The Therapeutic Potential of Zinc-Alpha2-Glycoprotein (AZGP1) in Fibrotic Kidney Disease

Chronic kidney disease (CKD) is characterized by a long-term loss of kidney function and, in most cases, by progressive fibrosis. Zinc-alpha2-glycoprotein (AZGP1) is a secreted protein, which is expressed in many different tissues and has been associated with a variety of functions. In a previous study, we have shown in cell culture and in AZGP1 deficient mice that AZGP1 has protective anti-fibrotic effects. In the present study, we tested the therapeutic potential of an experimental increase in AZGP1 using two different strategies. (1) C57Bl/6J mice were treated systemically with recombinant AZGP1, and (2) a transgenic mouse strain was generated to overexpress AZGP1 conditionally in proximal tubular cells. Mice underwent unilateral uretic obstruction as a pro-fibrotic kidney stress model, and kidneys were examined after 14 days. Recombinant AZGP1 treatment was accompanied by better preservation of tubular integrity, reduced collagen deposition, and lower expression of injury and fibrosis markers. Weaker but similar tendencies were observed in transgenic AZGP1 overexpressing mice. Higher AZGP1 levels led to a significant reduction in stress-induced accumulation of tubular lipid droplets, which was paralleled by improved expression of key players in lipid metabolism and fatty acid oxidation. Together these data show beneficial effects of elevated AZGP1 levels in fibrotic kidney disease and highlight a novel link to tubular cell lipid metabolism, which might open up new opportunities for CKD treatment.

Fibronectin Extra Domain A (FN-EDA) Causes Glaucomatous Trabecular Meshwork, Retina, and Optic Nerve Damage in Mice

Background: Elevated intraocular pressure (IOP) is a major risk factor for the development and progression of primary open angle glaucoma and is due to trabecular meshwork (TM) damage. Here, we investigate the role of an endogenous Toll-like receptor 4 (TLR4) ligand, FN-EDA, in the development of glaucoma utilizing a transgenic mouse strain (B6.EDA +/+ ) that constitutively expresses only FN containing the EDA isoform. Methods: Eyes from C57BL6/J (wild-type), B6.EDA +/+ (constitutively active EDA), B6.EDA -/- (EDA null) mice were processed for electron microscopy and consecutive images of the entire length of the TM and Schlemm’s canal (SC) from anterior to posterior were collected and montaged into a single image. ECM accumulation, basement membrane length, and size and number of giant vacuoles were quantified by ImageJ analysis. Tlr4 and Iba1 expression in the TM and ONH cells was conducted using RNAscope in situ hybridization and immunohistochemistry protocols. IOP was measured using a rebound tonometer, ON damage assessed by PPD stain, and RGC loss quantified in RBPMS labeled retina flat mounts. Results: Ultrastructure analyses show the TM of B6.EDA +/+ mice have significantly increased accumulation of ECM between TM beams with few empty spaces compared to C57BL/6J mice (P<0.05). SC basement membrane is thicker and more continuous in B6.EDA +/+ mice compared to C57BL/6J. No significant structural differences are detected in the TM of EDA null mice. Tlr4 and Iba1 expression is increased in the TM of B6.EDA +/+ mice compared to C57BL/6J eyes (p<0.05). IOP is significantly higher in B6.EDA +/+ mice compared to C57BL/6J eyes (p<0.001), and significant ON damage (p<0.001) and RGC loss (p<0.05) detected at one year of age. Tlr4 mRNA is expressed in mouse ONH cells, and is present in ganglion cell axons, microglia, and astrocytes. There is a significant increase in the area occupied by Iba-1 positive microglia cells in the ONH of B6.EDA +/+ mice compared to C57BL/6J control eyes (p<0.01). Conclusions: B6.EDA +/+ mice have increased ECM accumulation in the TM, elevated IOP, enhanced proinflammatory changes in the ONH, loss of RGCs, and ONH damage. These data suggest B6.EDA +/+ mice recapitulate many aspects of glaucomatous damage.

Low striatal T3 is implicated in inattention and memory impairment in an ADHD mouse model overexpressing thyroid hormone-responsive protein

Attention-deficit/hyperactivity disorder (ADHD) is a neurodevelopmental disorder, potentially with a biological basis; however, its exact cause remains unknown. Thyroid hormone (TH) abnormalities are more prevalent in patients with ADHD than in the general population, indicating a shared pathogenetic mechanism for these conditions. Previously, we identified that overexpression of thyroid hormone-responsive protein (THRSP), a gene highly responsive to TH status, induced inattention in male mice. Herein, we sought to explore whether TH function in THRSP-overexpressing (THRSP OE) mice influences ADHD-like (inattention) behavior. We now confirm that THRSP overexpression in male mice reproduces behavioral features of ADHD, including sustained inattention and memory impairment, accompanied by excessive theta waves that were found normal in both the THRSP-knockout and hetero groups. Physiological characterization revealed low striatal T3 levels in the THRSP OE mice due to reduced striatal T3-specific monocarboxylate transporter 8 (MCT8), indicating brain-specific hypothyroidism in this transgenic mouse strain. TH replacement for seven days rescued inattention and memory impairment and the normalization of theta waves. This study further supports the involvement of the upregulated THRSP gene in ADHD pathology and indicates that THRSP OE mice can serve as an animal model for the predominantly inattentive subtype of ADHD.

Brain and Lung Cross-Protection against Ancestral or Emerging SARS-CoV-2 by Intranasal Lentiviral Vaccination in a New hACE2 Transgenic Murine Model

COVID-19 vaccines already in use or in clinical development may have safety concerns, limited immunogenicity in high-risk groups or reduced efficacy against emerging SARS-CoV-2 variants. In addition, although the neurotropism of SARS-CoV-2 is well established, the vaccine strategies currently developed have not taken into account the protection of the central nervous system. Here, we generated a transgenic mouse strain expressing the human Angiotensin Converting Enzyme 2, with unprecedented brain as well as lung permissibility to SARS-CoV-2 replication. Using this stringent transgenic model, we demonstrated that a non-integrative lentiviral vector, encoding for the spike glycoprotein of the ancestral Wuhan SARS-CoV-2, used in intramuscular prime and intranasal boost elicits sterilizing protection of lung and brain against both the Wuhan and the most genetically distant Manaus P.1 SARS-CoV-2 variants. Beyond the induction of strong neutralizing antibodies, the mechanism underlying this broad protection spectrum involves a robust protective spike-specific CD8+ T-cell immunity, unaffected by the recent mutations accumulated in the emerging SARS-CoV-2 variants.

Full Brain and Lung Prophylaxis against SARS-CoV-2 by Intranasal Lentiviral Vaccination in a New hACE2 Transgenic Mouse Model or Golden Hamsters

SummaryNon-integrative, non-cytopathic and non-inflammatory lentiviral vectors are particularly suitable for mucosal vaccination and recently emerge as a promising strategy to elicit sterilizing prophylaxis against SARS-CoV-2 in preclinical animal models. Here, we demonstrate that a single intranasal administration of a lentiviral vector encoding a prefusion form of SARS-CoV-2 spike glycoprotein induces full protection of respiratory tracts and totally avoids pulmonary inflammation in the susceptible hamster model. More importantly, we generated a new transgenic mouse strain, expressing the human Angiotensin Converting Enzyme 2, with unprecedent brain permissibility to SARS-CoV-2 replication and developing a lethal disease in <4 days post infection. Even though the neurotropism of SARS-CoV-2 is now well established, so far other vaccine strategies under development have not taken into the account the protection of central nervous system. Using our highly stringent transgenic model, we demonstrated that an intranasal booster immunization with the developed lentiviral vaccine candidate achieves full protection of both respiratory tracts and brain against SARS-CoV-2.

A novel mouse model for pyridoxine-dependent epilepsy due to antiquitin deficiency

Pyridoxine-dependent epilepsy (PDE) is a rare autosomal recessive disease caused by mutations in the ALDH7A1 gene leading to blockade of the lysine catabolism pathway. PDE is characterized by recurrent seizures that are resistant to conventional anticonvulsant treatment but are well-controlled by pyridoxine (PN). Most PDE patients also suffer from neurodevelopmental deficits despite adequate seizure control with PN. To investigate potential pathophysiological mechanisms associated with ALDH7A1 deficiency, we generated a transgenic mouse strain with constitutive genetic ablation of Aldh7a1. We undertook extensive biochemical characterization of Aldh7a1-KO mice consuming a low lysine/high PN diet. Results showed that KO mice accumulated high concentrations of upstream lysine metabolites including ∆1-piperideine-6-carboxylic acid (P6C), α-aminoadipic semialdehyde (α-AASA) and pipecolic acid both in brain and liver tissues, similar to the biochemical picture in ALDH7A1-deficient patients. We also observed preliminary evidence of a widely deranged amino acid profile and increased levels of methionine sulfoxide, an oxidative stress biomarker, in the brains of KO mice, suggesting that increased oxidative stress may be a novel pathobiochemical mechanism in ALDH7A1 deficiency. KO mice lacked epileptic seizures when fed a low lysine/high PN diet. Switching mice to a high lysine/low PN diet led to vigorous seizures and a quick death in KO mice. Treatment with PN controlled seizures and improved survival of high-lysine/low PN fed KO mice. This study expands the spectrum of biochemical abnormalities that may be associated with ALDH7A1 deficiency and provides a proof-of-concept for the utility of the model to study PDE pathophysiology and to test new therapeutics.

Passive Transfer of Sera from ALS Patients with Identified Mutations Evokes an Increased Synaptic Vesicle Number and Elevation of Calcium Levels in Motor Axon Terminals, Similar to Sera from Sporadic Patients

Previously, we demonstrated increased calcium levels and synaptic vesicle densities in the motor axon terminals (MATs) of sporadic amyotrophic lateral sclerosis (ALS) patients. Such alterations could be conferred to mice with an intraperitoneal injection of sera from these patients or with purified immunoglobulin G. Later, we confirmed the presence of similar alterations in the superoxide dismutase 1 G93A transgenic mouse strain model of familial ALS. These consistent observations suggested that calcium plays a central role in the pathomechanism of ALS. This may be further reinforced by completing a similar analytical study of the MATs of ALS patients with identified mutations. However, due to the low yield of muscle biopsy samples containing MATs, and the low incidence of ALS patients with the identified mutations, these examinations are not technically feasible. Alternatively, a passive transfer of sera from ALS patients with known mutations was used, and the MATs of the inoculated mice were tested for alterations in their calcium homeostasis and synaptic activity. Patients with 11 different ALS-related mutations participated in the study. Intraperitoneal injection of sera from these patients on two consecutive days resulted in elevated intracellular calcium levels and increased vesicle densities in the MATs of mice, which is comparable to the effect of the passive transfer from sporadic patients. Our results support the idea that the pathomechanism underlying the identical manifestation of the disease with or without identified mutations is based on a common final pathway, in which increasing calcium levels play a central role.