Effects of Striatal Amyloidosis on the Dopaminergic System and Behavior: A Comparative Study in Male and Female 5XFAD Mice.

BACKGROUND: Nearly two-thirds of patients diagnosed with Alzheimer's disease (AD) are female. In addition, female patients with AD have more significant cognitive impairment than males at the same disease stage. This disparity suggests there are sex differences in AD progression. While females appear to be more affected by AD, most published behavioral studies utilize male mice. In humans, there is an association between antecedent attention-deficit/hyperactivity disorder and increased risk of dementia. Functional connectivity studies indicate that dysfunctional cortico-striatal networks contribute to hyperactivity in attention deficit hyperactivity disorder. Higher plaque density in the striatum accurately predicts the presence of clinical AD pathology. In addition, there is a link between AD-related memory dysfunction and dysfunctional dopamine signaling. OBJECTIVE: With the need to consider sex as a biological variable, we investigated the influence of sex on striatal plaque burden, dopaminergic signaling, and behavior in prodromal 5XFAD mice. METHODS: Six-month-old male and female 5XFAD and C57BL/6J mice were evaluated for striatal amyloid plaque burden, locomotive behavior, and changes in dopaminergic machinery in the striatum. RESULTS: 5XFAD female mice had a higher striatal amyloid plaque burden than male 5XFAD mice. 5XFAD females, but not males, were hyperactive. Hyperactivity in female 5XFAD mice was associated with increased striatal plaque burden and changes in dopamine signaling in the dorsal striatum. CONCLUSION: Our results indicate that the progression of amyloidosis involves the striatum in females to a greater extent than in males. These studies have significant implications for using male-only cohorts in the study of AD progression.

Endothelial Cells and the Cerebral Circulation.

Endothelial cells form the innermost layer of all blood vessels and are the only vascular component that remains throughout all vascular segments. The cerebral vasculature has several unique properties not found in the peripheral circulation; this requires that the cerebral endothelium be considered as a unique entity. Cerebral endothelial cells perform several functions vital for brain health. The cerebral vasculature is responsible for protecting the brain from external threats carried in the blood. The endothelial cells are central to this requirement as they form the basis of the blood-brain barrier. The endothelium also regulates fibrinolysis, thrombosis, platelet activation, vascular permeability, metabolism, catabolism, inflammation, and white cell trafficking. Endothelial cells regulate the changes in vascular structure caused by angiogenesis and artery remodeling. Further, the endothelium contributes to vascular tone, allowing proper perfusion of the brain which has high energy demands and no energy stores. In this article, we discuss the basic anatomy and physiology of the cerebral endothelium. Where appropriate, we discuss the detrimental effects of high blood pressure on the cerebral endothelium and the contribution of cerebrovascular disease endothelial dysfunction and dementia. © 2022 American Physiological Society. Compr Physiol 12:3449-3508, 2022.

Small Molecule 20S Proteasome Enhancer Regulates MYC Protein Stability and Exhibits Antitumor Activity in Multiple Myeloma.

Despite the addition of several new agents to the armamentarium for the treatment of multiple myeloma (MM) in the last decade and improvements in outcomes, the refractory and relapsing disease continues to take a great toll, limiting overall survival. Therefore, additional novel approaches are needed to improve outcomes for MM patients. The oncogenic transcription factor MYC drives cell growth, differentiation and tumor development in many cancers. MYC protein levels are tightly regulated by the proteasome and an increase in MYC protein expression is found in more than 70% of all human cancers, including MM. In addition to the ubiquitin-dependent degradation of MYC by the 26S proteasome, MYC levels are also regulated in a ubiquitin-independent manner through the REGγ activation of the 20S proteasome. Here, we demonstrate that a small molecule activator of the 20S proteasome, TCH-165, decreases MYC protein levels, in a manner that parallels REGγ protein-mediated MYC degradation. TCH-165 enhances MYC degradation and reduces cancer cell growth in vitro and in vivo models of multiple myeloma by enhancing apoptotic signaling, as assessed by targeted gene expression analysis of cancer pathways. Furthermore, 20S proteasome enhancement is well tolerated in mice and dogs. These data support the therapeutic potential of small molecule-driven 20S proteasome activation for the treatments of MYC-driven cancers, especially MM.

Chronic cerebral hypoperfusion in male rats results in sustained HPA activation and hyperinsulinemia.

Vascular contributions to cognitive impairment and dementia (VCID) is a spectrum of cognitive deficits caused by cerebrovascular disease, for which insulin resistance is a major risk factor. A major cause of VCID is chronic cerebral hypoperfusion (CCH). Under stress, sustained hypothalamic-pituitary-adrenal axis (HPA) activation can result in insulin resistance. Little is known about the effects of CCH on the HPA axis. We hypothesized that CCH causes sustained HPA activation and insulin resistance. Male rats were subjected to bilateral carotid artery stenosis (BCAS) for 12 wk to induce CCH and VCID. BCAS reduced cerebral blood flow and caused memory impairment. Plasma adrenocorticotropic hormone was increased in the BCAS rats (117.2 ± 9.6 vs. 88.29 ± 9.1 pg/mL, BCAS vs. sham, P = 0.0236), as was corticosterone (220 ± 21 vs. 146 ± 18 ng/g feces, BCAS vs. sham, P = 0.0083). BCAS rats were hypoglycemic (68.1 ± 6.1 vs. 76.5 ± 5.9 mg/dL, BCAS vs. sham, P = 0.0072), with increased fasting insulin (481.6 ± 242.6 vs. 97.94 ± 40.02 pmol/L, BCAS vs. sham, P = 0.0003) indicating that BCAS rats were insulin resistant [homeostasis model assessment of ß-cell function-insulin resistance (HOMA-IR): 11.71 ± 6.47 vs. 2.62 ± 0.93; BCAS vs. control, P = 0.0008]. Glucose tolerance tests revealed that BCAS rats had lower blood glucose areas under the curve (AUCs) than controls (250 ± 12 vs. 326 ± 20 mg/dL/h, BCAS vs. sham, P = 0.0075). These studies indicate that CCH causes sustained activation of the HPA and results in insulin resistance, a condition that is expected to worsen VCID.NEW & NOTEWORTHY Cerebrovascular disease and insulin resistance are two major risk factors for the development of dementia. Here, we demonstrate that chronic cerebral hypoperfusion results in glucocorticoid excess and hyperinsulinemia. This study indicates that chronic cerebral hypoperfusion, glucocorticoid excess, and insulin resistance participate in a detrimental cycle that could exacerbate cerebral vascular disease and dementia.

Soluble epoxide hydrolase inhibition improves cognitive function and parenchymal artery dilation in a hypertensive model of chronic cerebral hypoperfusion.

OBJECTIVE: Parenchymal arterioles (PAs) regulate perfusion of the cerebral microcirculation, and impaired PA endothelium-dependent dilation occurs in dementia models mimicking chronic cerebral hypoperfusion (CCH). Epoxyeicosatrienoic acids (EETs) are vasodilators; their actions are potentiated by soluble epoxide hydrolase (sEH) inhibition. We hypothesized that chronic sEH inhibition with trifluoromethoxyphenyl-3 (1-propionylpiperidin-4-yl) urea (TPPU) would prevent cognitive dysfunction and improve PA dilation in a hypertensive CCH model. METHODS: Bilateral carotid artery stenosis (BCAS) was used to induce CCH in twenty-week-old male stroke-prone spontaneously hypertensive rats (SHSRP) that were treated with vehicle or TPPU for 8 weeks. Cognitive function was assessed by novel object recognition. PA dilation and structure were assessed by pressure myography, and mRNA expression in brain tissue was assessed by qRT-PCR. RESULTS: TPPU did not enhance resting cerebral perfusion, but prevented CCH-induced memory deficits. TPPU improved PA endothelium-dependent dilation but reduced the sensitivity of PAs to a nitric oxide donor. TPPU treatment had no effect on PA structure or biomechanical properties. TPPU treatment increased brain mRNA expression of brain derived neurotrophic factor, doublecortin, tumor necrosis factor-alpha, sEH, and superoxide dismutase 3, CONCLUSIONS: These data suggest that sEH inhibitors may be viable treatments for cognitive impairments associated with hypertension and CCH.

Rs10230207 genotype confers changes in HDAC9 and TWIST1, but not FERD3L in lymphoblasts from patients with intracranial aneurysm.

Intracranial aneurysms (IA) are weakened outpouchings of the arterial wall in the cerebrovasculature. Rupture of an IA often leads to devastating consequences. The early identification of IA patients is crucial for management of their condition. A genetic variant at rs10230207, located nearby the HDAC9, TWIST1, and FERD3L genes, is associated with IA. HDAC9 is a class IIa histone deacetylase that mediates vascular smooth muscle cell dysfunction. TWIST1 is a mechanosensitive transcription factor and its expression is reduced in unstable carotid atherosclerotic plaques. In this study, the expression of the HDAC9, TWIST1, and FERD3L genes was characterized and associated with the presence of the rs10230207 genetic variant. Allelic discrimination and gene expression analysis were performed using lymphoblasts from 85 population controls and 109 IA patients. Subjects that were heterozygous (GT) within rs10230207 were 4.32 times more likely to have an IA than those that were homozygous for the reference allele (GG; 95%CI 1.23 to 14.16). Subjects that were homozygous (TT) were 8.27 times more likely to have an IA than those that were GG (95%CI 2.45 to 27.85). While the presence of the risk allele was not associated with changes in FERD3L gene expression, the risk allele was associated with increased HDAC9 and decrease in TWIST1 mRNA expression. The significant inverse correlation between HDAC9 and TWIST1 gene expression suggests that changes in the expression of both of genes may contribute to the formation of IAs.

Substituted quinolines as noncovalent proteasome inhibitors.

Screening of a library of diverse heterocyclic scaffolds identified substituted quinolines as inhibitors of the human proteasome. The heterocyclic library was prepared via a novel titanium-catalyzed multicomponent coupling reaction, which rendered a diverse set of isoxazoles, pyrimidines, pyrroles, pyrazoles and quinolines. SAR of the parent lead compound indicated that hydrophobic residues on the benzo-moiety significantly improved potency. Lead compound 25 inhibits the chymotryptic-like proteolytic activity of the proteasome (IC50 5.4µM), representing a new class of nonpeptidic, noncovalent proteasome inhibitors.

The effects of gestational and chronic atrazine exposure on motor behaviors and striatal dopamine in male Sprague-Dawley rats.

This study sought to investigate the effects of environmentally relevant gestational followed by continued chronic exposure to the herbicide, atrazine, on motor function, cognition, and neurochemical indices of nigrostriatal dopamine (DA) activity in male rats. Dams were treated with 100 µg/kg atrazine, 10mg/kg atrazine, or vehicle on gestational day 1 through postnatal day 21. Upon weaning, male offspring continued daily vehicle or atrazine gavage treatments for an additional six months. Subjects were tested in a series of behavioral assays, and 24h after the last treatment, tissue samples from the striatum were analyzed for DA and 3,4-dihydroxyphenylacetic acid (DOPAC). At 10mg/kg, this herbicide was found to produce modest disruptions in motor functioning, and at both dose levels it significantly lowered striatal DA and DOPAC concentrations. These results suggest that exposures to atrazine have the potential to disrupt nigrostriatal DA neurons and behaviors associated with motor functioning.

Indolo-phakellins as ß5-specific noncovalent proteasome inhibitors.

The proteasome represents an invaluable target for the treatment of cancer and autoimmune disorders. The application of proteasome inhibitors, however, remains limited to blood cancers because their reactive headgroups and peptidic scaffolds convey unfavorable pharmacodynamic properties. Thus, the discovery of more drug-like lead structures is indispensable. In this study, we present the first structure of the proteasome in complex with an indolo-phakellin that exhibits a unique noncovalent binding mode unparalleled by all hitherto reported inhibitors. The natural product inspired pentacyclic alkaloid binds solely and specificially into the spacious S3 subpocket of the proteasomal ß5 substrate binding channel, gaining major stabilization through halogen bonding with the protein backbone. The presented compound provides an ideal scaffold for the structure-based design of subunit-specific nonpeptidic proteasome-blockers.

Inhibition of the human proteasome by imidazoline scaffolds.

The proteasome has emerged as the primary target for the treatment of multiple myeloma. Unfortunately, nearly all patients develop resistance to competitive-type proteasome inhibitors such as bortezomib. Herein, we describe the optimization of noncompetitive proteasome inhibitors to yield derivatives that exhibit nanomolar potency (compound 49, IC50 130 nM) toward proteasome inhibition and overcome bortezomib resistance. These studies illustrate the feasibility of the development of noncompetitive proteasome inhibitors as additives and/or alternatives to competitive proteasome inhibitors.

Noncompetitive modulation of the proteasome by imidazoline scaffolds overcomes bortezomib resistance and delays MM tumor growth in vivo.

Multiple myeloma (MM) is a malignant disorder of differentiated B-cells for which standard care involves the inhibition of the proteasome. All clinically used proteasome inhibitors, including the chemotherapeutic drug bortezomib, target the catalytic active sites of the proteasome and inhibit protein proteolysis by competing with substrate binding. However, nearly all (~97%) patients become intolerant or resistant to treatments within a few years, after which the average survival time is less than 1 year. We describe herein the inhibition of the human proteasome via a noncompetitive mechanism by the imidazoline scaffold, TCH-13. Consistent with a mechanism distinct from that of competitive inhibitors, TCH-013 acts additively with and overcomes resistance to bortezomib. Importantly, TCH-013 induces apoptosis in a panel of myeloma and leukemia cell lines, but in contrast, normal lymphocytes, primary bone marrow stromal cells (hBMSC), and macrophages are resistant to its cytotoxic effects. TCH-013 was equally effective in blocking MM cell growth in co-cultures of MM cells with hBMSC isolated from CD138 negative bone marrow (BM) samples of MM patients. The cellular activity translated well in vivo where TCH-013 delayed tumor growth in an MM xenograft model to a similar extent as bortezomib.

Reprint of BMCL_19101.

The pathogenesis of rheumatoid arthritis is mainly driven by NF-κB-mediated production of cytokines, such as TNF-α. We report herein that the orally available imidazoline-based NF-κB inhibitor, TCH-013, was found to significantly reduce TNF-α signaling and attenuate collagen antibody induced arthritis in BALB/c mice.

Attenuation of collagen-induced arthritis by orally available imidazoline-based NF-κB inhibitors.

The pathogenesis of rheumatoid arthritis is mainly driven by NF-κB-mediated production of cytokines, such as TNF-α. We report herein that the orally available imidazoline-based NF-κB inhibitor, TCH-013, was found to significantly reduce TNF-α signaling and attenuate collagen antibody induced arthritis in BALB/c mice.

Palau'amine and related oroidin alkaloids dibromophakellin and dibromophakellstatin inhibit the human 20S proteasome.

We report herein that the oroidin-derived alkaloids palau'amine (1), dibromophakellin (2), and dibromophakellstatin (3) inhibit the proteolytic activity of the human 20S proteasome as well as the (i)20S immunoproteasome catalytic core. Palau'amine is found to prevent the degradation of ubiquitinylated proteins, including IκBα, in cell culture, which may be indicative of the potential mechanism by which these agents exhibit their exciting cytotoxic and immunosuppressive properties.

Characterization of the biosynthetic operon for the antibacterial peptide herbicolin in Pantoea vagans biocontrol strain C9-1 and incidence in Pantoea species.

Pantoea vagans C9-1 is a biocontrol strain that produces at least two antibiotics inhibiting the growth of Erwinia amylovora, the causal agent of fire blight disease of pear and apple. One antibiotic, herbicolin I, was purified from culture filtrates of P. vagans C9-1 and determined to be 2-amino-3-(oxirane-2,3-dicarboxamido)-propanoyl-valine, also known as N(ß)-epoxysuccinamoyl-DAP-valine. A plasposon library was screened for mutants that had lost the ability to produce herbicolin I. It was shown that mutants had reduced biocontrol efficacy in immature pear assays. The biosynthetic gene cluster in P. vagans C9-1 was identified by sequencing the flanking regions of the plasposon insertion sites. The herbicolin I biosynthetic gene cluster consists of 10 coding sequences (CDS) and is located on the 166-kb plasmid pPag2. Sequence comparisons identified orthologous gene clusters in Pantoea agglomerans CU0119 and Serratia proteamaculans 568. A low incidence of detection of the biosynthetic cluster in a collection of 45 Pantoea spp. from biocontrol, environmental, and clinical origins showed that this is a rare trait among the tested strains.

Synthesis and evaluation of debromohymenialdisine-derived Chk2 inhibitors.

Natural products have been the subject of interest for drug discovery and as tools for understanding the underlying cellular pathways in various diseases. We present herein the synthesis and evaluation of new analogs of the marine sponge metabolite, debromohymenialdisine, as checkpoint kinase 2 (Chk2) inhibitors. We illustrate herein that slight modifications to the natural product scaffold can induce strong selectivity for Chk2 over Chk1. These Chk2 inhibitors can serve as drug templates or molecular tools to gain insight in Chk2 mediated radioprotection.

Structural-activity relationship study of highly-functionalized imidazolines as potent inhibitors of nuclear transcription factor-kappaB mediated IL-6 production.

We herein describe the synthesis and anti-inflammatory properties of a small library of imidazoline-based NF-kappaB inhibitors. The structure-activity relationship of various substituents on an imidazoline core structure was evaluated for the ability to inhibit NF-kappaB mediated IL-6 production. Optimization of the scaffolds was pursued by correlating luciferase-based NF-kappaB reporter assays with inhibition of IL-6 production in IL-1beta stimulated human blood. Several derivatives were found to inhibit NF-kappaB mediated IL-6 production in the nanomolar range in IL-1beta stimulated human blood.

Nuclear factor-kappaB mediated inhibition of cytokine production by imidazoline scaffolds.

The mammalian nuclear transcription factor NF-kappaB is responsible for the transcription of multiple cytokines, including the pro-inflammatory cytokines tumor necrosis factor alpha (TNF-alpha) and interleukin 6 (IL-6). Elevated levels of pro-inflammatory cytokines play an important role in the pathogenesis of inflammatory disorders such as rheumatoid arthritis (RA). Inhibition of the pro-inflammatory transcription factor NF-kappaB has therefore been identified as a possible therapeutic treatment for RA. We describe herein the synthesis and biological activity of a series of imidazoline-based scaffolds as potent inhibitors of NF-kappaB mediated gene transcription in cell culture as well as inhibitors of TNF-alpha and IL-6 production in interleukin 1 beta (IL-1beta) stimulated human blood.

Sensitization of tumor cells toward chemotherapy: enhancing the efficacy of camptothecin with imidazolines.

Activation of nuclear transcription factor kappaB (NF-kappaB) by chemotherapeutic agents was found to protect cells from apoptosis. In light of its central role in regulating the cellular resistance to apoptotic agents, inhibition of NF-kappaB-mediated gene transcription may sensitize tumor cells to chemotherapeutic agents and enhance their efficacy. We describe herein a noncytotoxic imidazoline scaffold that sensitizes leukemia T cells to the chemotherapeutic agent camptothecin. No significant induction of apoptosis was found when cells were treated with the imidazoline; however, pretreatment of cells with this agent resulted in a drastic enhancement in efficacy of camptothecin (approximately 75-fold). Elucidation of the potential cellular mechanism revealed that the imidazoline prevents nuclear translocation of NF-kappaB. These findings indicate that inhibition of NF-kappaB by this imidazoline may present improved strategies in the chemotherapeutic treatment of cancer.

Inhibition of cytokine production by hymenialdisine derivatives.

We describe herein the synthesis and biological activity of two indoloazepines that are structurally related to the marine sponge metabolite hymenialdisine. The natural product hymenialdisine was found to be a potent inhibitor of interleukin-2 (IC(50) = 2.4 microM) and tumor necrosis factor alpha (IC(50) = 1.4 microM) production. One of the hymenialdisine derived indoloazepines was found to also inhibit interleukin-2 (IC(50) = 3.5 microM) and tumor necrosis factor alpha (IC(50) = 8.2 microM) production.