Lewy bodies, the defining pathological feature of Parkinson's disease, are intraneuronal inclusions enriched in aggregated alpha-synuclein (αSyn). We used correlative light and electron microscopy to selectively investigate phosphorylated αSyn (αSynpS129)-positive inclusions in the substantia nigra of end-stage postmortem Parkinson's disease brain. Here we show that somatic αSynpS129 inclusions in nigral dopaminergic neurons are consistently fibrillar, whereas the membranous-type inclusions are restricted to neuritic processes. These neuritic inclusions displayed marked ultrastructural heterogeneity, ranging from predominantly membranous to mixed membranous-fibrillar forms. The selective targeting of defined inclusions enabled detailed structural characterization of Lewy pathology, rather than quantitative or disease-stage comparisons. Our findings highlight clear ultrastructural differences between somatic and neuritic αSynpS129 pathology and demonstrate the structural complexity and heterogeneity of Lewy pathology in human Parkinson's disease brain.
Methotrexate (MTX) is a well-known medication for the treatment of different cancer types and autoimmune diseases. The current study target was to measure the capability of human placental extract (HPE) to ameliorate the nephrotoxicity induced by MTX in male albino rats. In the present study, rats were distributed into four groups; a control group (each rat was intraperitoneally injected with 0.5 ml of 0.9% NaCl daily for five days), HPE-treated group (HPE, 10.08 mg/Kg b.w/day, was subcutaneously injected for two weeks), MTX-treated group (MTX, 5 mg/Kg b.w/day, was intraperitoneally injected for five days) and MTX and HPE-treated group (Both MTX and HPE were injected to rats at the same time with the same doses, duration and injection routes in MTX and HPE groups). During the experimental period, clinical observations and body weights of rats were recorded. Rats were dissected after twenty-four hours from the last dose of each group, blood samples were collected for relative blood viscosity measurements and kidneys were also collected for biochemical, ultrastructural and dielectric properties (dielectric constant, dielectric loss and conductivity) investigations. MTX treatment resulted in a highly significant decrease in rat body weights, a highly significant decrease in glutathione (GSH) level and catalase (CAT) activity, a significant decrease in superoxide dismutase (SOD) and glutathione peroxidase (GPx) activities and a highly significant increase in the malondialdehyde (MDA) level and relative blood viscosity compared to the control group. Besides, obvious ultrastructural changes and pronounced decrease in the dielectric properties of kidney tissues were noticed. While HPE treatment with MTX improved body weight, biochemical, ultrastructural and biophysical changes comparing to the MTX group. Human placental extract can reduce MTX-induced nephrotoxicity in rats through boosting oxidative stress/anti-oxidant balance as it is rich with essential elements.
Inflammation within the central nervous system (CNS) plays a pivotal role in neuronal survival and degeneration. Lipopolysaccharide (LPS) is a widely used agent for inducing systemic and localized inflammation in mammals, providing a model for studying neurodegenerative processes. While previous research has documented neuronal loss due to LPS-induced neurodegeneration, the progressive morphological changes in neurons remain insufficiently characterized, particularly in retinal tissues. This study addresses this gap by establishing acute and chronic retinal inflammation models in mice using single and repeated intraperitoneal LPS injections. Through ultrastructural analyses using electron microscopy, we observed significant pathological changes in retinal neurons, glial cells, and blood-retinal barrier (BRB) components. Acute LPS exposure resulted in lipid droplet accumulation and membrane disruption in retinal pigment epithelium (RPE), as well as abnormal neuronal and vascular ultrastructures. Chronic LPS exposure amplified these effects, causing more pronounced damage to neurons and exacerbating BRB dysfunction. This study provides, for the first time, detailed ultrastructural insights into LPS-induced acute and chronic retinal inflammation. These findings advance our understanding of retinal pathology in inflammatory conditions and support the development of novel therapeutic strategies for retinal and CNS neurodegenerative diseases.
Astrocytes are key regulators of neuronal, metabolic, and vascular homeostasis, yet their morphological diversity and involvement in alcohol-related brain pathology remain incompletely characterized. In this study, we investigated astrocytic morphology in the human striatum of control individuals and subjects with short- and long-term alcohol exposure using immunohistochemistry combined with Sholl-based morphometric analysis, and ultrastructural assessment. GFAP immunohistochemistry was used to identify astrocytes, assess their morphology, and manually quantify GFAP+ cells in gray and white matter, followed by Sholl-based morphometric analysis to characterize astrocytic branching architecture and spatial organization. The number of GFAP+ astrocytes differed between tissue compartments, with a significant increase in white matter in alcohol-exposed individuals and no detectable change in gray matter. Morphometric analysis revealed pronounced astrocytic heterogeneity across all study groups. Sholl-derived metrics supported the distinction of six recurrent astrocytic morphometric profiles in the human striatum, distinguished by soma size, branching complexity, process length, and cell territory size. These profiles were present across gray and white matter, indicating intrinsic astrocytic structural diversity. Ultrastructural analysis further revealed alcohol-associated alterations at the astrocyte-vascular interface, including swelling of perivascular astrocytic endfeet, accumulation of intermediate filaments, and focal reductions in vascular wall coverage. Together, these findings demonstrate substantial astrocytic structural diversity in the human striatum accompanied by alcohol-related gliovascular remodeling.
Autism spectrum disorder (ASD) is a neurodevelopmental disorder that typically appears in early childhood, and there is a link between ASD and zinc deficiency. Neither the potential protective effects of the trace element zinc on the cerebellum against ultrastructural damage in animal models of ASD, nor its ability to protect against the modulation of the oxidative stress/gliosis/neuronal loss axis implicated in autism, have been previously investigated. Young rats were injected with the short-chain fatty acid produced by gut bacteria that is known to induce ASD, propionic acid (PPA) (500 mg/kg) for 5 days (model group) and the protection group was treated with 2 mg/kg zinc sulfate for two weeks following PPA injections. Cerebellum damage and behavioral impairment developed in the model group of rats as demonstrated by profound cerebellar ultrastructural alterations such as degeneration of Purkinje neurons and fragmented endoplasmic reticulum, as well as anxiety-like behavior and social interaction deficits. PPA also significantly (p < .05) modulated the cerebellar protein levels of the gliosis biomarker GFAP and the neuronal loss biomarker calbindin D28K, as well as the cerebellar tissue levels of zinc, glutathione, and the oxidative stress biomarker malondialdehyde (MDA). All these parameters were significantly (p < .05) protected by zinc treatment. Additionally, we observed a significant (p < .001) correlation between the degree of Purkinje cell damage and these parameters associated with autism. In conclusion, PPA-induced autistic behaviors in rats are associated with alteration to the cerebellar ultrastructure, modulation of the oxidative stress/gliosis/neuronal loss axis of ASD, and a decline in zinc and glutathione, which were protected by zinc treatment.
Desmin-related cardiomyopathy is a rare disorder caused by pathogenic variants in desmin and associated protein genes. We aimed to describe the clinical, histopathological, immunohistochemical, ultrastructural, and molecular features in a pediatric cohort. This retrospective descriptive study reviewed pediatric cases diagnosed over a 30-year period. Clinical data were extracted from electronic medical records. Archived histologic slides (H&E, desmin immunostains, and special stains) and electron photomicrographs were re-evaluated. Key features were documented and summarized. Four pediatric patients (3 males and 1 female) presented between ages 2 and 14 years with restrictive cardiomyopathy and conduction abnormalities. Endomyocardial biopsies and/or explanted heart specimens showed eosinophilic cytoplasmic inclusions that were desmin-immunoreactive, PAS-negative, and highlighted by toluidine blue. Electron microscopy revealed dense cytoplasmic granulofilamentous aggregates that were frequently continuous with the sarcoplasmic membrane, Z-bands, and intercalated discs. Two patients harbored the same pathogenic variant: DES c.1360T>C (p.Arg454Trp). Desmin-related cardiomyopathy should be considered in any child presenting with restrictive cardiomyopathy and/or rhythm disturbances. Distinctive light microscopic and ultrastructural features can aid in confirming the diagnosis. Certain pathogenic variants are increasingly linked to more severe phenotypes, highlighting the importance of genetic evaluation and its implications for family counseling.
Sperm cryopreservation is a common procedure in assisted reproduction (ART) laboratories. Although generally regarded as an efficient and safe technique, a decrease in the fertilization potential has been described. Hence, a detailed ultrastructural analysis of human spermatozoa might provide important insights into cellular alterations occurring during cryopreservation. To address this question, the ultrastructure of human spermatozoa was analysed, before and after cryopreservation, for the time periods of one month (1-Mo) and seven months (7-Mo), using paired, repeated measures. Semen samples were cryopreserved with a rapid freezing protocol, using a glycerol- and raffinose-containing cryopreservation medium, allocated into cryo-straws and stored in liquid nitrogen. Ultrastructural results indicate a significant increase in anomalous sperm, with changes in the head membrane and acrosomal vesicle, after both cryopreservation periods (fresh:26.8% vs. 1-Mo:52.2%, 7-Mo:57.4%, P < 0.001, partial η²=0.849), suggesting a concurrent cryo-damage to these structures. Concerning the sperm head membrane, a significant increase in total detachment was observed, after 1 and 7 months of cryopreservation (fresh:9.3%, 1-Mo:26.3%, 7-Mo:34.5%, P < 0.001, partial η²=0.727); for the acrosomal vesicle, a significant increase was observed in apical detachment after 1 month (fresh:11.4%, 1-Mo:22.9%, 7-Mo:17.1%, P = 0.006, partial η²=0.433) and in total detachment after 7 months (fresh:24.9%, 1-Mo:31.8%, 7-Mo:43.6%, P < 0.001, partial η²=0.636). In conclusion, our results indicate that both the sperm head membrane and the acrosomal vesicle are affected by cryopreservation, with both potentially decreasing the functional competence of the spermatozoa. These data adds information about cryopreservation, highlighting the importance of detailed inspection for sperm selection during ART. (244/250).
Cerebral toxoplasmosis is a common opportunistic parasitic infection of the CNS caused by the Toxoplasma gondii parasite. Host immunosuppression can affect disease outcomes. To explore the changes in the cerebral cortical ultrastructure accompanying the infection in different immune-altered models and to find an effective treatment against the infection, we tested the possible therapeutic effect of clofazimine (CFZ) (the FDA-approved antimycobacterial drug) against the infection using 60 male CD1 Swiss Albino mice divided into 6 groups: 3 dexamethasone (DEX)- treated groups (DEX-only, DEX-infected, and DEX-infected-treated), and 3 streptozotocin (STZ)-induced type 1 diabetic groups (STZ-only, STZ-infected, STZ-infected-treated). The worst ultrastructural changes were observed in the diabetic and diabetic-infected groups, characterized by a significant increase in neuronal apoptotic and necrotic nuclei (P < 0.05) and changes in the numbers and structure of glial cells compared to the DEX and DEX-infected groups. CFZ (at a dose of 10 mg/kg/day for 3 days starting on 45th day post infection) significantly improved cortical neuronal ultrastructural changes in both models (P < 0.05), reduced microglial numbers, increased astrocyte numbers, and restored brain capillary integrity and axonal growth, in addition to significantly reducing mature cyst numbers in both models (P < 0.05). However, the drug didn't reduce the number of atrophic and necrotic cysts in the infected-treated groups. So, in our study, CFZ showed preclinical promise in treating experimental cerebral toxoplasmosis and reducing the parasitic cyst burden, highlighting the adverse impact of the host's altered immune status on brain tissue and the course of the infection, especially in diabetes.
Adult neural stem cells (NSCs) are located in the sub-granular zone (SGZ) of the dentate gyrus (DG) of the hippocampus, one of the prominent regions of neurogenesis in the adult brain. Activation of endogenous stem cells may provide a therapeutic approach in demyelinating diseases. Due to their minuscule size, nanoparticles (AuNPs) can cross the blood-brain barrier. To evaluate the effect of AuNPs on adult NSCs and the progression of demyelination in the hippocampus in a model of demyelination, thirty-two adult mice were divided into: control group I, AuNPs group II (10 mg/kg BW/day), cuprizone (CPZ) group III (0.2% orally for 8 weeks to induce demyelination), and CPZ & AuNPs group IV. Hippocampus was prepared for light microscopy, nestin immunohistochemical staining (NSCs' immune marker), and electron microscopy. In groups I and II, spindle-shaped adult NSCs in the SGZ of DG showed a positive nestin immunoreaction. In group III, most of the nerves were demyelinated. Pyramidal and granular neurons showed degenerative changes. Nestin-immune-positive cells were significantly decreased. In group IV, preservation of the myelin, less degenerative changes, and a significant increase in nestin-positive cells were detected. In conclusion, AuNPs activated adult NSCs in the mouse hippocampus, partially ameliorated degenerative changes, and enhanced partial remyelination in the CPZ demyelination model.
C3 glomerulopathy (C3G) is a rare type of glomerular disease characterized by predominant deposits of C3 complement. Although dominant C3 immunofluorescent (IF) staining has become well known as a diagnostic criterion, the findings of electron microscopy (EM) are not well specified. The goal of this study was to scrutinize the characteristic features of C3 deposits on EM and to correlate with C3 IF staining patterns. We examined the EM images of a cohort of 24 cases of C3G (22 C3 dominant glomerulonephritis [C3GN] and 2 dense deposit disease [DDD]) to determine their characteristic features when compared to a cohort of patients with immune complex-mediated glomerulonephritis. In our patients with C3G, the C3 deposits were present in all three glomerular compartments (mesangial, subendothelial, and subepithelial spaces) and they had the following features: (1) Smear pattern of C3 deposits along glomerular basement membranes (GBM) and subepithelial spaces; (2) C3 deposits were mostly lighter in gray colors as opposed to the dark black appearance of immune complex deposits (ICD); (3) C3 aggregates revealed smooth contours with a homogeneous fine granular appearance when compared to the humpy and bumpy appearance of ICD, and (4) C3 deposits rarely showed either retraction artifacts around the deposits or vacuolization within the GBM or mesangial areas. In our cohort, C3 deposits exhibit reproducible EM features that are well correlated with clinical data and dominant C3 staining by IF and are different from ICD upon securitized review.
Ischemic stroke triggers severe disruption of the neurovascular unit (NVU), with pathology evident at the ultrastructural level. Hypoxia-inducible factor-1α (HIF-1α) activators such as Prolyl hydroxylase inhibitors e.g., dimethyloxalylglycine (DMOG) and histone deacetylase inhibitors (e.g., Apicidin) are promising therapeutic candidates, but their specific impact on subcellular architecture remains poorly characterized. Endothelin-1 (ET-1) model was used to induce ischemic stroke. Animals were treated intraperitoneally with either DMOG (40 mg/kg), Apicidin (1 mg/kg), or vehicle at reperfusion onset. After 7 days, behavioral analysis was conducted, Cytochrome-C immunohistochemistry and cortical tissue was processed for transmission electron microscopy (TEM) to assess ultrastructural integrity. Severe ultrastructural pathology, such as pyknotic neuronal soma, broken axons, enlarged mitochondria, synaptic disintegration, and reactive microglia and oligodendrocytes, was seen in model animals. With relatively slight improvements in synaptic and glial function and ongoing somatic, axonal, and mitochondrial damage, DMOG therapy offered little protection for neurons. With intact soma and axons, decreased mitochondrial swelling, increased synaptic vesicle density, and enhanced oligodendrocyte and microglial morphology, Apicidin therapy, on the other hand, showed strong neuronal preservation. While Apicidin is more effective at maintaining the integrity of neurons and synapses, DMOG mainly stabilizes the vascular compartment. These results imply that better neuroprotection may be provided by combination therapy that targets both vascular and neuronal components.
Natural products have historically been integral to pharmacotherapy, attributed to their remarkable structural diversity and evolutionary refinement for biological interactions. Nonetheless, traditional natural product-based drug discovery has faced challenges such as mechanistic ambiguity, scalability limitations, and inadequate translational predictability. Concurrently, reductionist single-target approaches have been insufficient for addressing complex diseases characterized by network-level dysregulations. Recent advancements in analytical chemistry, genomics, and data-driven methodologies have rejuvenated natural product research by facilitating rapid structural elucidation, systematic exploration of biosynthetic diversity, and rational prioritization of bioactive compounds. Notably, many natural products exhibit multitarget effects that necessitate interpretation beyond isolated molecular interactions. Systems pharmacology offers a quantitative framework to analyze such network-level perturbations by integrating omics data, computational modeling, and experimental validation. However, molecular and computational predictions alone do not suffice to establish therapeutic relevance. Experimental pathology, encompassing histopathology, immunohistochemistry, spatial analysis, and ultrastructural evaluation, remains essential for validating efficacy and safety at tissue and organ levels. This review synthesizes technological innovation, systems pharmacology, and pathological validation to reposition natural products as mechanistically grounded and translationally robust resources for contemporary drug discovery.
Brazilian porcupinepox virus (BPoPV) has been associated with fatal infections in free-ranging Brazilian porcupines (Coendou longicaudatus), raising concerns regarding its epidemiology and conservation impact. This study comprehensively investigates BPoPV infection, pathology, and mortality in a free-living population from the Federal District, Brazil. Between 2021 and 2024, 30 porcupines submitted for postmortem examination were evaluated using gross and histopathology, PCR, epidermal morphometric analysis, and transmission electron microscopy. BPoPV infection was detected in 70% of animals (21/30), including all ten fatal cases and eleven individuals that died from unrelated traumatic injuries. Six PCR-positive animals without gross lesions showed significant epidermal thickening on microscopy, revealing a previously unrecognized subclinical infection. Fatal cases consistently exhibited severe proliferative and degenerative cutaneous lesions, intracytoplasmic inclusions, marked dermal inflammation, and visceral involvement, particularly interstitial pneumonia, esophageal pustules, and lymphoid depletion, suggesting systemic dissemination. Ultrastructural analysis confirmed abundant poxvirus-like particles within keratinocytes. Traumatic injuries, mainly dog attacks and electrocution, represented the most frequent noninfectious cause of death. These findings demonstrate a high frequency of clinical and subclinical BPoPV infection, identify the virus as a major infectious threat, and provide essential baseline data to support wildlife disease surveillance and conservation strategies for C. longicaudatus.
The blood-brain barrier (BBB) plays a critical role in maintaining brain homeostasis by tightly regulating molecular transport. However, its integrity is often compromised with aging and in neurodegenerative diseases, contributing to disease pathology. Studying the biological consequences of BBB dysfunction independent of concomitant pathology remains challenging, largely due to the absence of reliable and inducible animal models that avoid unintended side effects such as osmotic effects, neuroinflammation, or vascular damage. In this study, we evaluated the use of Power Doppler ultrasound (PDUS) combined with microbubbles to induce widespread, bilateral BBB opening in the mouse brain. Mice received intravenous infusions of SonoMAC microbubbles during transcranial PDUS application. BBB permeability was assessed via Evans Blue dye extravasation and immunofluorescence analysis of extravasated immunoglobulins. Vessel integrity was evaluated at the ultrastructural level using transmission electron microscopy (TEM). PDUS combined with microbubbles successfully induced widespread BBB opening, as evidenced by diffuse Evans Blue staining and immunoglobulin extravasation in coronal sections. Immunoglobulin leakage was detected in all analyzed brain regions, with lower levels in white matter, likely reflecting its lower vascular density. Leakage appeared to primarily originate from capillaries while TEM analysis revealed no overt vascular damage. These findings support PDUS with microbubbles as a non-destructive, reproducible method to model widespread BBB dysfunction. This approach offers an in vivo platform to study BBB-related pathophysiological processes such as impaired clearance, protein aggregation, and neurotoxicity, as well as for investigation of therapeutic delivery to the brain parenchyma.
Statins are a class of drugs commonly prescribed to lower cholesterol and low-density lipoprotein (LDL) cholesterol levels, as well as to reduce morbidity and mortality associated with cardiovascular illnesses. However, statins are associated with potential side effects, such as muscle pain, liver problems, and neurological or psychiatric disturbances. Therefore, a protective medication is required to mitigate the harmful effects of statins. Thymoquinone (THQ), found in Nigella sativa, has several medicinal benefits and protective effects. The current study aimed to investigate the effects of simvastatin on cerebellar and hepatic tissues, focusing on cellular apoptosis, using light and electron microscopy, morphometric analysis, and immunohistochemical techniques. Additionally, the study evaluates whether thymoquinone can protect against simvastatin-induced histological and ultrastructural damage in hepatic and cerebellar tissues. Thirty adult male Wistar albino rats were divided into three equal groups. Group I was the control group (Ctrl group). Group II (statin group) received simvastatin (20 mg/kg/day) orally for 8 weeks. Group III (statin + THQ group) received concomitant treatment with simvastatin (20 mg/kg/day) and thymoquinone (10 mg/kg/day) for 8 weeks. Tissue specimens were taken from the liver and cerebellum of all animal groups and processed for light and electron microscopic examination. An immunohistochemical investigation for caspase-3 was performed. The molecular and granular layer thickness, number of Purkinje cells, central vein diameter, hepatocyte nuclear diameter, and optical density of anti-caspase-3-positive cells were measured in both the cerebellum and the liver in all experimental groups. Results showed that cerebellum from simvastatin-treated animals showed perinuclear halos in cells within the molecular layer. Purkinje cells appeared disorganized, irregularly arranged, shrunken, with irregular outlines, hyperchromatic nuclei, and vacuolated cytoplasm. Moreover, the granular cell layer appeared shrunken with irregular hyperchromatic nuclei. In addition, decreased thickness of both the molecular and granular layers, as well as a reduction in the number and diameter of Purkinje cells, was observed and confirmed by morphometric analysis. Liver tissue showed shrunken hepatocytes, widened blood sinusoids, widened central veins, and inflammatory cell infiltration. Furthermore, strong positive expression of caspase-3 was observed in both the cerebellum and liver. However, all these histological, ultrastructural, and morphometric changes were markedly decreased after the addition of thymoquinone. It can be concluded that simvastatin induces significant degeneration in both the cerebellar cortex and liver tissue, primarily due to increased oxidative stress and reduced antioxidant protection. Thymoquinone exerts protective effects against this oxidative damage in both organs and alleviates degenerative changes, suggesting that it may be recommended for hypercholesterolemic patients on statins.
Alzheimer's disease (AD) lacks effective early-stage non-pharmacological interventions capable of simultaneously targeting neurotrophic signaling and neurofunctional decline. This study examined the early-stage effects of tactile stimulation (TS), aerobic exercise (Ex), and their combination (TS + Ex) on behavioral, molecular, and histopathological outcomes in 5xFAD mouse model of AD. Twenty-eight male transgenic 5xFAD mice (7-8 weeks old) were randomly assigned to four groups: control (n = 7), TS (n = 7), Ex (n = 7), and TS + Ex (n = 7). Interventions were applied for eight weeks, 5 days per week, with daily 30-minute sessions. Behavioral assessments were conducted, and biochemical analyses measured brain-derived neurotrophic factor (BDNF), tropomyosin receptor kinase B (TrkB), fibroblast growth factor-2 (FGF-2), irisin, lactate, total antioxidant status (TAS), total oxidant status (TOS), and myeloperoxidase (MPO) levels in brain, muscle, skin, and serum tissues. Histological, immunofluorescent, and ultrastructural evaluations were performed in the hippocampus. Compared to control, the TS + Ex group showed significant improvements in motor performance, neurotrophic factor levels, irisin and lactate concentrations, and antioxidant capacity in 5xFAD mice (p = 0.05-<0.001, partial η²=0.498-0.902). Additionally, increased doublecortin expression (p = 0.032, partial η²=0.571) and improved histopathological architecture were observed. Aerobic exercise alone improved motor function, anxiety-related behaviors, modulated several biochemical and histological outcomes compared with the control (p = 0.05 - 0.004, partial η²=0.332-0.498). TS alone produced limited effects, with notable increases only in hippocampal BDNF (p < 0.001, partial η²=0.712) and lactate levels (p = 0.015, partial η²=0.605) compared with the control group. The combination of TS and Ex may exert synergistic neuroprotective effects, suggesting that multimodal non-pharmacological strategies could represent promising early-stage interventions for mitigating AD-related pathology.
In Alzheimer's disease (AD), endogenous tau undergoes a pathogenic transition to form paired helical filaments (PHFs), but the cellular mechanisms driving this process have been elusive. Here, we identify the neuron-specific plasma membrane proteasome ('neuroproteasome') as a critical determinant of tau proteostasis. Selective inhibition of neuroproteasome function rapidly triggers the de novo formation of endogenous, sarkosyl-insoluble tau PHFs in primary neurons and mouse brain, which share key biochemical and ultrastructural features with PHFs from human AD brains. The APOE gene has three isoforms (E2, E3 and E4), with APOE4 being the largest genetic risk factor for AD. Neuroproteasome abundance at the plasma membrane is differentially modulated by ApoE isoforms (E2 > E3 > E4) and declines with age. ApoE4 neurons accumulate tau aggregates following modest neuroproteasome disruption, whereas ApoE2 neurons remain resistant. Our findings delineate a neuron-specific mechanism linking genetic and age-related risk factors to the formation of AD-relevant tau pathology, and position neuroproteasome function as a potential target to preserve proteostasis.
Cerebral small vessel disease is a leading cause of cognitive decline and stroke in the elderly, with cerebral microbleeds (CMBs) serving as a key imaging biomarker. Despite their clinical significance, the pathophysiological mechanisms underlying cerebral small vessel disease remain poorly understood owing to a lack of appropriate animal models. We performed targeted deletion of Col4a1 in brain microvessels of adult mice using brain endothelium-specific adeno-associated virus (AAV)-BR1 vectors with clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9). Eight-week-old Cas9 transgenic mice received retro-orbital injections of AAV-BR1 containing single guide RNA (sgRNA) targeting Col4a1 or control Rosa26 sequences. Animals underwent longitudinal behavioural testing, including novel object recognition, Y-maze and rotarod tests, over 6 months. Brain pathology was assessed using T2*-weighted MRI, histological analysis and electron microscopy. For human studies, we analysed MRI and genomic data from 836 participants from the BICWALZS biobank, examining associations between genetic variants and CMB burden using linear regression and χ2 analyses. T2*-weighted MRI revealed numerous CMBs with distributions remarkably similar to human CMBs, appearing within 3 months post-injection. CMB burden increased progressively over 6 months in a dose-dependent manner. Behaviourally, mice exhibited progressive cognitive decline and motor incoordination. Histological examinations revealed haemosiderin deposits corresponding to MRI-detected CMBs, without macroscopic intracerebral haemorrhage or white matter changes. Ultrastructural analysis demonstrated significant basement membrane thinning in Col4a1-depleted microvessels. CMB accumulation was associated with widespread astrocytic reactivity extending beyond microbleed sites, whereas microglial activation remained localized. In human subjects, we identified significant associations between four genetic variants of TIMP2, an endogenous inhibitor of the matrix-degrading enzyme MMP2 and CMB burden, with odds ratios of 1.50-1.96 for increased microbleed susceptibility. This work provides the first animal model demonstrating that selective disruption of collagen IV in adult brain microvessels is sufficient to generate CMBs with high penetrance and dose-dependent tunability. Our findings establish that basement membrane integrity is critical for preventing microbleed formation and suggest that dysregulated collagen IV homeostasis underlies sporadic human CMB development.
Chronic intermittent hypoxia (CIH), the cardinal pathophysiological feature of obstructive sleep apnea, is increasingly recognized as an important modifier of metabolic dysfunction-associated steatotic liver disease (MASLD), but the underlying mechanisms remain incompletely understood. In this study, male C57BL/6 mice were fed a standard diet or a high-fat diet (HFD) and exposed to normoxia or CIH for 8 weeks. Histological, ultrastructural, biochemical, transcriptomic, proteomic, and metabolomic analyses were integrated to characterize hepatic alterations induced by CIH under metabolic stress. CIH markedly aggravated HFD-induced liver injury, as evidenced by increased body fat, hepatomegaly, serum transaminases, steatosis, mitochondrial ultrastructural alterations, and inflammatory infiltration. Mechanistically, CIH promoted hepatic lipid metabolic reprogramming by suppressing the PPARα/CPT1A fatty acid β-oxidation axis while enhancing the SREBP-1c/FASN/PLIN2 lipogenic pathway, impaired the Nrf2/HO-1/SLC7A11/GPX4 antioxidant defense system, increased lipid peroxidation and iron accumulation, and activated NF-κB/NLRP3 signaling. These findings support a multifactorial model in which CIH functions as an additional hypoxic stressor that exacerbates HFD-induced MASLD-like liver injury through coordinated metabolic, oxidative, and inflammatory dysregulation.
Pathogenic (P) variants in COL4A3/A4/A5 genes are known to cause thin glomerular basement membrane (GBM) or Alport-related kidney disease; however, the exact diagnostic yield of genetic testing remains unknown. In this retrospective genotype-phenotype correlation study, we screened the patient populations of 2 major US medical centers for individuals who underwent kidney biopsy, and who had documented genetic testing results on a large 385-kidney disease gene panel. We correlated GBM thickness, estimated glomerular filtration rate, proteinuria, and hematuria with genotyping results. We identified 115 patients with coexisting histopathology and genetic testing data, of which 49 had ultrastructural abnormalities of the GBM. Among those 49 cases, 9 had a heterozygous pathogenic or likely pathogenic (P/LP) variant in one of the COL4A genes, and 9 additional patients had COL4A variants of uncertain significance (VUS). Thirty-one patients with thin GBM were COL4A3/A4/A5 wildtype. One patient with a P variant in COL4A4 had no GBM abnormalities. Three COL4A VUS were upgraded to P/LP through experimental testing. Presence of P/LP variants in COL4A genes correlated with GBM thickness, but not with other clinical parameters. Among 31 thin GBM cases with no COL4A variant, we found variants in steroid-resistant nephrotic syndrome-, congenital anomalies of the kidneys and urinary tract (CAKUT)-, and autosomal dominant tubulointerstitial kidney disease (ADTKD) genes characterized as P/LP or as "high-risk VUS." Immune-mediated glomerular injury was as frequent in biopsy specimens with thin GBM as with normal GBM. In our study, almost two-thirds of patients with thin GBM have no variant in COL4A3/A4/A5 genes. Our data suggest that genetic testing may not obviate the need for kidney biopsy.