KA enhanced the colocalization of free Fe2+ with lysosomes in neurons. DFO intervention could effectively rescue cell damage, reduce intracellular lipid peroxidation, and decrease the increased transcript levels of PTGS2 caused by KA. Pretreatment with 3-MA effectively reversed KA-induced ferritinophagy and ferroptosis. Endogenous interference with NCOA4 significantly improved cell viability and reduced intracellular free Fe2+ levels in KA-treated cells. CONCLUSION: KA-induced excitotoxicity activates ferritinophagy, and targeting ferritinophagy effectively inhibits downstream ferroptosis. Interference with NCOA4 effectively attenuates KA-induced neuronal damage. This study provides a potential therapeutic target for excitotoxicity related disease conditions.

Nr2f1 enhancers have distinct functions in controlling Nr2f1 expression during cortical development.

There is evidence that transcription factor (TF) encoding genes, which temporally control development in multiple cell types, can have tens of enhancers that regulate their expression. The NR2F1 TF developmentally promotes caudal and ventral cortical regional fates. Here, we epigenomically compared the activity of Nr2f1's enhancers during mouse cortical development with their activity in a transgenic assay. We identified at least six that are likely to be important in prenatal cortical development, with three harboring de novo mutants identified in ASD individuals. We chose to study the function of two of the most robust enhancers by deleting them singly or together. We found that they have distinct and overlapping functions in driving Nr2f1's regional and laminar expression in the developing cortex. Thus, these two enhancers, probably in combination with the others that we defined epigenetically, precisely tune Nr2f1's regional, cell type, and temporal expression during corticogenesis.

High Impact AMPAkines Induce a Gq-Protein Coupled Endoplasmic Calcium Release in Cortical Neurons: A Possible Mechanism for Explaining the Toxicity of High Impact AMPAkines.

α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) positive allosteric modulators (AMPAkines) have a multitude of promising therapeutic properties. The pharmaceutical development of high impact AMPAkines has, however, been limited by the appearance of calcium-dependent neuronal toxicity and convulsions in vivo. Such toxicity is not observed at exceptionally high concentrations of low impact AMPAkines. Because most AMPAR are somewhat impermeable to calcium, the current study sought to examine the extent to which different mechanisms contribute to the rise in intracellular calcium in the presence of high impact ampakines. In the presence of AMPA alone, cytosolic calcium elevation is shown to be sodium-dependent. In the presence of high impact AMPAkines such as cyclothiazide (CTZ) or CX614, however, AMPAR potentiation also activates an additional mechanism that induces calcium release from endoplasmic reticular (ER) stores. The pathway that connects AMPAR to the ER system involves a Gq-protein, phospholipase Cß-mediated inositol triphosphate (InsP3) formation, and ultimately stimulation of InsP3-receptors located on the ER. The same linkage was not observed using high concentrations of the low impact AMPAkines, CX516 (Ampalex), and CX717. We also demonstrate that CX614 produces neuronal hyper-excitability at therapeutic doses, whereas the newer generation low impact AMPAkine CX1739 is safe at exceedingly high doses. Although earlier studies have demonstrated a functional linkage between AMPAR and G-proteins, this report demonstrates that in the presence of high impact AMPAkines, AMPAR also couple to a Gq-protein, which triggers a secondary calcium release from the ER and provides insight into the disparate actions of high and low impact AMPAkines.

Unveiling MRI markers for Parkinson's Disease: GABAergic dysfunction and cortical changes.

OBJECTIVE: The study aimed to investigate changes in basal levels of the inhibitory γ-aminobutyric acid (GABA) neurotransmitter in the sensorimotor cortex (SMC) and cortical gyrification in patients with Parkinson's disease (PD), which could further identify potential imaging biomarkers for PD, particularly in patients with early-onset Parkinson's disease (EOPD). METHOD: Fifty patients with PD (EOPD: 10, late-onset Parkinson's disease [LOPD]: 40) and fifty-two age- and gender-matched healthy controls (HC) underwent GABA-edited 1H MRS of the SMC and high-resolution 3D T1-weighted brain imaging. GABA levels and local gyrification index (LGI) were calculated to assess GABAergic and cortical gyrification deficits in PD. RESULT: The Pearson correlation coefficients revealed significant negative associations between eight indicators, including GABA/Cr level and local gyrification index (LGI) of specific cortical regions (precentral, postcentral, entorhinal, superiortemporal, posteriorcingulate, cuneus, and transversetemporal cortex), and the likelihood of Parkinson's disease (r < -0.4, p < 0.001). Additionally, GABA levels were significantly lower in the SMC region of both EOPD and LOPD patients compared to healthy controls (mean ± SD [u.i.]: EOPD=0.081 ± 0.022 vs. Young-HC=0.112 ± 0.021, p = 0.003; LOPD=0.054 ± 0.024 vs. Old-HC=0.099 ± 0.021, p < 0.001). The logistic regression model was established by using multivariate analysis, identifying two statistically significant indicators: GABA/Cr and LGI of the transversetemporal. The combined model exhibited the highest AUC values in both younger and older populations. CONCLUSION: GABAergic dysfunction may play an important role in the pathogenesis of PD patients. Changes in neurotransmitter and morphological may serve as potential markers for the preclinical diagnosis and progression of PD, including EOPD.

Regional patterns of human cortex development correlate with underlying neurobiology.

Human brain morphology undergoes complex changes over the lifespan. Despite recent progress in tracking brain development via normative models, current knowledge of underlying biological mechanisms is highly limited. We demonstrate that human cortical thickness development and aging trajectories unfold along patterns of molecular and cellular brain organization, traceable from population-level to individual developmental trajectories. During childhood and adolescence, cortex-wide spatial distributions of dopaminergic receptors, inhibitory neurons, glial cell populations, and brain-metabolic features explain up to 50% of the variance associated with a lifespan model of regional cortical thickness trajectories. In contrast, modeled cortical thickness change patterns during adulthood are best explained by cholinergic and glutamatergic neurotransmitter receptor and transporter distributions. These relationships are supported by developmental gene expression trajectories and translate to individual longitudinal data from over 8000 adolescents, explaining up to 59% of developmental change at cohort- and 18% at single-subject level. Integrating neurobiological brain atlases with normative modeling and population neuroimaging provides a biologically meaningful path to understand brain development and aging in living humans.

Lrig1 regulates cell fate specification of glutamatergic neurons via FGF-driven Jak2/Stat3 signaling in cortical progenitors.

The cell-intrinsic mechanisms underlying the decision of a stem/progenitor cell to either proliferate or differentiate remain incompletely understood. Here, we identify the transmembrane protein Lrig1 as a physiological homeostatic regulator of FGF2-driven proliferation and self-renewal of neural progenitors at early-to-mid embryonic stages of cortical development. We show that Lrig1 is expressed in cortical progenitors (CPs), and its ablation caused expansion and increased proliferation of radial/apical progenitors and of neurogenic transit-amplifying Tbr2+ intermediate progenitors. Notably, our findings identify a previously unreported EGF-independent mechanism through which Lrig1 negatively regulates neural progenitor proliferation by modulating the FGF2-induced IL6/Jak2/Stat3 pathway, a molecular cascade that plays a pivotal role in the generation and maintenance of CPs. Consistently, Lrig1 knockout mice showed a significant increase in the density of pyramidal glutamatergic neurons placed in superficial layers 2 and 3 of the postnatal neocortex. Together, these results support a model in which Lrig1 regulates cortical neurogenesis by influencing the cycling activity of a set of progenitors that are temporally specified to produce upper layer glutamatergic neurons.

Dysregulated Purinergic Signalling in Fragile X Syndrome Cortical Astrocytes.

The symptoms of fragile X syndrome (FXS), caused by a single gene mutation to Fmr1, have been increasingly linked to disordered astrocyte signalling within the cerebral cortex. We have recently demonstrated that the purinergic signalling pathway, which utilizes nucleoside triphosphates and their metabolites to facilitate bidirectional glial and glial-neuronal interactions, is upregulated in cortical astrocytes derived from the Fmr1 knockout (KO) mouse model of FXS. Heightened Fmr1 KO P2Y purinergic receptor levels were correlated with prolonged intracellular calcium release, elevated synaptogenic protein secretion, and hyperactivity of developing circuits. However, due to the relative lack of sensitive and reproducible quantification methods available for measuring purines and pyrimidines, determining the abundance of these factors in Fmr1 KO astrocytes was limited. We therefore developed a hydrophilic interaction liquid chromatography protocol coupled with mass spectrometry to compare the abundance of intracellular and extracellular purinergic molecules between wildtype and Fmr1 KO mouse astrocytes. Significant differences in the concentrations of UDP, ATP, AMP, and adenosine intracellular stores were found within Fmr1 KO astrocytes relative to WT. The extracellular level of adenosine was also significantly elevated in Fmr1 KO astrocyte-conditioned media in comparison to media collected from WT astrocytes. Glycosylation of the astrocyte membrane-bound CD39 ectonucleotidase, which facilitates ligand breakdown following synaptic release, was also elevated in Fmr1 KO astrocyte cultures. Together, these differences demonstrated further dysregulation of the purinergic signalling system within Fmr1 KO cortical astrocytes, potentially leading to significant alterations in FXS purinergic receptor activation and cellular pathology.

Phosphorylation of the DNA damage repair factor 53BP1 by ATM kinase controls neurodevelopmental programs in cortical brain organoids.

53BP1 is a well-established DNA damage repair factor that has recently emerged to critically regulate gene expression for tumor suppression and neural development. However, its precise function and regulatory mechanisms remain unclear. Here, we showed that phosphorylation of 53BP1 at serine 25 by ATM is required for neural progenitor cell proliferation and neuronal differentiation in cortical brain organoids. Dynamic phosphorylation of 53BP1-serine 25 controls 53BP1 target genes governing neuronal differentiation and function, cellular response to stress, and apoptosis. Mechanistically, ATM and RNF168 govern 53BP1's binding to gene loci to directly affect gene regulation, especially at genes for neuronal differentiation and maturation. 53BP1 serine 25 phosphorylation effectively impedes its binding to bivalent or H3K27me3-occupied promoters, especially at genes regulating H3K4 methylation, neuronal functions, and cell proliferation. Beyond 53BP1, ATM-dependent phosphorylation displays wide-ranging effects, regulating factors in neuronal differentiation, cytoskeleton, p53 regulation, as well as key signaling pathways such as ATM, BDNF, and WNT during cortical organoid differentiation. Together, our data suggest that the interplay between 53BP1 and ATM orchestrates essential genetic programs for cell morphogenesis, tissue organization, and developmental pathways crucial for human cortical development.

TRPM4 inhibition slows neuritogenesis progression of cortical neurons.

TRPM4 is a non-selective cation channel activated by intracellular Ca2+ but only permeable to monovalent cations, its activation regulates membrane potential and intracellular calcium. This channel participates in the migration and adhesion of non-excitable cells and forms an integral part of the focal adhesion complex. In neurons, TRPM4 expression starts before birth and its function at this stage is not clear, but it may function in processes such as neurite development. Here we investigate the role of TRPM4 in neuritogenesis. We found that neurons at DIV 0 express TRPM4, the inhibition of TRPM4 using 9-Ph reduces neurite number and slows the progression of neurite development, keeping neurons in stage 1. The genetic suppression of TRPM4 using an shRNA at later stages (DIV2) reduces neurite length. Conversely, at DIV 0, TRPM4 inhibition augments the Cch-induced Ca2 + i increase, altering the calcium homeostasis. Together, these results show that TRPM4 participates in progression of neurite development and suggest a critical role of the calcium modulation during this stage of neuronal development.

Cortexa: a comprehensive resource for studying gene expression and alternative splicing in the murine brain.

BACKGROUND: Gene expression and alternative splicing are strictly regulated processes that shape brain development and determine the cellular identity of differentiated neural cell populations. Despite the availability of multiple valuable datasets, many functional implications, especially those related to alternative splicing, remain poorly understood. Moreover, neuroscientists working primarily experimentally often lack the bioinformatics expertise required to process alternative splicing data and produce meaningful and interpretable results. Notably, re-analyzing publicly available datasets and integrating them with in-house data can provide substantial novel insights. However, such analyses necessitate developing harmonized data handling and processing pipelines which in turn require considerable computational resources and in-depth bioinformatics expertise. RESULTS: Here, we present Cortexa-a comprehensive web portal that incorporates RNA-sequencing datasets from the mouse cerebral cortex (longitudinal or cell-specific) and the hippocampus. Cortexa facilitates understandable visualization of the expression and alternative splicing patterns of individual genes. Our platform provides SplicePCA-a tool that allows users to integrate their alternative splicing dataset and compare it to cell-specific or developmental neocortical splicing patterns. All standardized gene expression and alternative splicing datasets can be downloaded for further in-depth downstream analysis without the need for extensive preprocessing. CONCLUSIONS: Cortexa provides a robust and readily available resource for unraveling the complexity of gene expression and alternative splicing regulatory processes in the mouse brain. The data portal is available at https://cortexa-rna.com/.

Protein Kinase C-Delta Mediates Cell Cycle Reentry and Apoptosis Induced by Amyloid-Beta Peptide in Post-Mitotic Cortical Neurons.

Amyloid-beta peptide (Aß) is a neurotoxic constituent of senile plaques in the brains of Alzheimer's disease (AD) patients. The detailed mechanisms by which protein kinase C-delta (PKCδ) contributes to Aß toxicity is not yet entirely understood. Using fully differentiated primary rat cortical neurons, we found that inhibition of Aß25-35-induced PKCδ increased cell viability with restoration of neuronal morphology. Using cyclin D1, proliferating cell nuclear antigen (PCNA), and histone H3 phosphorylated at Ser-10 (p-Histone H3) as the respective markers for the G1-, S-, and G2/M-phases, PKCδ inhibition mitigated cell cycle reentry (CCR) and subsequent caspase-3 cleavage induced by both Aß25-35 and Aß1-42 in the post-mitotic cortical neurons. Upstream of PKCδ, signal transducers and activators of transcription (STAT)-3 mediated PKCδ induction, CCR, and caspase-3 cleavage upon Aß exposure. Downstream of PKCδ, aberrant neuronal CCR was triggered by overactivating cyclin-dependent kinase-5 (CDK5) via calpain2-dependent p35 cleavage into p25. Finally, PKCδ and CDK5 also contributed to Aß25-35 induction of p53-upregulated modulator of apoptosis (PUMA) in cortical neurons. Together, we demonstrated that, in the post-mitotic neurons exposed to Aßs, STAT3-dependent PKCδ expression triggers calpain2-mediated p35 cleavage into p25 to overactivate CDK5, thus leading to aberrant CCR, PUMA induction, caspase-3 cleavage, and ultimately apoptosis.

Comparing Viral Vectors and Fate Mapping Approaches for Astrocyte-to-Neuron Reprogramming in the Injured Mouse Cerebral Cortex.

Direct neuronal reprogramming is a promising approach to replace neurons lost due to disease via the conversion of endogenous glia reacting to brain injury into neurons. However, it is essential to demonstrate that the newly generated neurons originate from glial cells and/or show that they are not pre-existing endogenous neurons. Here, we use controls for both requirements while comparing two viral vector systems (Mo-MLVs and AAVs) for the expression of the same neurogenic factor, the phosphorylation-resistant form of Neurogenin2. Our results show that Mo-MLVs targeting proliferating glial cells after traumatic brain injury reliably convert astrocytes into neurons, as assessed by genetic fate mapping of astrocytes. Conversely, expressing the same neurogenic factor in a flexed AAV system results in artefactual labelling of endogenous neurons fatemapped by birthdating in development that are negative for the genetic fate mapping marker induced in astrocytes. These results are further corroborated by chronic live in vivo imaging. Taken together, the phosphorylation-resistant form of Neurogenin2 is more efficient in reprogramming reactive glia into neurons than its wildtype counterpart in vivo using retroviral vectors (Mo-MLVs) targeting proliferating glia. Conversely, AAV-mediated expression generates artefacts and is not sufficient to achieve fate conversion.

Memantine and the Kynurenine Pathway in the Brain: Selective Targeting of Kynurenic Acid in the Rat Cerebral Cortex.

Cytoprotective and neurotoxic kynurenines formed along the kynurenine pathway (KP) were identified as possible therapeutic targets in various neuropsychiatric conditions. Memantine, an adamantane derivative modulating dopamine-, noradrenaline-, serotonin-, and glutamate-mediated neurotransmission is currently considered for therapy in dementia, psychiatric disorders, migraines, or ischemia. Previous studies have revealed that memantine potently stimulates the synthesis of neuroprotective kynurenic acid (KYNA) in vitro via a protein kinase A-dependent mechanism. Here, the effects of acute and prolonged administration of memantine on brain kynurenines and the functional changes in the cerebral KP were assessed in rats using chromatographic and enzymatic methods. Five-day but not single treatment with memantine selectively activated the cortical KP towards neuroprotective KYNA. KYNA increases were accompanied by a moderate decrease in cortical tryptophan (TRP) and L-kynurenine (L-KYN) concentrations without changes in 3-hydroxykynurenine (3-HK) levels. Enzymatic studies revealed that the activity of cortical KYNA biosynthetic enzymes ex vivo was stimulated after prolonged administration of memantine. As memantine does not directly stimulate the activity of KATs' proteins, the higher activity of KATs most probably results from the increased expression of the respective genes. Noteworthy, the concentrations of KYNA, 3-HK, TRP, and L-KYN in the striatum, hippocampus, and cerebellum were not affected. Selective cortical increase in KYNA seems to represent one of the mechanisms underlying the clinical efficacy of memantine. It is tempting to hypothesize that a combination of memantine and drugs could strongly boost cortical KYNA and provide a more effective option for treating cortical pathologies at early stages. Further studies should evaluate this issue in experimental animal models and under clinical scenarios.

Modeling Lewy body disease with SNCA triplication iPSC-derived cortical organoids and identifying therapeutic drugs.

Aggregated α-synuclein (α-SYN) proteins, encoded by the SNCA gene, are hallmarks of Lewy body disease (LBD), affecting multiple brain regions. However, the specific mechanisms underlying α-SYN pathology in cortical neurons, crucial for LBD-associated dementia, remain unclear. Here, we recapitulated α-SYN pathologies in human induced pluripotent stem cells (iPSCs)-derived cortical organoids generated from patients with LBD with SNCA gene triplication. Single-cell RNA sequencing, combined with functional and molecular validation, identified synaptic and mitochondrial dysfunction in excitatory neurons exhibiting high expression of the SNCA gene, aligning with observations in the cortex of autopsy-confirmed LBD human brains. Furthermore, we screened 1280 Food and Drug Administration-approved drugs and identified four candidates (entacapone, tolcapone, phenazopyridine hydrochloride, and zalcitabine) that inhibited α-SYN seeding activity in real-time quaking-induced conversion assays with human brains, reduced α-SYN aggregation, and alleviated mitochondrial dysfunction in SNCA triplication organoids and excitatory neurons. Our findings establish human cortical LBD models and suggest potential therapeutic drugs targeting α-SYN aggregation for LBD.

[Construction and analysis of brain metabolic network in temporal lobe epilepsy patients based on 18F-FDG PET].

The establishment of brain metabolic network is based on 18fluoro-deoxyglucose positron emission computed tomography ( 18F-FDG PET) analysis, which reflect the brain functional network connectivity in normal physiological state or disease state. It is now applied to basic and clinical brain functional network research. In this paper, we constructed a metabolic network for the cerebral cortex firstly according to 18F-FDG PET image data from patients with temporal lobe epilepsy (TLE).Then, a statistical analysis to the network properties of patients with left or right TLE and controls was performed. It is shown that the connectivity of the brain metabolic network is weakened in patients with TLE, the topology of the network is changed and the transmission efficiency of the network is reduced, which means the brain metabolic network connectivity is extensively impaired in patients with TLE. It is confirmed that the brain metabolic network analysis based on 18F-FDG PET can provide a new perspective for the diagnose and therapy of epilepsy by utilizing PET images.

The Role of Nde1 phosphorylation in interkinetic nuclear migration and neural migration during cortical development.

Nde1 is a cytoplasmic dynein regulatory protein with important roles in vertebrate brain development. One noteworthy function is in the nuclear oscillatory behavior in neural progenitor cells, the control and mechanism of which remain poorly understood. Nde1 contains multiple phosphorylation sites for the cell cycle-dependent protein kinase CDK1, though the function of these sites is not well understood. To test their role in brain development, we expressed phosphorylation-state mutant forms of Nde1 in embryonic rat brains using in utero electroporation. We find that Nde1 T215 and T243 phosphomutants block apical interkinetic nuclear migration (INM) and, consequently, mitosis in radial glial progenitor cells. Another Nde1 phosphomutant at T246 also interfered with mitotic entry without affecting INM, suggesting a more direct role for Nde1 T246 in mitotic regulation. We also found that the Nde1 S214F mutation, which is associated with schizophrenia, inhibits Cdk5 phosphorylation at an adjacent residue which causes alterations in neuronal lamination. These results together identify important new roles for Nde1 phosphorylation in neocortical development and disease, and represent the first evidence for Nde1 phosphorylation roles in INM and neuronal lamination.

Increased excitatory connectivity and epileptiform activity in thrombospondin1/2 knockout mice following cortical trauma.

Thrombospondins (TSPs) are astrocyte-secreted extracellular matrix proteins that play key roles as regulators of synaptogenesis in the central nervous system. We previously showed that TSP1/2 are upregulated in the partial neocortical isolation model ("undercut" or "UC" below) of posttraumatic epileptogenesis and may contribute to abnormal axonal sprouting, aberrant synaptogenesis and epileptiform discharges in the UC cortex. These results led to the hypothesis that posttraumatic epileptogeneis would be reduced in TSP1/2 knockout (TSP1/2 KO) mice. To test the hypothesis, we made UC lesions at P21, and subsequent experiments were conducted 14d later at P35. Ex vivo extracellular single or multi-electrode field potential recordings were obtained from layer V in cortical slices at P35 and in vivo video-EEGs of spontaneous epileptiform bursts were recorded to examine the effect of TSP1/2 deletion on epileptogenesis following cortical injury. Immunohistochemical experiments were performed to assess the effect of TSP1/2 KO + UC on the number of putative excitatory synapses and the expression of TSP4 and HEVIN, other astrocytic proteins known to up-regulate excitatory synapse formation. Unexpectedly, our results showed that, compared with WT + UC mice, TSP1/2 KO + UC mice displayed increased epileptiform activity, as indicated by 1) increased incidence and more rapid propagation of evoked and spontaneous epileptiform discharges in UC neocortical slices; 2) increased occurrence of spontaneous epileptiform discharges in vivo. There was an associated increase in the density of VLUT1/PSD95-IR colocalizations (putative excitatory synapses) and significantly upregulated TSP4- and HEVIN-IR in TSP1/2 KO + UC versus WT + UC mice. Results suggest that TSP1/2 deletion plays a potential epileptogenic role following neocortical injury, associated with compensatory upregulation of TSP4 and HEVIN, which may contribute to the increase in the density of excitatory synapses and resulting neural network hyperexcitability.

Cortical neurodegeneration caused by Psen1 mutations is independent of Aß.

Mutations in the PSEN genes are the major cause of familial Alzheimer's disease, and presenilin (PS) is the catalytic subunit of γ-secretase, which cleaves type I transmembrane proteins, including the amyloid precursor protein (APP) to release Aß peptides. While PS plays an essential role in the protection of neuronal survival, PSEN mutations also increase the ratio of Aß42/Aß40. Thus, it remains unresolved whether PSEN mutations cause AD via a loss of its essential function or increases of Aß42/Aß40. Here, we test whether the knockin (KI) allele of Psen1 L435F, the most severe FAD mutation located closest to the active site of γ-secretase, causes age-dependent cortical neurodegeneration independent of Aß by crossing various Psen mutant mice to the App-null background. We report that removing Aß completely through APP deficiency has no impact on the age-dependent neurodegeneration in Psen mutant mice, as shown by the absence of effects on the reduced cortical volume and decreases of cortical neurons at the ages of 12 and 18 mo. The L435F KI allele increases Aß42/Aß40 in the cerebral cortex while decreasing de novo production and steady-state levels of Aß42 and Aß40 in the presence of APP. Furthermore, APP deficiency does not alleviate elevated apoptotic cell death in the cerebral cortex of Psen mutant mice at the ages of 2, 12, and 18 mo, nor does it affect the progressive microgliosis in these mice. Our findings demonstrate that Psen1 mutations cause age-dependent neurodegeneration independent of Aß, providing further support for a loss-of-function pathogenic mechanism underlying PSEN mutations.

Loss of Cldn5 -and increase in Irf7-in the hippocampus and cerebral cortex of diabetic mice at the early symptomatic stage.

Analyzing changes in gene expression within specific brain regions of individuals with Type 2 Diabetes (T2DM) who do not exhibit significant cognitive deficits can yield valuable insights into the mechanisms underlying the progression towards a more severe phenotype. In this study, transcriptomic analysis of the cortex and hippocampus of mice with long-term T2DM revealed alterations in the expression of 28 genes in the cerebral cortex and 15 genes in the hippocampus. Among these genes, six displayed consistent changes in both the cortex and hippocampus: Interferon regulatory factor 7 (Irf7), Hypoxia-inducible factor 3 alpha (Hif-3α), period circadian clock 2 (Per2), xanthine dehydrogenase (Xdh), and Transforming growth factor ß-stimulated clone 22/TSC22 (Tsc22d3) were upregulated, while Claudin-5 (Cldn5) was downregulated. Confirmation of these changes was achieved through RT-qPCR. At the protein level, CLDN5 and IRF7 exhibited similar alterations, with CLDN5 being downregulated and IRF7 being upregulated. In addition, the hippocampus and cortex of the T2DM mice showed decreased levels of IκBα, implying the involvement of NF-κB pathways as well. Taken together, these results suggest that the weakening of the blood-brain barrier and an abnormal inflammatory response via the Interferon 1 and NF-κB pathways underlie cognitive impairment in individuals with long-standing T2DM.

Cross-site reproducibility of human cortical organoids reveals consistent cell type composition and architecture.

While guided human cortical organoid (hCO) protocols reproducibly generate cortical cell types at one site, variability in hCO phenotypes across sites using a harmonized protocol has not yet been evaluated. To determine the cross-site reproducibility of hCO differentiation, three independent research groups assayed hCOs in multiple differentiation replicates from one induced pluripotent stem cell (iPSC) line using a harmonized miniaturized spinning bioreactor protocol across 3 months. hCOs were mostly cortical progenitor and neuronal cell types in reproducible proportions that were consistently organized in cortical wall-like buds. Cross-site differences were detected in hCO size and expression of metabolism and cellular stress genes. Variability in hCO phenotypes correlated with stem cell gene expression prior to differentiation and technical factors associated with seeding, suggesting iPSC quality and treatment are important for differentiation outcomes. Cross-site reproducibility of hCO cell type proportions and organization encourages future prospective meta-analytic studies modeling neurodevelopmental disorders in hCOs.