Rat CNS Research Articles

Abstract B055: Characterization of highly selective and CNS-penetrant PARP1 inhibitors

Abstract First-generation PARP1/2 inhibitors are FDA-approved for cancers such as breast, ovarian prostate and pancreas and provide significant therapeutic benefit to patients whose tumors harbor BRCA1 or BRCA2 mutations or homologous recombination deficiency (HRD). However, their use is associated with hematological toxicities that narrow their therapeutic index and restrict their application, particularly in combination with standard-of-care chemotherapy. All four FDA-approved PARP1 inhibitors are largely non-selective for the closely related enzyme PARP2, inhibition of which has been shown to drive hematotoxicity. Hence, the development of second-generation molecules highly selective for PARP1 over PARP2 offers a significant opportunity to 1) dramatically enhance therapeutic index, 2) enable additional precision medicine/combination approaches with chemotherapy, radiotherapy, immunotherapy and targeted agents and 3) expand the addressable patient population to those whose tumors harbour additional DDR defects. Utilising X-ray crystallography and structure-based drug design, we describe the characterization of exquisitely selective PARP1 inhibitors. These molecules demonstrate high potency in multiple biochemical and cell-based assays, including viability and colony forming unit read-outs in BRCA-deficient cancer cells, whilst sparing HR-proficient cancer cell lines and primary cells. The molecules are potent PARP1-DNA trappers and exhibit high selectivity across the PARP family and wider secondary pharmacology targets. The molecules potentiate the activity of ATR inhibitors when tested in ATM-deficient cancer cell lines. This chemical series generally exhibits highly desirable physico-chemical and in vitro ADME properties, which translate to high oral bioavailability and low clearance in rodent PK studies. Exemplar molecules also demonstrate high rat CNS penetrance using in vivo microdialysis. Lead molecules yield deep and durable dose-dependent anti-tumor efficacy in a variety of BRCAm xenograft models with responses continuing after cessation of treatment. The compounds demonstrate strong PK/PD relationships which correlate with anti-tumor efficacy using inhibition of tumor PARylation as a PD read-out. Strikingly, the molecules demonstrate compelling anti-tumor efficacy in BRCA-deficient xenograft models that are known to be largely resistant to FDA-approved non-selective PARP1/2 inhibitors. Taken together, these data predict low once daily therapeutic oral dosing based on a highly favorable pre-clinical PK profile and potent in vivo efficacy, with the potential to demonstrate improved efficacy and tolerability compared to marketed PARP inhibitors. In summary, we describe the characterization of next generation potent and selective CNS-penetrant PARP1 inhibitors. These molecules have the potential for an improved safety profile and greater combination potential with standard-of-care and targeted anti-cancer therapies. Citation Format: Phillip M Cowley, Barry E McGuinness, Gillian M Campbell, Alan Wise. Characterization of highly selective and CNS-penetrant PARP1 inhibitors [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr B055.

Propentofylline Improves Thiol-Based Antioxidant Defenses and Limits Lipid Peroxidation following Gliotoxic Injury in the Rat Brainstem.

Propentofylline (PROP) is a methylated xanthine compound that diminishes the activation of microglial cells and astrocytes, which are neuronal cells strongly associated with many neurodegenerative diseases. Based on previously observed remyelination and neuroprotective effects, PROP has also been proposed to increment antioxidant defenses and to prevent oxidative damage in neural tissues. Since most neurodegenerative processes have free radicals as molecular pathological agents, the aim of this study was to evaluate the antioxidant effects of 12.5 mg·kg-1·day-1 PROP in plasma and the brainstem of Wistar rats exposed to the gliotoxic agent 0.1% ethidium bromide (EB) for 7-31 days. The bulk of the data here demonstrates that, after 7 days of EB treatment, TBARS levels were 2-fold higher in the rat CNS than in control, reaching a maximum of 2.4-fold within 15 days. After 31 days of EB treatment, lipoperoxidation in CNS was still 65% higher than that in the control. Clearly, PROP treatment limited the progression of lipoperoxidation in EB-oxidized CNS: it was, for example, 76% lower than in the EB-treated group after 15 days. Most of these effects were associated with PROP-induced activity of glutathione reductase in the brainstem: the EB + PROP group showed 59% higher GR activity than that of the EB or control groups within 7 days. In summary, aligning with previous studies from our group and with literature about MTXs, we observed that propentofylline (PROP) improved the thiol-based antioxidant defenses in the rat brainstem by the induction of the enzymatic activity of glutathione reductase (GR), which diminished lipid oxidation progression and rebalanced the redox status in the CNS.

Protocatechuic Acid Prevents Some of the Memory-Related Behavioural and Neurotransmitter Changes in a Pyrithiamine-Induced Thiamine Deficiency Model of Wernicke-Korsakoff Syndrome in Rats.

The purpose of this research was to investigate the effects of protocatechuic acid (PCA) at doses of 50 and 100 mg/kg on the development of unfavourable changes in cognitive processes in a pyrithiamine-induced thiamine deficiency (PTD) model of the Wernicke-Korsakoff syndrome (WKS) in rats. The effects of PCA were assessed at the behavioural and biochemical levels. Behavioural analysis was conducted using the Foot Fault test (FF), Bar test, Open Field test, Novel Object Recognition test (NOR), Hole-Board test and Morris Water Maze test (MWM). Biochemical analysis consisting of determination of concentration and turnover of neurotransmitters in selected structures of the rat CNS was carried out using high-performance liquid chromatography. PTD caused catalepsy (Bar test) and significantly impaired motor functions, leading to increased ladder crossing time and multiplied errors due to foot misplacement (FF). Rats with experimentally induced WKS showed impaired consolidation and recall of spatial reference memory in the MWM test, while episodic memory related to object recognition in the NOR was unimpaired. Compared to the control group, rats with WKS showed reduced serotonin levels in the prefrontal cortex and changes in dopamine and/or norepinephrine metabolites in the prefrontal cortex, medulla oblongata and spinal cord. PTD was also found to affect alanine, serine, glutamate, and threonine levels in certain areas of the rat brain. PCA alleviated PTD-induced cataleptic symptoms in rats, also improving their performance in the Foot Fault test. In the MWM, PCA at 50 and 100 mg/kg b.w. improved memory consolidation and the ability to retrieve acquired information in rats, thereby preventing unfavourable changes caused by PTD. PCA at both tested doses was also shown to have a beneficial effect on normalising PTD-disrupted alanine and glutamate concentrations in the medulla oblongata. These findings demonstrate that certain cognitive deficits in spatial memory and abnormalities in neurotransmitter levels persist in rats that have experienced an acute episode of PTD, despite restoration of thiamine supply and long-term recovery. PCA supplementation largely had a preventive effect on the development of these deficits, to some extent also normalising neurotransmitter concentrations in the brain.

Mitochondrial formation and degradation in the CNS of male and female rats subjected to a two-hit model and paliperidone treatment

Mitochondrial formation and degradation in the CNS of male and female rats subjected to a two-hit model and paliperidone treatment

CENTRAL AND PERIPHERAL EFFECT OF MPTP VIA DOSE-DEPENDENT MAGNESIUM MODULATION.

Recent studies suggested that MPTP could cause gastrointestinal motility deficits additionally to its nonconclusive and controverted effects on the CNS (behavior and brain oxidative stress) in rats. A possible interaction between MPTP typical impairments and magnesium modulatory potential was previously suggested, as magnesium role was described in neuroprotection, gastrointestinal function, and oxidative stress. To investigate the possible modulatory effect of several magnesium intake formulations (via drinking water) in MPTP neurotoxicity and functional gastrointestinal impairment induction. Adult male Wistar rats were subjected to 3-week magnesium intake-controlled diets (magnesium depleted food and magnesium enriched drinking water) previously to acute subcutaneous MPTP treatment (30 mg/ kg body weight). Gastrointestinal motility (one hour stool collection test), and behavioral patterns (Y maze task, elevated plus maze test, open field test, forced swim test) were evaluated. Followingly, brain and bowel samples were collected, and oxidative stress was evaluated (glutathione peroxidase activity, malondial-dehyde concentrations). MPTP could lead to magnesium intake-dependent constipation-like gastrointestinal motility impairments, anxiety- and depressive-like affective behavior changes, and mild pain tolerance defects. Also, we found similar brain and intestinal patterns in magnesium-dependent oxidative stress. While the MPTP effects in normal magnesium intake could be regarded as not fully relevant in rat models and limited to the current experimental conditions, the abnormalities observed in the affective behavior, gastrointestinal status, pain tolerance, peripheric and central oxidative status could be indicative of the extent of the systemic effects of MPTP that are not restricted to the CNS level, but also to gastro-intestinal system.

Region-specific changes in expression and activity of calpains in the CNS of native rats.

Introduction and Aim: It has been proposed that µ-calpain is responsible for neuronal survival, while m-calpain – for the degeneration. It can be assumed that the "susceptibility" to the damage factor for neurons in different CNS regions depends on the content/activity of calpain isoforms. We analyzed the mRNA levels and the activity of µ-and m-calpain in the different CNS structures of rats. Materials and Methods: After decapitation intact male Wistar rats the prefrontal cortex, striatum, hippocampus, midbrain, brainstem, cerebellum, and spinal cord were removed. Each structure was divided into two parts: casein zymography was performed to determine the activity and real-time RT–PCR - to determine the level of expression mRNA of µ-and m-calpains. Results: We have shown that m-calpain mRNA predominates in the striatum, midbrain and brainstem, while µ-calpain mRNA enrichment was noticed for the hippocampus and cerebellum. The highest µ-calpain activity was in the cervical spinal cord, the lowest - in the striatum. The m-calpain activity was relatively high in the midbrain, striatum, hippocampus and brainstem, while in the cervical spinal cord and cerebellum it was moderate. Conclusion: The selective neuronal death observed during neurodegeneration can be partially determined by the initial level of calpains expression and/or activity.

Abstract 2675: Discovery of potent CNS-penetrant covalent KRAS G12C inhibitors

Abstract The RAS family of GTPases, which include KRAS, NRAS, and HRAS, are key regulators of cellular signaling, acting as molecular switches downstream of receptor tyrosine kinases (RTKs) and as key regulators of the RAS/MAPK pathway. RAS cycles between inactive (GDP) and active (GTP) states to regulate the activity of the RAS/MAPK pathway. In many cancers, activating KRAS mutations enable KRAS to persist in the active GTP-bound state, resulting in hyperactive RAS/MAPK pathway signaling that drives cell growth and survival. The KRAS G12C mutation occurs in 14% of lung adenocarcinoma, a cancer type that frequently metastasizes to the brain (40%). To address the high prevalence of KRAS G12C mutant CNS metastases in lung adenocarcinoma, we are developing CNS-penetrant covalent KRAS G12C inhibitors (“ERAS G12Ci’s”) to treat both CNS and systemic disease. ERAS G12Ci’s rapidly form covalent adducts with the KRAS G12C protein in its inactive GDP state and exhibit low nanomolar IC50’s in a nucleotide exchange assay. ERAS G12Ci’s demonstrate high selectivity in a cell-based proteome selectivity assay and inhibit the growth of RAS Initiative KRAS G12C mutant cells, and not RAS Initiative KRAS WT cells. ERAS G12Ci’s potently inhibit cell proliferation in 3-dimensional Cell-Titer Glo (3D-CTG) assays in KRAS G12C mutant lung and pancreatic cell lines (NCI-H1373, NCI-H2122, and MIA PaCa-2). Mechanistically, ERAS G12Ci’s blocked RAS-RAF complex formation and inhibited ERK1/2 phosphorylation. This in vitro activity translates in vivo where ERAS G12Ci’s induce pharmacodynamic modulation in the pancreatic cancer MIA PaCa-2 model. ERAS G12Ci’s significantly inhibit tumor growth in NCI-H1373 and NCI-H2122 CDX lung adenocarcinoma and MIA PaCa-2 PDAC models. No meaningful body weight loss or clinical adverse events were observed with any of these compounds. Human efflux transporter substrate assessments indicate that ERAS G12Ci’s are either not substrates or weak/modest substrates of P-gp, a crucial efflux transporter that can limit CNS penetration. In rat CNS studies, these inhibitors exhibit good CNS penetration performance as measured by brain-to-plasma partition coefficients, which are comparable to those of approved CNS-active small molecule inhibitors. Daily oral administration demonstrates dose-dependent tumor regression in intracranial and intra-carotid injection (ICA) KRAS G12C CDX models. We are optimizing multiple covalent CNS-penetrant KRAS G12C inhibitors that exhibit both CNS and systemic activity in vivo. Citation Format: Jae Hyun Bae, Erin D. Lew, Jun Feng, Marcos Gonzalez-Lopez, Joanne Oh, Patrick Fagan, Matt Salie, Nick Isley, Richard Lam, Adriana Irimia, Robin Nevarez, Bingzhen Lin, Taylor Congdon, Jingchuan Zhang, Dawei Xuan, Ping Chen, Jean-Michel Vernier, Robert Shoemaker. Discovery of potent CNS-penetrant covalent KRAS G12C inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2675.

Abstract 3261: LNG-451, a potent inhibitor of EGFR exon 20 insertion mutations with high CNS exposure

Abstract Exon 20 insertion mutations (Ex20ins) are the 3rd most common type of EGFR oncogenic mutation in lung cancer patients and are often associated with brain metastases. While several small molecules have been approved (mobocertinib) or are in clinical development (e.g. CLN-081, BDTX-189) none have demonstrated meaningful CNS activity and they can be associated with treatment-limiting adverse events, including wild-type (WT) EGFR-mediated toxicities. LNG-451 is a potent covalent inhibitor of EGFR Ex20ins (IC50 7 - 78 nM) and other EGFR oncogenic mutations (e.g L858R IC50 = 3 nM, exon 19 deletion IC50 = 24 nM) while being selective against WT EGFR (IC50 = 1630 nM). LNG-451 causes tumor regression in both cell line-derived and patient-derived EGFR Ex20ins-dependent xenograft tumor models. In a rat CNS PK model, a single oral dose of 30 mg/kg LNG-451 achieved high exposures in plasma (AUC0 - 8h = 9771 ng•h/mL) and brain (AUC0 - 8h = 10,041 ng•h/g). In a rat steady-state CNS PK model, unbound partition coefficient for LNG-451 was determined to be Kpu,u = 0.66. To evaluate the potential for LNG-451 to treat metastatic brain disease driven by EGFR mutations, an intracranial PC9-luc xenograft mouse model harboring an EGFR exon 19 deletion mutation was used (LNG-451 cellular EGFR Del19 IC50 = 10.6 nM). LNG-451 had dose-dependent exposures in the plasma and brain with both high mean brain exposure (e.g 1074 ng/g with 25 mg/kg LNG-451) and high mean brain to plasma ratios (e.g. 0.451 for 25 mg/kg LNG-451). LNG-451 treatments were well-tolerated and produced significant anti-tumor activities with 99.5% (2.5 mg/kg LNG-451) and 99.9% (25 mg/kg LNG-451) reductions in total bioluminescence signal (BLI) and regression of tumor BLI (2.5 mg/kg LNG-451 28.8% regression; 25 mg/kg LNG-451, 80.3% regression). An ex vivo imaging analysis of the brain and spinal cords from all animals at the end of the study showed a dose-dependent decrease in the bioluminescent signal in both brain and spinal cords from mice treated with LNG-451 relative to the vehicle control animals. Taken together, LNG-451 is a CNS-penetrant, wild-type EGFR-sparing, EGFR Ex20ins inhibitor that is expected to provide strong anti-tumor efficacy for patients with advanced/metastatic solid cancers harboring oncogenic EGFR exon 20 insertions with reduced WT EGFR driven toxicities. Citation Format: Paul G. Pearson, Anjali Pandey, Bruce Roth, Tracy Saxton, Daniel J. Estes, Ravi Trivedi, Himanshu Agrawal, Gurulingappa Hallur, Ishtiyaque Ahmad, Helen Jenkins, Brion W. Murray. LNG-451, a potent inhibitor of EGFR exon 20 insertion mutations with high CNS exposure [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3261.

Sulforaphane Ameliorates Metabolic Changes Associated With Status Epilepticus in Immature Rats.

Status epilepticus (SE) is a common paediatric emergency with the highest incidence in the neonatal period and is a well-known epileptogenic insult. As previously established in various experimental and human studies, SE induces long-term alterations to brain metabolism, alterations that directly contribute to the development of epilepsy. To influence these changes, organic isothiocyanate compound sulforaphane (SFN) has been used in the present study for its known effect of enhancing antioxidative, cytoprotective, and metabolic cellular properties via the Nrf2 pathway. We have explored the effect of SFN in a model of acquired epilepsy induced by Li-Cl pilocarpine in immature rats (12 days old). Energy metabolites PCr, ATP, glucose, glycogen, and lactate were determined by enzymatic fluorimetric methods during the acute phase of SE. Protein expression was evaluated by Western blot (WB) analysis. Neuronal death was scored on the FluoroJadeB stained brain sections harvested 24 h after SE. To assess the effect of SFN on glucose metabolism we have performed a series of 18F-DG μCT/PET recordings 1 h, 1 day, and 3 weeks after the induction of SE. Responses of cerebral blood flow (CBF) to electrical stimulation and their influence by SFN were evaluated by laser Doppler flowmetry (LDF). We have demonstrated that the Nrf2 pathway is upregulated in the CNS of immature rats after SFN treatment. In the animals that had undergone SE, SFN was responsible for lowering glucose uptake in most regions 1 h after the induction of SE. Moreover, SFN partially reversed hypometabolism observed after 24 h and achieved full reversal at approximately 3 weeks after SE. Since no difference in cell death was observed in SFN treated group, these changes cannot be attributed to differences in neurodegeneration. SFN per se did not affect the glucose uptake at any given time point suggesting that SFN improves endogenous CNS ability to adapt to the epileptogenic insult. Furthermore, we had discovered that SFN improves blood flow and accelerates CBF response to electrical stimulation. Our findings suggest that SFN improves metabolic changes induced by SE which have been identified during epileptogenesis in various animal models of acquired epilepsy.

Why is motilin active in some studies with mice, rats, and guinea pigs, but not in others? Implications for functional variability among rodents.

The gastrointestinal (GI) hormone motilin helps control human stomach movements during hunger and promotes hunger. Although widely present among mammals, it is generally accepted that in rodents the genes for motilin and/or its receptor have undergone pseudonymization, so exogenous motilin cannot function. However, several publications describe functions of low concentrations of motilin, usually within the GI tract and CNS of mice, rats, and guinea pigs. These animals were from institute‐held stocks, simply described with stock names (e.g., “Sprague–Dawley”) or were inbred strains. It is speculated that variation in source/type of animal introduces genetic variations to promote motilin‐sensitive pathways. Perhaps, in some populations, motilin receptors exist, or a different functionally‐active receptor has a good affinity for motilin (indicating evolutionary pressures to retain motilin functions). The ghrelin receptor has the closest sequence homology, yet in non‐rodents the receptors have a poor affinity for each other's cognate ligand. In rodents, ghrelin may substitute for certain GI functions of motilin, but no good evidence suggests rodent ghrelin receptors are highly responsive to motilin. It remains unknown if motilin has functional relationships with additional bioactive molecules formed from the ghrelin and motilin genes, or if a 5‐TM motilin receptor has influence in rodents (e.g., to dimerize with GPCRs and create different pharmacological profiles). Is the absence/presence of responses to motilin in rodents’ characteristic for systems undergoing gene pseudonymization? What are the consequences of rodent supplier‐dependent variations in motilin sensitivity (or other ligands for receptors undergoing pseudonymization) on gross physiological functions? These are important questions for understanding animal variation.

Piperine Improves Experimental Autoimmune Encephalomyelitis (EAE) in Lewis Rats Through its Neuroprotective, Anti-inflammatory, and Antioxidant Effects.

Inflammation, demyelination, glial activation, and oxidative damage are the most pathological hallmarks of multiple sclerosis (MS). Piperine, a main bioactive alkaloid of black pepper, possesses antioxidant, anti-inflammatory, and neuroprotective properties whose therapeutic potential has been less studied in the experimental autoimmune encephalomyelitis (EAE) models. In this study, the efficiency of piperine on progression of EAE model and myelin repair mechanisms was investigated. EAE was induced in female Lewis rats and piperine and its vehicle were daily administrated intraperitoneally from day 8 to 29 post immunization. We found that piperine alleviated neurological deficits and EAE disease progression. Luxol fast blue and H&E staining and immunostaining of lumbar spinal cord cross sections confirmed that piperine significantly reduced the extent of demyelination, inflammation, immune cell infiltration, microglia, and astrocyte activation. Gene expression analysis in lumbar spinal cord showed that piperine treatment decreased the level of pro-inflammatory cytokines (TNF-α, IL-1β) and iNOS and enhanced IL-10, Nrf2, HO-1, and MBP expressions. Piperine supplementation also enhanced the total antioxidant capacity (FRAP) and reduced the level of oxidative stress marker (MDA) in the CNS of EAE rats. Finally, we found that piperine has anti-apoptotic and neuroprotective effect in EAE through reducing caspase-3 (apoptosis marker) and enhancing BDNF and NeuN expressing cells. This study strongly indicates that piperine has a beneficial effect on the EAE progression and could be considered as a potential therapeutic target for MS treatment. Upcoming clinical trials will provide a deeper understanding of piperine's role for the treatment of the MS.

Neuronal and Glial Distribution of Tau Protein in the Adult Rat and Monkey.

Tau is a microtubule-associated protein for which the physiological functions remain a topic of vigorous investigation. Additionally, tau is a central player in the pathogenesis of several diseases such as Alzheimer’s disease and several frontotemporal dementias. A critical variable to understanding tau in physiological and disease contexts is its normal localization within cells of the adult CNS. Tau is often described as an axon-specific (or enriched) and neuron-specific protein with little to no expression in glial cells, all of which are untrue. Understanding normal tau distribution also impacts interpretation of experimental results and hypotheses regarding its role in disease. Thus, we set out to help clarify the normal localization of tau in the adult CNS of middle-aged rats and rhesus macaque using the hippocampus as a representative brain structure. The physiological concentration of tau in the rat hippocampus was 6.6 μM and in white matter was 3.6 μM as determined by quantitative sandwich ELISAs. We evaluated the cellular localization of tau using multiple tau-specific antibodies with epitopes to different regions, including Tau1, Tau5, Tau7, R1, and two novel primate-specific antibodies NT9 and NT15. In the rat and monkey, tau was localized within the somatodendritic and axonal compartments, as well as a subset of neuronal nuclei. Semi-quantitative fluorescence intensity measurements revealed that depending on the specific reagent used the somatodendritic tau is relatively equal to, higher than, or lower than axonal tau, highlighting differential labeling of tau with various antibodies despite its distribution throughout the neuron. Tau was strongly expressed in mature oligodendrocytes and displayed little to no expression in oligodendrocyte precursor cells, astrocytes or microglia. Collectively, the data indicate tau is ∼3 – 7 μM under physiological conditions, is not specifically enriched in axons, and is normally found in both neurons and mature oligodendrocytes in the adult CNS. The full landscape of tau distribution is not revealed by all antibodies suggesting availability of the epitopes is different within specific neuronal compartments. These findings set the stage for better understanding normal tau distributions and interpreting data regarding the presence of tau in different compartments or cell types within disease conditions.

Crossing the blood-brain barrier with carbon dots: uptake mechanism and in vivo cargo delivery.

The blood–brain barrier (BBB) is a major obstacle for drug delivery to the central nervous system (CNS) such that most therapeutics lack efficacy against brain tumors or neurological disorders due to their inability to cross the BBB. Therefore, developing new drug delivery platforms to facilitate drug transport to the CNS and understanding their mechanism of transport are crucial for the efficacy of therapeutics. Here, we report (i) carbon dots prepared from glucose and conjugated to fluorescein (GluCD-F) cross the BBB in zebrafish and rats without the need of an additional targeting ligand and (ii) uptake mechanism of GluCDs is glucose transporter-dependent in budding yeast. Glucose transporter-negative strain of yeast showed undetectable GluCD accumulation unlike the glucose transporter-positive yeast, suggesting glucose-transporter-dependent GluCD uptake. We tested GluCDs' ability to cross the BBB using both zebrafish and rat models. Following the injection to the heart, wild-type zebrafish showed GluCD-F accumulation in the central canal consistent with the transport of GluCD-F across the BBB. In rats, following intravenous administration, GluCD-F was observed in the CNS. GluCD-F was localized in the gray matter (e.g. ventral horn, dorsal horn, and middle grey) of the cervical spinal cord consistent with neuronal accumulation. Therefore, neuron targeting GluCDs hold tremendous potential as a drug delivery platform in neurodegenerative disease, traumatic injury, and malignancies of the CNS.

Preparation of Neural Stem Cells and Progenitors: Neuronal Production and Grafting Applications.

Neural stem cells (NSCs) are a valuable tool for the study of neural development and function as well as an important source of cell transplantation strategies for neural disease. NSCs can be used to study how neurons acquire distinct phenotypes and how the interactions between neurons and glial cells in the developing nervous system shape the structure and function of the CNS. NSCs can also be used for cell replacement therapies following CNS injury targeting astrocytes, oligodendrocytes, and neurons. With the availability of patient-derived induced pluripotent stem cells (iPSCs), neurons prepared from NSCs can be used to elucidate the molecular basis of neurological disorders leading to potential treatments. Although NSCs can be derived from different species and many sources, including embryonic stem cells (ESCs), iPSCs, adult CNS, and direct reprogramming of nonneural cells, isolating primary NSCs directly from fetal tissue is still the most common technique for preparation and study of neurons. Regardless of the source of tissue, similar techniques are used to maintain NSCs in culture and to differentiate NSCs toward mature neural lineages. This chapter will describe specific methods for isolating and characterizing multipotent NSCs and neural precursor cells (NPCs) from embryonic rat CNS tissue (mostly spinal cord) and from human ESCs and iPSCs as well as NPCs prepared by reprogramming. NPCs can be separated into neuronal and glial restricted progenitors (NRP and GRP, respectively) and used to reliably produce neurons or glial cells both in vitro and following transplantation into the adult CNS. This chapter will describe in detail the methods required for the isolation, propagation, storage, and differentiation of NSCs and NPCs isolated from rat and mouse spinal cords for subsequent in vitro or in vivo studies as well as new methods associated with ESCs, iPSCs, and reprogramming.

Effect of avandamet on conditioned food reflex in rats of different age groups

The prevalence of Alzheimer’s disease (AD) is increasing in the absence of truly effective treatments. The recently discovered causal link between AD and type 2 diabetes mellitus (T2DM) (non-insulin-dependent diabetes mellitus) suggests that an increase in insulin receptor resistance may affect brain aging and lead to AD. In our experiments, we tested Avandamet (AVD), the antidiabetic drug used for the treatment of T2DM, on temporary parameters of conditioning food reflex in rats. We measured the individual components of the sensorimotor reaction (SMR) of rats, which reflect CNS’s functional state. Important tha7t rosiglitazone is the main component of AVD. It acts by activating the nuclear peroxisome proliferator-activated receptor (PPAR) gamma. Our data showed that this drug influences the brain function of both young and old rats. It is proved that AVD affects the CNS of rats by increasing their excitability. Considering the obtained data from the point of view of AD, the time parameter, which changed more significantly in old rats, was responsible for analyzing the situation and speed of action. Under AVD’s influence, old rats became more agile and had increased motor activity, also the success of rats in performing a conditioned reflex was increased. Thus, the drug AVD and its active component rosiglitazone can be perspective for its testing in AD treatment.

Lingering Effects of Prenatal Alcohol Exposure on Basal and Ethanol-Evoked Expression of Inflammatory-Related Genes in the CNS of Adolescent and Adult Rats.

Emerging data suggest that alcohol’s effects on central inflammatory factors are not uniform across the lifespan. In particular, prenatal alcohol exposure (PAE) significantly alters steady-state levels of neuroimmune factors, as well as subsequent reactivity to later immune challenge. Thus, the current experiment investigated developmental sensitivities to, and long-lasting consequences of, PAE on ethanol-evoked cytokine expression in male and female adolescent and adult rats. Pregnant dams received either an ad libitum ethanol liquid diet (2.2% GD 6–8; 4.5% GD 9–10; 6.7% GD11–20; 35% daily calories from ethanol) or free-choice access to a control liquid diet and water. At birth, offspring were fostered to dams given free-choice access to the control liquid diet. Pups then matured until mid-adolescence [postnatal day (PD) 35] or adulthood (PD90), at which time they were challenged with either a binge-like dose of ethanol (4 g/kg; intragastrically) or tap water. During intoxication (3 h post-ethanol challenge), brains and blood were collected for assessment of neuroimmune gene expression (reverse transcription-polymerase chain reaction; RT-PCR) in the hippocampus, amygdala, and PVN, as well as for blood ethanol concentrations (BEC) and plasma corticosterone levels. Results revealed that rats challenged with ethanol at either PD35 or PD90 generally exhibited a characteristic cytokine signature of acute intoxication that we have previously reported: increased Il-6 and IkBα expression, with decreased Il-1β and Tnfα gene expression. With a few exceptions, this pattern of gene changes was observed in all three structures examined, at both ages of postnatal ethanol challenge, and in both sexes. While few significant effects of PAE were observed for ethanol-induced alterations in cytokine expression, there was a consistent (but nonsignificant) trend for PAE to potentiate the expression of Il-6 and IkBα in all groups except adult females. Although these data suggest that later-life ethanol challenge was a far greater driver of inflammatory signaling than PAE, the current results demonstrate PAE resulted in subtle long-term alterations in the expression of many key neuroinflammatory factors associated with NF-κB signaling. Such long-lasting impacts of PAE that may engender vulnerability to later environmental events triggering neuroinflammatory processes, such as chronic ethanol exposure or stress, could contribute to heightened vulnerability for PAE-related alterations and deficits.

Ciliary melanin-concentrating hormone receptor 1 (MCHR1) is widely distributed in the murine CNS in a sex-independent manner.

Melanin-concentrating hormone (MCH) is a ubiquitous vertebrate neuropeptide predominantly synthesized by neurons of the diencephalon that can act through two G protein-coupled receptors, called MCHR1 and MCHR2. The expression of Mchr1 has been investigated in both rats and mice, but its synthesis remains poorly described. After identifying an antibody that detects MCHR1 with high specificity, we employed immunohistochemistry to map the distribution of MCHR1 in the CNS of rats and mice. Multiple neurochemical markers were also employed to characterize some of the neuronal populations that synthesize MCHR1. Our results show that MCHR1 is abundantly found in a subcellular structure called the primary cilium, which has been associated, among other functions, with the detection of free neurochemical messengers present in the extracellular space. Ciliary MCHR1 was found in a wide range of areas, including the olfactory bulb, cortical mantle, striatum, hippocampal formation, amygdala, midline thalamic nuclei, periventricular hypothalamic nuclei, midbrain areas, and in the spinal cord. No differences were observed between male and female mice, and interspecies differences were found in the caudate-putamen nucleus and the subgranular zone. Ciliary MCHR1 was found in close association with several neurochemical markers, including tyrosine hydroxylase, calretinin, kisspeptin, estrogen receptor, oxytocin, vasopressin, and corticotropin-releasing factor. Given the role of neuronal primary cilia in sensing free neurochemical messengers in the extracellular fluid, the widespread distribution of ciliary MCHR1, and the diverse neurochemical populations who synthesize MCHR1, our data indicate that nonsynaptic communication plays a prominent role in the normal function of the MCH system.

Chronic Stress Alters Expression of Vomiting‐related Receptors in CNS in SD Rats

Chemotherapy‐induced nausea and vomiting (CINV) are extremely painful side effects that occur frequently in cancer patients during chemotherapy. Our previous study found that cancer patients have high incidence of depression that correlate positively with CINV. However, whether depression aggravate the CINV is not clear up to now. In this report, we established chronic unpredictable mild stress‐induced depression model in SD male rats. The vomiting‐related receptors including serotonin 3 receptor (5‐HT3R), dopamine receptor (DA2R), endogenous cannabinoid receptor (CB1R) and substance P receptor (NK1R) in the posterior region of the fourth ventricle and in the medulla were studied by qRT‐PCT and Western blotting analysis. Blood levels of 5‐HT, DA, AEA and substance P were measured by ELISA. There were significant difference in behavioral evaluation scores between the model rats and control rats, indicating depression model was successfully built up in model rats. Compared with control rats, blood levels of 5‐HT, DA and substance P were significantly decreased while AEA was significantly increased in model rats, the mRNA and protein of expression levels of 5‐HT3R, DA2R, NK1R were increased while CB1R were decreased in model rats. In conclusion, chronic stress altered expression of vomiting‐related receptors in CNS in SD rats.Support or Funding InformationShanghai Science and Technology Commision Plan Project (No. 19411971700)

Exposure to Acute, High Dose Cadmium Significantly Downregulates Postnatal Expression of the Vascular Endothelial Barrier Antigen in the Rat Central Nervous System as Quantitated by Immunofluorescent Microscopy

Clinical and experimental studies have demonstrated the neurotoxic and behavioral effects of cadmium. The exact mechanism(s) of cadmium neurotoxicity on the human central nervous system is not completely understood. It has been suggested that the metal exerts its effect via oxidative stress‐induced morphological and functional disruption of blood‐brain barrier (BBB) structures. A sensitive and specific rat BBB marker, the endothelial barrier antigen (EBA), has been previously identified and characterized by lymphocyte hybridoma technology. Alterations in EBA expression have been reported to occur during infectious and non‐infectious central and peripheral nervous system (CNS and PNS) pathologies. We have previously demonstrated that in the rat, EBA first becomes detectable during the first week postnatally and is progressively expressed along with the biochemical and hemodynamic changes that accompany normal brain maturation and barriergenesis. We have also shown that this anti‐EBA IgG1 antibody exclusively recognizes barrier‐competent microvessels in rat central and peripheral nervous system (CNS and PNS). Endothelial cells of peripheral tissues such as the liver and kidney and brain circumventricular organs possessing fenestrated microvascular endothelia, do not display immunoreactivity for EBA.In the present study, a double, sequential immunofluorescent protocol was employed to colocalize EBA‐specific microvessels and astrocytes, using anti‐EBA and anti‐GFAP antibodies, respectively. Sections were examined with an Olympus BX63 Fluorescent microscope outfitted with a DP80 Camera and cellSense software. The EBA‐immunoreactive microvessels were quantitated by measuring the areal fraction of the microvessels per field using an algorithm that allowed for the microvessels to be segmented from the surrounding background within the region of interest (ROI) using a global threshold. The grand mean areal fractions of all microscopic fields captured from the normal control brain sections were compared for statistical significance with the areal fraction grand mean for the cadmium‐treated brain sections using a two‐sample t‐test at a 95% confidence interval. Positive staining was not detected in any of the tissue sections in which the primary antibodies were omitted. Also, EBA expression was not detected in sections from the kidney, known to be vascularized by fenestrated capillaries lacking a barrier function. Statistical analysis (two‐sample t‐test) of the Grand Mean Areal Fractions (μm2) for all microscopic fields evaluated for control and experimental groups yielded a p‐value of less than 0.001 (t = 5.8507, df = 1789, p‐value = 5.808e‐09; p < 0.001).This study indicates that acute exposure to a single, high dose of cadmium significantly downregulates the expression of EBA in the developing rat CNS. The results suggest that EBA is involved in the pathogenesis of cadmium neurotoxicity and could serve as a useful tool for studying the extent of cadmium‐induced impact on BBB structures of the central nervous system in the rat model system.Support or Funding InformationWe express our thanks and sincere appreciation of the research funding provided by the Alabama College of Osteopathic Medicine.

AAV Capsid-Promoter Interactions Determine CNS Cell-Selective Gene Expression InVivo.

Cell-selective gene expression comprises a critical element of many adeno-associated virus (AAV) vector-based gene therapies, and to date achieving this goal has focused on AAV capsid engineering, cell-specific promoters, or cell-specific enhancers. Recently, we discovered that the capsid of AAV9 exerts a differential influence on constitutive promoters of sufficient magnitude to alter cell type gene expression in the rat CNS. For AAV9 vectors chicken β-actin (CBA) promoter-driven gene expression exhibited a dominant neuronal gene expression in the rat striatum. Surprisingly, for otherwise identical AAV9 vectors, the truncated CBA hybrid (CBh) promoter shifted gene expression toward striatal oligodendrocytes. In contrast, AAV2 vector gene expression was restricted to striatal neurons, regardless of the constitutive promoter used. Furthermore, a six-glutamate residue insertion immediately after the VP2 start residue shifted CBA-driven cellular gene expression from neurons to oligodendrocytes. Conversely, a six-alanine insertion in the same AAV9 capsid region reversed the CBh-mediated oligodendrocyte expression back to neurons without changing AAV9 capsid access to oligodendrocytes. Given the preponderance of AAV9 in ongoing clinical trials and AAV capsid engineering, this AAV9 capsid-promoter interaction reveals apreviously unknown novel contribution to cell-selective AAV-mediated gene expression in the CNS.