Subchapter 4.1

Perspective: Run1 Eval1 Synthesis

Evidence Sub-Set: Unknown Evidence
Alignment Score: 5/7 | Consilience Score: 6/7

Even though this fact check looked at unique up-to-date abstracts, new evidence may refute this answer in the future. Although 'Zero Hallucinated Moneyshot Quotes' is programmatically enforced, AI is not always immune to inadvertently/erroneously misinterpreting data. This is not medical or professional advice, but instead, is an opinion calculated by AI based on the literature evaluated.

CLAIM EVALUATED AND ANSWER TO USER

"How do electrophilic binding and charge polarity modulate proteinopathy in Alzheimer's Disease, specifically regarding BBB interactions?"

ABSTRACT & REWRITTEN CLAIM

The interplay between protein solubility and blood-brain barrier (BBB) permeability is heavily modulated by metabolite-protein interactions, covalent modifications, and electrostatic properties. While exogenous molecules like β-hydroxybutyrate (βHB) and polyamine modifications regulate protein solubility and trans-barrier transit, these processes are distinct from traditional covalent enzyme-inhibitor paradigms, relying instead on selective, non-covalent, and metabolically regulated mechanisms.

INTRODUCTION & JUSTIFICATION

In the context of Alzheimer's Disease (AD), proteinopathy—specifically the aggregation of amyloid-β (Aβ)—represents a critical failure of proteostasis. Research indicates that metabolic regulators such as β-hydroxybutyrate (βHB) act as metabolic regulators of protein solubility. βHB is a small molecule metabolite which primarily provides an oxidative substrate for ATP during hypoglycemic conditions, and also regulates other cellular processes through covalent and noncovalent protein interactions. Notably, the mechanism by which βHB affects protein insolubility does not depend on pH, solute load, or covalent modification; rather, it is observable in mouse brain in vivo after delivery of a ketone ester. This mechanism is selective for pathological proteins such as amyloid-β, and exogenous βHB ameliorates pathology in nematode models of amyloid-β aggregation toxicity. Furthermore, βHB facilitates the clearance of neurodegeneration-related proteins from the mouse brain, likely via βHB-induced autophagy.

Concurrently, the transport of therapeutic agents across the BBB is governed by charge polarity and covalent modification strategies. The permeability of Abeta at the blood-brain barrier can be increased by at least twofold through covalent modification with the naturally occurring polyamine, putrescine. This effect is not limited to amylopeptides; the permeability of the blood-nerve barrier (BNB) and the blood-brain barrier (BBB) to superoxide dismutase (SOD), insulin, albumin, and IgG in normal adult rats was quantified by measuring the permeability coefficient-surface area product (PS) with the intravenous bolus injection technique before and after covalent protein modification with naturally occurring polyamines-putrescine (PUT), spermidine (SPD), and spermine (SPM). The PS value of the BNB for PUT-SOD was 21.1-fold greater than the native SOD, and the PS values of the BBB for PUT-SOD ranged from 17.6-fold greater for the thalamus to 23.6-fold greater for the caudate-putamen compared with native SOD. However, electrostatic charge is not a monolithic determinant of permeability. Although cationic proteins are known to interact with fixed anionic charges on the lumen of the microvascular endothelium, this observation of decreased permeability with increased positive charge distribution along the aliphatic carbon chain of the polyamines implies mechanisms other than simple electrostatic interaction involving charge density. It is surprising that the PS values of the BNB and BBB decreased with the increasing number of positive charges of the protonated amino groups on the polyamines (PUT>SPD>SPM). This suggests that specific transporters or physico-chemical properties, rather than simple charge-based electrostatic adsorption, drive the enhanced flux across the BBB.

DISCUSSION: NOVEL & OVERLOOKED

* βHB-induced protein insolubility is a specific regulatory mechanism for pathological proteins, distinct from general pH-driven aggregation.
* Modification with naturally occurring polyamines increases protein permeability at the blood-brain barrier by up to several hundred-fold in the case of IgG.
* There is a counter-intuitive inverse relationship between the number of positive charges on polyamine-modified proteins and their BBB permeability.
* The selectivity of cysteine protease inhibitors is inherently problematic due to the limitations of targeting isozymes within protease families.
* Nanoparticle surface charge and morphology (e.g., negative zeta potential) significantly influence binding interactions with regulatory proteins like Tyrosine Hydroxylase.
* Low-voltage electromembrane extraction efficiency is highly dependent on both logP values and the number of basic functional groups on the analyte.
* Protein retention in RP-HPLC is significantly altered by intermediate pH mobile phases through shifts in overall charge, polarity, and hydrophobicity.

EVIDENCE, METHODOLOGY & CITATIONS

1. PMID: 37461525- "βHB is a small molecule metabolite which primarily provides an oxidative substrate for ATP during hypoglycemic conditions, and also regulates other cellular processes through covalent and noncovalent protein interactions."
2. PMID: 39626664- "βHB-induced protein insolubility is not dependent on covalent protein modification, pH, or solute load, and is observable in mouse brain in vivo after delivery of a ketone ester."
3. PMID: 39626664- "This mechanism is selective for pathological proteins such as amyloid-β, and exogenous βHB ameliorates pathology in nematode models of amyloid-β aggregation toxicity."
4. PMID: 37461525- "Finally, we show enrichment of neurodegeneration-related proteins among βHB targets and the clearance of these targets from mouse brain, likely via βHB-induced autophagy."
5. PMID: 10932157- "the permeability of Abeta at the blood-brain barrier can be increased by at least twofold through covalent modification with the naturally occurring polyamine, putrescine."
6. PMID: 8627316- "The PS value of the BNB for PUT-SOD was 21.1-fold greater than the native SOD, and the PS values of the BBB for PUT-SOD ranged from 17.6-fold greater for the thalamus to 23.6-fold greater for the caudate-putamen compared with native SOD."
7. PMID: 8627316- "It is surprising that the PS values of the BNB and BBB decreased with the increasing number of positive charges of the protonated amino groups on the polyamines (PUT>SPD>SPM)."
8. PMID: 8627316- "Although cationic proteins are known to interact with fixed anionic charges on the lumen of the microvascular endothelium, this observation of decreased permeability with increased positive charge distribution along the aliphatic carbon chain of the polyamines implies mechanisms other than simple electrostatic interaction involving charge density."
9. PMID: 26713267- "obtaining selectivity within families of cysteine proteases and their isozymes is problematic."
10. PMID: 33417459- "However, as seen by thioflavin-T fluorescence, TH aggregated at this more acidic pH."
11. PMID: 32661727- "RVG29-nanoparticles clearly increased in 1.5-fold the permeability across the in vitro model of blood-brain barrier after 4 h of incubation compared with non-functionalized nanoparticles."
12. PMID: 32661727- "Zeta potential values were between -20 and -25 mV."
13. PMID: 22704880- "Drug substances with logP≥2.3 and with one basic group were all extracted at low voltages and with a strong compound selectivity which appeared to have some influence from the polar surface area of the compound."
14. PMID: 19285675- "the increase in the pH with the 20 mM ammonium formate mobile phase led to significant changes in both peptide retention to the reversed-phase column and the collision-induced dissociation at the MS/MS stage as a consequence of the changes in the physico-chemical properties of these peptides, such as their overall charge, polarity and relative hydrophobicity."
15. PMID: 33417459- "As pSiNPs are negatively charged, we also investigated the binding at pH 6, which makes TH less negatively charged than at pH 7."
16. PMID: 21981972- "In the PVOH, the polar liqudiffuses into the polymer network, whereas in the SA latex the hydrophobic nature prevents the diffusion into the polymer matrix and there exists surface diffusion."
17. PMID: 37461525- "This activity is shared by select structurally similar metabolites, is not dependent on covalent protein modification, pH, or solute load, and is observable in mouse brain in vivo after delivery of a ketone ester."
18. PMID: 39626664- "βHB primarily provides ATP substrate during periods of reduced glucose availability, and regulates other cellular processes through protein interactions."
19. PMID: 10363910- "By switching to the all D-enantiomers of YiAbeta11 and PUT-YiAbeta11, significant protection from degradation by proteases in rat plasma was obtained with only 1.9% and 5.7% degradation at 15 min after intravenous bolus injection, respectively."
20. PMID: 26713267- "Cysteine proteases continue to provide validated targets for treatment of human diseases."

Systemic Logic Chain Framework

Metabolic State --> βHB Production (Align: 6)
Rationale: Metabolic shifts under hypoglycemic conditions drive ketone body production.
3-Hydroxybutyric Acid --> Protein Aggregates (Align: 6)
Rationale: βHB regulates the solubility of neurodegeneration-related proteins.
Protein Aggregates --> Autophagy (Align: 6)
Rationale: βHB promotes clearance of targeted proteins via autophagic pathways.
Polyamines --> Blood-Brain Barrier (Align: 6)
Rationale: Covalent polyamine modification facilitates transit across the BBB.

Gap Analysis Audit

Study Type/Intent: in_vivo/in_vitro / permeability/proteostasis
Justification: Evidence is robust for βHB and polyamine effects, but the specific molecular mechanism for how charge polarity modulates the 'non-electrostatic' permeability component remains partially gap-filled by the polyamine transporter hypothesis.
Predicted Result: Polyamines utilize specific transport proteins rather than simple electrostatic adsorption.

Chapter 5

Verbatim Quote Audit Log

The following excerpts represent direct, character-for-character verifications from the raw source material. PathMap guarantees 100% fidelity on these passed citations.

VERIFIED VERBATIM (PMID: 39626664)

"βHB-induced protein insolubility is not dependent on covalent protein modification, pH, or solute load, and is observable in mouse brain in vivo after delivery of a ketone ester."

VERIFIED VERBATIM (PMID: 37461525)

"This activity is shared by select structurally similar metabolites, is not dependent on covalent protein modification, pH, or solute load, and is observable in mouse brain in vivo after delivery of a ketone ester."

VERIFIED VERBATIM (PMID: 8627316)

"It is surprising that the PS values of the BNB and BBB decreased with the increasing number of positive charges of the protonated amino groups on the polyamines (PUT>SPD>SPM)."

VERIFIED VERBATIM (PMID: 8627316)

"Although cationic proteins are known to interact with fixed anionic charges on the lumen of the microvascular endothelium, this observation of decreased permeability with increased positive charge distribution along the aliphatic carbon chain of the polyamines implies mechanisms other than simple electrostatic interaction involving charge density."

VERIFIED VERBATIM (PMID: 8627316)

"The PS value of the BNB for PUT-SOD was 21.1-fold greater than the native SOD, and the PS values of the BBB for PUT-SOD ranged from 17.6-fold greater for the thalamus to 23.6-fold greater for the caudate-putamen compared with native SOD."

VERIFIED VERBATIM (PMID: 33417459)

"However, as seen by thioflavin-T fluorescence, TH aggregated at this more acidic pH."

VERIFIED VERBATIM (PMID: 32661727)

"Zeta potential values were between -20 and -25 mV."

VERIFIED VERBATIM (PMID: 32661727)

"RVG29-nanoparticles clearly increased in 1.5-fold the permeability across the in vitro model of blood-brain barrier after 4 h of incubation compared with non-functionalized nanoparticles."

VERIFIED VERBATIM (PMID: 26713267)

"obtaining selectivity within families of cysteine proteases and their isozymes is problematic."

VERIFIED VERBATIM (PMID: 22704880)

"Drug substances with logP≥2.3 and with one basic group were all extracted at low voltages and with a strong compound selectivity which appeared to have some influence from the polar surface area of the compound."

VERIFIED VERBATIM (PMID: 19285675)

"the increase in the pH with the 20 mM ammonium formate mobile phase led to significant changes in both peptide retention to the reversed-phase column and the collision-induced dissociation at the MS/MS stage as a consequence of the changes in the physico-chemical properties of these peptides, such as their overall charge, polarity and relative hydrophobicity."

VERIFIED VERBATIM (PMID: 37461525)

"Finally, we show enrichment of neurodegeneration-related proteins among βHB targets and the clearance of these targets from mouse brain, likely via βHB-induced autophagy."

VERIFIED VERBATIM (PMID: 21981972)

"In the PVOH, the polar liqudiffuses into the polymer network, whereas in the SA latex the hydrophobic nature prevents the diffusion into the polymer matrix and there exists surface diffusion."

VERIFIED VERBATIM (PMID: 10932157)

"the permeability of Abeta at the blood-brain barrier can be increased by at least twofold through covalent modification with the naturally occurring polyamine, putrescine."

VERIFIED VERBATIM (PMID: 39626664)

"This mechanism is selective for pathological proteins such as amyloid-β, and exogenous βHB ameliorates pathology in nematode models of amyloid-β aggregation toxicity."

VERIFIED VERBATIM (PMID: 33417459)

"As pSiNPs are negatively charged, we also investigated the binding at pH 6, which makes TH less negatively charged than at pH 7."

VERIFIED VERBATIM (PMID: 37461525)

"βHB is a small molecule metabolite which primarily provides an oxidative substrate for ATP during hypoglycemic conditions, and also regulates other cellular processes through covalent and noncovalent protein interactions."

VERIFIED VERBATIM (PMID: 37461525)

VERIFIED VERBATIM (PMID: 39626664)

"βHB-induced protein insolubility is not dependent on covalent protein modification, pH, or solute load, and is observable in mouse brain in vivo after delivery of a ketone ester."

VERIFIED VERBATIM (PMID: 39626664)

"This mechanism is selective for pathological proteins such as amyloid-β, and exogenous βHB ameliorates pathology in nematode models of amyloid-β aggregation toxicity."

VERIFIED VERBATIM (PMID: 37461525)

"Finally, we show enrichment of neurodegeneration-related proteins among βHB targets and the clearance of these targets from mouse brain, likely via βHB-induced autophagy."

VERIFIED VERBATIM (PMID: 10932157)

"the permeability of Abeta at the blood-brain barrier can be increased by at least twofold through covalent modification with the naturally occurring polyamine, putrescine."

VERIFIED VERBATIM (PMID: 8627316)

"It is surprising that the PS values of the BNB and BBB decreased with the increasing number of positive charges of the protonated amino groups on the polyamines (PUT>SPD>SPM)."

VERIFIED VERBATIM (PMID: 8627316)

VERIFIED VERBATIM (PMID: 26713267)

"obtaining selectivity within families of cysteine proteases and their isozymes is problematic."

VERIFIED VERBATIM (PMID: 33417459)

"However, as seen by thioflavin-T fluorescence, TH aggregated at this more acidic pH."

VERIFIED VERBATIM (PMID: 32661727)

"RVG29-nanoparticles clearly increased in 1.5-fold the permeability across the in vitro model of blood-brain barrier after 4 h of incubation compared with non-functionalized nanoparticles."

VERIFIED VERBATIM (PMID: 32661727)

"Zeta potential values were between -20 and -25 mV."

VERIFIED VERBATIM (PMID: 22704880)

VERIFIED VERBATIM (PMID: 19285675)

VERIFIED VERBATIM (PMID: 33417459)

"As pSiNPs are negatively charged, we also investigated the binding at pH 6, which makes TH less negatively charged than at pH 7."

VERIFIED VERBATIM (PMID: 21981972)

"In the PVOH, the polar liqudiffuses into the polymer network, whereas in the SA latex the hydrophobic nature prevents the diffusion into the polymer matrix and there exists surface diffusion."

VERIFIED VERBATIM (PMID: 37461525)

VERIFIED VERBATIM (PMID: 39626664)

"βHB primarily provides ATP substrate during periods of reduced glucose availability, and regulates other cellular processes through protein interactions."

VERIFIED VERBATIM (PMID: 10363910)

"By switching to the all D-enantiomers of YiAbeta11 and PUT-YiAbeta11, significant protection from degradation by proteases in rat plasma was obtained with only 1.9% and 5.7% degradation at 15 min after intravenous bolus injection, respectively."

VERIFIED VERBATIM (PMID: 26713267)

"Cysteine proteases continue to provide validated targets for treatment of human diseases."

VERIFIED VERBATIM (PMID: 9751199)

"Previous investigations from our laboratory have demonstrated that the covalent modification of a variety of proteins, including antioxidant enzymes, with the naturally occurring polyamines--putrescine (PUT), spermidine, and spermine--dramatically increases their permeability coefficient-surface area product (PS) at the blood-brain and blood-nerve barriers after parenteral administration."

VERIFIED VERBATIM (PMID: 8149897)

"However, avidin is a cationic protein, which causes a marked increase in the systemic clearance of avidin-based conjugates from the plasma compartment."

VERIFIED VERBATIM (PMID: 37461525)

"Overall, these data indicate a new metabolically regulated mechanism of proteostasis relevant to aging and AD."

VERIFIED VERBATIM (PMID: 39626664)

"We demonstrate βHB-induced protein insolubility is not dependent on covalent protein modification, pH, or solute load, and is observable in mouse brain in vivo after delivery of a ketone ester."

VERIFIED VERBATIM (PMID: 1602382)

"rCD4 is a cationic protein and the mechanism of rCD4 transport through the BBB may be analogous to the absorptive-mediated transcytosis of other polycationic proteins."

VERIFIED VERBATIM (PMID: 3097421)

"These data link rapchanges in ODC and polyamines to blood-brain barrier breakdown, and suggest that the abnormal permeability is associated with an acute, polyamine-mediated stimulation of microvillus formation, endocytosis, and vesicular transport in capillary endothelium."

VERIFIED VERBATIM (PMID: 18726697)

"Adsorptive-mediated transcytosis (AMT) provides a means for brain delivery of medicines across the blood-brain barrier (BBB). The BBB is readily equipped for the AMT process: it provides both the potential for binding and uptake of cationic molecules to the luminal surface of endothelial cells"

VERIFIED VERBATIM (PMID: 41961384)

"Spermidine treatment also improved gut epithelial barrier integrity and reduced epithelial release of high-mobility group box 1 (HMGB1) into systemic circulation."

VERIFIED VERBATIM (PMID: 38666466)

"Mechanistically, it is unveiled that BHB infusion is taken up by monocarboxylic actransporter 1 (MCT1) specifically expressed in cerebral endothelium and upregulated the expression of tight junction protein ZO-1 by enhancing local β-hydroxybutyrylation of H3K9 at the promoter of TJP1 gene."

VERIFIED VERBATIM (PMID: 35002279)

"Genetic silencing of VPS35 in human brain endothelial cells; measurement of retromer complex system proteins, autophagy and ubiquitin-proteasome systems."

VERIFIED VERBATIM (PMID: 2600816)

"These studies demonstrate that histone is transported through the BBB in vivo via absorptive-mediated transport."

VERIFIED VERBATIM (PMID: 16529872)

"Polyethylenimine is a chemically synthesized small molecule cationization agent; the charge density is greater than a peptide-based cationic cluster such as trans-acting factor, and it is resistant to proteolysis in vivo."

VERIFIED VERBATIM (PMID: 9365024)

"Modification of CAT with PUT increased its permeability 2-3-fold and preserved 67% of its enzymatic activity compared to native CAT"

VERIFIED VERBATIM (PMID: 34544422)

"Using CRISPR/Cas9 gene editing in BBB organoarrays, we showed that clathrin, but not caveolin, is required for transferrin receptor-dependent transcytosis."

VERIFIED VERBATIM (PMID: 41213123)

"This study identifies Kbhb modification of mitochondrial proteins as a potential mechanism linking ketone body availability to mitochondrial function in DbCM."

VERIFIED VERBATIM (PMID: 42498022)

"Path modeling revealed consistent positive associations between Calditerricola enrichment and polyamine synthesis, membrane stability, and proteostasis."

VERIFIED VERBATIM (PMID: 41828397)

"The convergence of their actions on energy restoration, reactive oxygen species reduction, and epigenetic modulation of protective pathways suggests their synergistic potential."

VERIFIED VERBATIM (PMID: 42012729)

"Preclinical studies consistently show that spermidine enhances proteostasis, reduces soluble amyloid-beta, improves microglial phagocytic activity, preserves mitochondrial fitness, and attenuates inflammatory responses in models of aging and neurodegeneration."

VERIFIED VERBATIM (PMID: 18726697)

VERIFIED VERBATIM (PMID: 9365024)

"Modification of CAT with PUT increased its permeability 2-3-fold and preserved 67% of its enzymatic activity compared to native CAT"

VERIFIED VERBATIM (PMID: 38666466)

VERIFIED VERBATIM (PMID: 39626664)

"We demonstrate βHB-induced protein insolubility is not dependent on covalent protein modification, pH, or solute load, and is observable in mouse brain in vivo after delivery of a ketone ester."

VERIFIED VERBATIM (PMID: 1602382)

"rCD4 is a cationic protein and the mechanism of rCD4 transport through the BBB may be analogous to the absorptive-mediated transcytosis of other polycationic proteins."

VERIFIED VERBATIM (PMID: 3097421)

VERIFIED VERBATIM (PMID: 2600816)

"These studies demonstrate that histone is transported through the BBB in vivo via absorptive-mediated transport."

VERIFIED VERBATIM (PMID: 16529872)

VERIFIED VERBATIM (PMID: 35002279)

"Genetic silencing of VPS35 in human brain endothelial cells; measurement of retromer complex system proteins, autophagy and ubiquitin-proteasome systems."

VERIFIED VERBATIM (PMID: 41213123)

"This study identifies Kbhb modification of mitochondrial proteins as a potential mechanism linking ketone body availability to mitochondrial function in DbCM."

VERIFIED VERBATIM (PMID: 42498022)

"Path modeling revealed consistent positive associations between Calditerricola enrichment and polyamine synthesis, membrane stability, and proteostasis."

VERIFIED VERBATIM (PMID: 41828397)

"The convergence of their actions on energy restoration, reactive oxygen species reduction, and epigenetic modulation of protective pathways suggests their synergistic potential."

VERIFIED VERBATIM (PMID: 42012729)

VERIFIED VERBATIM (PMID: 34544422)

"Using CRISPR/Cas9 gene editing in BBB organoarrays, we showed that clathrin, but not caveolin, is required for transferrin receptor-dependent transcytosis."

VERIFIED VERBATIM (PMID: 8149897)

"However, avidin is a cationic protein, which causes a marked increase in the systemic clearance of avidin-based conjugates from the plasma compartment."

VERIFIED VERBATIM (PMID: 37461525)

"Overall, these data indicate a new metabolically regulated mechanism of proteostasis relevant to aging and AD."

VERIFIED VERBATIM (PMID: 41961384)

"Spermidine treatment also improved gut epithelial barrier integrity and reduced epithelial release of high-mobility group box 1 (HMGB1) into systemic circulation."

VERIFIED VERBATIM (PMID: 34579098)

"We found no change in MCT1 and MCT4 expression."

VERIFIED VERBATIM (PMID: 34668776)

"The interaction of p17 with chemokine receptor 2 (CXCR2) at the surface of brain endothelial cells triggers transcytosis."

VERIFIED VERBATIM (PMID: 39587264)

"Loss of Mettl1 results in aberrant metabolic reprogramming and cardiomyocyte immaturity, leading to heart failure, although some clinical features can be rescued by β-hydroxybutyrate supplementation."

Chapter 8

Abstract Repository

Raw text abstracts programmatically cached during the evaluation phase. Only those cited within the active verification paths are included below.

PMID: 1602382 Mapped to Reference [15]

ID: 1602382 Title: Transport of recombinant CD4 through the rat blood-brain barrier in vivo. Abstract: One class of potential acquired immunodeficiency syndrome therapeutics are derivatives of recombinant CD4 (rCD4). Therefore, the present investigations use in vivo techniques to measure the rate at which [3H]rCD4 is transported through the blood-brain barrier (BBB). In addition, the binding of labeled rCD4 to isolated human and bovine brain capillaries is measured. These studies show that [3H]CD4 is removed rapidly from the bloodstream with a half-time of 12.6 +/- 0.9 min. The volume of distribution (Vd) of the protein in brain increases with time and reaches a Vd that is 11.1 +/- 1.1-fold greater than the brain Vd of plasma marker, native rat serum albumin. In addition, [3H]rCD4 is extracted rapidly by the kidney and the ratio of rCD4 Vd to native rat serum albumin Vd in the rat kidney reaches 99 +/- 5 at 60 min after i.v. injection. rCD4 is shown to undergo transcytosis through the BBB using an internal carotid artery perfusion/capillary depletion method coupled with gel filtration fast protein liquid chromatography. In conclusion, these studies report the unexpected finding that rCD4 is transportable through the BBB. rCD4 is a cationic protein and the mechanism of rCD4 transport through the BBB may be analogous to the absorptive-mediated transcytosis of other polycationic proteins.

PMID: 2600816 Mapped to Reference [17]

ID: 2600816 Title: Transport of histone through the blood-brain barrier. Abstract: The present studies were designed to determine if the endogenous cationic protein, e.g., histone, is capable of penetrating the blood-brain barrier (BBB) in vivo. Calf thymus histone was iodinated with [125I]iodine and was found to be taken up rapidly by isolated bovine brain capillaries used as an in vitro model system of the BBB via a time- and temperature-dependent mechanism. The binding was saturable and a Scatchard plot of the binding data was linear, yielding a KD = 15.2 +/- 2.8 microM and a maximal binding = 7.7 +/- 1.0 nmol/mg of protein. Other polycations such as protamine or polylysine markedly inhibited uptake of [125I] histone, but cationized albumin demonstrated minimal inhibition and cationized immunoglobulin caused no inhibition of bovine brain capillary uptake of [125I]histone. The in vivo brain VD of [125I] histone reached 159 +/- 70 microliters/g by 10 min of carotid arterial perfusion as compared to the 10-min VD for [3H]albumin, 17 +/- 7 microliter/g. Most of this uptake represented sequestration by the vasculature, but approximately 8% of the total histone taken up by brain was found to be transported unmetabolized (based on trichloroacetic acid precipitability of brain supernatant [( 125I]) into brain interstitium. These studies demonstrate that histone is transported through the BBB in vivo via absorptive-mediated transport. Thus, histone is an endogenous protein that is capable of transport through the BBB and may be a potential vector for pharmaceutical delivery through the BBB.

PMID: 3097421 Mapped to Reference [16]

ID: 3097421 Title: Blood-brain barrier breakdown by cold injury. Polyamine signals mediate acute stimulation of endocytosis, vesicular transport, and microvillus formation in rat cerebral capillaries. Abstract: Polyamines have been previously implicated in the mediation of blood-brain barrier breakdown induced by cryogenic injury (H Koenig, AD Goldstone, CY Lu, Biochem Biophys Res Commun 116:1039, 1983). We studied acute (less than 5 minute) changes in capillary ultrastructure, microvascular permeability, and the levels of polyamines and their rate regulating synthetic enzyme ornithine decarboxylase (ODC) in rat cerebral cortex after focal cold injury. Microvascular permeability was measured by relative transport of intravenously administered fluorescein. Capillary ultrastructure was studied by quantitative stereology and morphometry after intravenous administration of horseradish peroxidase. Focal cold injury induced a 2.5-, 3.8-, 1.7-, and 1.4-fold increase in the levels of ODC, putrescine, spermidine and spermine, and a 46-fold increase in fluorescein uptake in perilesional cortex. Few capillaries in control cortex contained endocytic pits or horseradish peroxidase-positive vesicles, whereas most capillaries near lesions showed these structures. Cryoinjury induced a 5-fold increase in the relative volume of microvilli and horseradish peroxidase vesicles, a 2.3-fold increase in area of luminal endocytic pits, and a 6.3-fold increase in area of abluminal exocytic pits. The ODC inhibitor alpha-difluoromethylornithine blocked the cryoinjury-induced changes in ODC, polyamines, fluorescein uptake, and capillary ultrastructure. Putrescine negated the effect of alpha-difluoromethylornithine or capillary ultrastructure, and was previously shown to nullify the alpha-difluoromethylornithine effects on polyamines and fluorescein permeability (cited above). These data link rapid changes in ODC and polyamines to blood-brain barrier breakdown, and suggest that the abnormal permeability is associated with an acute, polyamine-mediated stimulation of microvillus formation, endocytosis, and vesicular transport in capillary endothelium.

PMID: 8149897 Mapped to Reference [25]

ID: 8149897 Title: Pharmacokinetics and saturable blood-brain barrier transport of biotin bound to a conjugate of avidin and a monoclonal antibody to the transferrin receptor. Abstract: The delivery of biotinylated therapeutics through the blood-brain barrier (BBB) may be facilitated by the use of avidin-based chimeric peptide conjugates. The latter are formed by conjugating avidin to a BBB drug delivery vector, which is a protein that undergoes receptor-mediated transcytosis through the BBB. The murine OX26 monoclonal antibody to the rat transferrin receptor undergoes receptor-mediated transport through the BBB, and previous studies have shown that a [3H]biotin/avidin-OX26 conjugate is effectively transported through the BBB. However, avidin is a cationic protein, which causes a marked increase in the systemic clearance of avidin-based conjugates from the plasma compartment. The present studies describe attempts to elevate the reduced plasma area under the curve (AUC) of [3H]biotin/avidin-OX26 by preloading or coloading with unconjugated OX26 antibody or unconjugated avidin. Both systemic clearance and BBB transport of avidin-OX26 were equally affected by OX26 preloading or coloading; this had inverse effects on the plasma AUC and the BBB permeability surface area product with no resulting change in the fractional delivery of [3H]biotin to brain. Conversely, avidin coloading preferentially reduced brain clearance of the [3H]biotin/avidin-OX26 conjugate, without substantial alteration in the plasma AUC and greatly reduced the fractional delivery of [3H]biotin to brain. In summary, these studies show that the use of avidin-based vectors results in rapid systemic clearance, which causes a reduction in the delivery of [3H]biotin to brain, despite a comparable BBB permeability coefficient for either the unconjugated OX26 antibody or the avidin-OX26 conjugate.

PMID: 8627316 Mapped to Reference [4]

ID: 8627316 Title: Polyamine modification increases the permeability of proteins at the blood-nerve and blood-brain barriers. Abstract: The permeability of the blood-nerve barrier (BNB) and the blood-brain barrier (BBB) to superoxide dismutase (SOD), insulin, albumin, and IgG in normal adult rats was quantified by measuring the permeability coefficient-surface area product (PS) with the intravenous bolus injection technique before and after covalent protein modification with naturally occurring polyamines-putrescine (PUT), spermidine (SPD), and spermine (SPM). The PS value of the BNB for PUT-SOD was 21.1-fold greater than the native SOD, and the PS values of the BBB for PUT-SOD ranged from 17.6-fold greater for the thalamus to 23.6-fold greater for the caudate-putamen compared with native SOD. In a similar manner, polyamine-modified insulin showed a 1.7-2.0-fold increase in PS of the BNB and BBB compared with the high values of native insulin. Polyamine-modified albumin showed a remarkable 54-165-fold increase in PS of the BNB and BBB compared with native albumin, whereas PUT-IgG resulted in an even higher increase in the PS that ranged from 111- to 349-fold for nerve and different brain regions compared with native IgG. Polyamine modification of proteins, therefore, can dramatically increase the permeability at the BNB and BBB of a variety of proteins with widely differing M(r) and function. It is surprising that the PS values of the BNB and BBB decreased with the increasing number of positive charges of the protonated amino groups on the polyamines (PUT>SPD>SPM). Although cationic proteins are known to interact with fixed anionic charges on the lumen of the microvascular endothelium, this observation of decreased permeability with increased positive charge distribution along the aliphatic carbon chain of the polyamines implies mechanisms other than simple electrostatic interaction involving charge density. It is suggested that the polyamine transporter may be responsible for the transport of these polyamine-modified proteins. Systemic administration of polyamine-modified peptides and proteins might prove to be an efficient approach to deliver therapeutic agents into the CNS and PNS for the treatment of a variety of neurological diseases.

PMID: 9365024 Mapped to Reference [13]

ID: 9365024 Title: Putrescine-modified catalase with preserved enzymatic activity exhibits increased permeability at the blood-nerve and blood-brain barriers. Abstract: Much evidence exists in support of the hypothesis that free radicals contribute to the pathogenesis of several neurodegenerative disorders and that mechanisms of free radical generation occur both intracellularly and extracellularly. Previous studies in this laboratory have shown that covalent modification of growth factors and antioxidant enzymes with the naturally occurring polyamine, putrescine, increases their permeability at the blood-nerve and blood-brain barriers (BNB and BBB), but does not significantly inhibit bioactivity. Furthermore, putrescine-modified superoxide dismutase (SOD) was shown to reduce neurodegeneration in a rat model of global cerebral ischemia. The purpose of the present study was to modify the antioxidant enzyme, catalase (CAT), with putrescine (PUT) at carboxylic acid groups whose ionization, and hence reactivity, was controlled with pH and investigate the effects on permeability and enzymatic activity. Modification of CAT with PUT increased its permeability 2-3-fold and preserved 67% of its enzymatic activity compared to native CAT and 137% compared to lyophilized CAT. The results of this study indicate that modification of CAT with putrescine increases its permeability while preserving enzymatic activity. PUT-SOD administered in combination with PUT-CAT may eliminate both the superoxide radical and the H2O2 produced from the dismutation of superoxide, respectively, and thus prevent the formation of hydroxyl radicals. This combination may exhibit increased neuroprotective effects, compared to native enzymes, following systemic administration for the treatment of free radical associated neurodegenerative disorders.

PMID: 10363910 Mapped to Reference [11]

ID: 10363910 Title: Beta-sheet breaker peptide inhibitor of Alzheimer's amyloidogenesis with increased blood-brain barrier permeability and resistance to proteolytic degradation in plasma. Abstract: Short synthetic peptides homologous to the central region of Abeta but bearing proline residues as beta-sheet blockers have been shown in vitro to bind to Abeta with high affinity, partially inhibit Abeta fibrillogenesis, and redissolve preformed fibrils. While short peptides have been used extensively as therapeutic drugs in medicine, two important problems associated with their use in central nervous system diseases have to be addressed: (a) rapid proteolytic degradation in plasma, and (b) poor blood-brain barrier (BBB) permeability. Recently, we have demonstrated that the covalent modification of proteins with the naturally occurring polyamines significantly increases their permeability at the BBB. We have extended this technology to iAbeta11, an 11-residue beta-sheet breaker peptide that inhibits Abeta fibrillogenesis, by covalently modifying this peptide with the polyamine, putrescine (PUT), and evaluating its plasma pharmacokinetics and BBB permeability. After a single intravenous bolus injection in rats, both 125I-YiAbeta11 and 125I-PUT-YiAbeta11 showed rapid degradation in plasma as determined by trichloroacetic acid (TCA) precipitation and paper chromatography. By switching to the all D-enantiomers of YiAbeta11 and PUT-YiAbeta11, significant protection from degradation by proteases in rat plasma was obtained with only 1.9% and 5.7% degradation at 15 min after intravenous bolus injection, respectively. The permeability coefficient x surface area product at the BBB was five- sevenfold higher in the cortex and hippocampus for the 125I-PUT-D-YiAbeta11 compared to the 125I-D-YiAbeta11, with no significant difference in the residual plasma volume. In vitro assays showed that PUT-D-YiAbeta11 retains its ability to partially inhibit Abeta fibrillogenesis and dissolve preformed amyloid fibrils. Because of its five- to sevenfold increase in permeability at the BBB and its resistance to proteolysis in the plasma, this polyamine-modified beta-sheet breaker peptide may prove to be an effective inhibitor of amyloidogenesis in vivo and, hence, an important therapy for Alzheimer's disease.

PMID: 10932157 Mapped to Reference [3]

ID: 10932157 Title: Targeting alzheimer amyloid plaques in vivo. Abstract: The only definitive diagnosis for Alzheimer disease (AD) at present is postmortem observation of neuritic plaques and neurofibrillary tangles in brain sections. Radiolabeled amyloid-beta peptide (Abeta), which has been shown to label neuritic plaques in vitro, therefore could provide a diagnostic tool if it also labels neuritic plaques in vivo following intravenous injection. In this study, we show that the permeability of Abeta at the blood-brain barrier can be increased by at least twofold through covalent modification with the naturally occurring polyamine, putrescine. We also show that, following intravenous injection, radiolabeled, putrescine-modified Abeta labels amyloid deposits in vivo in a transgenic mouse model of AD, as well as in vitro in human AD brain sections. This technology, when applied to humans, may be used to detect plaques in vivo, allowing early diagnosis of the disease and therapeutic intervention before cognitive decline occurs.

PMID: 16529872 Mapped to Reference [18]

ID: 16529872 Title: In vivo protein transduction to the CNS. Abstract: Proteins and peptides are useful research and therapeutic tools, however applications are limited because delivery to the desired location is not easily achievable. There are two hurdles in protein/peptide delivery to the brain: the blood-brain barrier and intracellular penetration. Penetration to both brain and the intracellular space can be achieved by adjusting hydrophilicity, and small molecule pharmacological agents have been successfully developed using this approach. But with proteins and peptides, it is difficult to modify the hydrophilicity without influencing biological functions. Trans-acting factor protein from the human immunodeficiency virus contains a highly conserved cationic peptide sequence necessary for transduction across the cell membrane. While trans-acting factor peptide has been used for in vitro protein transduction, its in vivo application is very limited because it is rapidly degraded by proteolysis. Polyethylenimine is a chemically synthesized small molecule cationization agent; the charge density is greater than a peptide-based cationic cluster such as trans-acting factor, and it is resistant to proteolysis in vivo. We first tested intracellular protein transduction following direct brain injection in mice using polyethylenimine-conjugated green fluorescence protein and beta-galactosidase (molecular weights 29 and 540 kDa, respectively). Polyethylenimine-conjugates penetrated to the intracellular space immediately surrounding the injection site within one hour. We further tested polyethylenimine-mediated protein transduction following intranasal administration, which bypasses the blood-brain barrier. Polyethylenimine-conjugates in pH 7.5 solution did not reach the brain, probably because the polyethylenimine-conjugates penetrated into the intracellular space where first exposed to the tissue, i.e. at the nasal mucosae. We temporarily reduced the electrostatic interaction between cationized polyethylenimine-conjugates and cellular surfaces by adjusting the pH to 4.5; solution rapidly reached the brain and penetrated to the intracellular space. This study suggests that polyethylenimine is a useful protein transduction agent in the brain in vivo, and adjusting cationic charge interaction can determine the extent of brain penetration.

PMID: 18726697 Mapped to Reference [12]

ID: 18726697 Title: CNS delivery via adsorptive transcytosis. Abstract: Adsorptive-mediated transcytosis (AMT) provides a means for brain delivery of medicines across the blood-brain barrier (BBB). The BBB is readily equipped for the AMT process: it provides both the potential for binding and uptake of cationic molecules to the luminal surface of endothelial cells, and then for exocytosis at the abluminal surface. The transcytotic pathways present at the BBB and its morphological and enzymatic properties provide the means for movement of the molecules through the endothelial cytoplasm. AMT-based drug delivery to the brain was performed using cationic proteins and cell-penetrating peptides (CPPs). Protein cationization using either synthetic or natural polyamines is discussed and some examples of diamine/polyamine modified proteins that cross BBB are described. Two main families of CPPs belonging to the Tat-derived peptides and Syn-B vectors have been extensively used in CPP vector-mediated strategies allowing delivery of a large variety of small molecules as well as proteins across cell membranes in vitro and the BBB in vivo. CPP strategy suffers from several limitations such as toxicity and immunogenicity--like the cationization strategy--as well as the instability of peptide vectors in biological media. The review concludes by stressing the need to improve the understanding of AMT mechanisms at BBB and the effectiveness of cationized proteins and CPP-vectorized proteins as neurotherapeutics.

PMID: 19285675 Mapped to Reference [9]

ID: 19285675 Title: Peptide mapping with mobile phases of intermediate pH value using capillary reversed-phase high-performance liquid chromatography/electrospray ionisation tandem mass spectrometry. Abstract: This investigation describes the separation of tryptic peptides by capillary reversed-phase high-performance liquid chromatography (RP-HPLC) with eluents in the intermediate pH range, followed by in-line electrospray ionisation tandem mass spectrometry (ESI-MS/MS) analysis. For these purposes, gradient elution procedures with an aqueous eluent containing 20 mM ammonium formate, and an increasing content of acetonitrile or methanol, were employed. Compared to the analysis of the same tryptic peptides under low-pH conditions with an ion-pairing reagent, the increase in the pH with the 20 mM ammonium formate mobile phase led to significant changes in both peptide retention to the reversed-phase column and the collision-induced dissociation at the MS/MS stage as a consequence of the changes in the physico-chemical properties of these peptides, such as their overall charge, polarity and relative hydrophobicity. Thus, improved selectivity for the peptide separation and favourable tandem mass spectrometry analysis could be obtained with eluents in this intermediate pH range. The number of tryptic peptides identified by the new approach for the proteins investigated were significantly higher than that obtained by the conventional low-pH methods. Moreover, analysis of protein digests at very low concentrations was also performed under both acidic and intermediate pH conditions and similar improvements in selectivity and MS/MS detection limits were observed, i.e. identification of more distinct peptides and higher sequence coverage of the protein was obtained when eluents of intermediate pH were employed. This study therefore highlights the potential of conducting peptide mapping in the intermediate pH range to achieve more reliable and sensitive protein identifications with capillary RP-HPLC-ESI-MS/MS.

PMID: 21981972 Mapped to Reference [10]

ID: 21981972 Title: Short timescale inkjet ink component diffusion: an active part of the absorption mechanism into inkjet coatings. Abstract: The structures of inkjet coatings commonly contain a high concentration of fine diameter pores together with a large pore volume capacity. To clarify the interactive role of the porous structure and the coincidentally occurring swelling of binder during inkjet ink vehicle imbibition, coating structures were studied in respect to their absorption behaviour for polar and non-polar liquid. The absorption measurement was performed using compressed pigment tablets, based on a range of pigment types and surface charge polarity, containing either polyvinyl alcohol (PVOH) or styrene acrylic latex (SA) as the binder, by recording the liquid uptake with a microbalance. The results indicate that, at the beginning of liquid uptake, at times less than 2 s, the small pores play the dominant role with respect to the inkjet ink vehicle imbibition. Simultaneously, water molecules diffuse into and within the hydrophilic PVOH binder causing binder swelling, which diminishes the number of active small pores and reduces the diameter of remaining pores, thus slowing the capillary flow as a function of time. The SA latex does not absorb the vehicle, and therefore the dominating phenomenon is then capillary absorption. However, the diffusion coefficient of the water vapour across separately prepared PVOH and SA latex films seems to be quite similar. In the PVOH, the polar liquid diffuses into the polymer network, whereas in the SA latex the hydrophobic nature prevents the diffusion into the polymer matrix and there exists surface diffusion. At longer timescale, permeation flow into the porous coating dominates as the resistive term controlling the capillary driven liquid imbibition rate.

PMID: 22704880 Mapped to Reference [8]

ID: 22704880 Title: Selective electromembrane extraction at low voltages based on analyte polarity and charge. Abstract: Electromembrane extraction (EME) at low voltage (0-15 V) of 29 different basic model drug substances was investigated. The drug substances with logP<2.3 were not extracted at voltages less than 15 V. Extraction of drug substances with logP≥2.3 and with two basic groups were also effectively suppressed by the SLM at voltages less than 15 V. Drug substances with logP≥2.3 and with one basic group were all extracted at low voltages and with a strong compound selectivity which appeared to have some influence from the polar surface area of the compound. For this group of substances, recoveries varied between 0 and 23% at 5 V, whereas, recoveries varied between 5.5 and 51% at 15 V. Based on mass transfer differences related to charge, polarity, and polar surface, highly selective extractions of drug substances were demonstrated from human plasma, urine, and breast milk. An initial evaluation at low voltage (5 V) was compared with similar extractions at a more normal voltage level (50 V), and this supported that reliable data can be obtained under these low-voltage (mild) conditions by EME.

PMID: 26713267 Mapped to Reference [5]

ID: 26713267 Title: Cysteine proteases as therapeutic targets: does selectivity matter? A systematic review of calpain and cathepsin inhibitors. Abstract: Cysteine proteases continue to provide validated targets for treatment of human diseases. In neurodegenerative disorders, multiple cysteine proteases provide targets for enzyme inhibitors, notably caspases, calpains, and cathepsins. The reactive, active-site cysteine provides specificity for many inhibitor designs over other families of proteases, such as aspartate and serine; however, a) inhibitor strategies often use covalent enzyme modification, and b) obtaining selectivity within families of cysteine proteases and their isozymes is problematic. This review provides a general update on strategies for cysteine protease inhibitor design and a focus on cathepsin B and calpain 1 as drug targets for neurodegenerative disorders; the latter focus providing an interesting query for the contemporary assumptions that irreversible, covalent protein modification and low selectivity are anathema to therapeutic safety and efficacy.

PMID: 32661727 Mapped to Reference [7]

ID: 32661727 Title: RVG29-Functionalized Lipid Nanoparticles for Quercetin Brain Delivery and Alzheimer's Disease. Abstract: Lipid nanoparticles (SLN and NLC) were functionalized with the RVG29 peptide in order to target the brain and increase the neuronal uptake through the nicotinic acetylcholine receptors. These nanosystems were loaded with quercetin to take advantage of its neuroprotective properties mainly for Alzheimer's disease. The functionalization of nanoparticles with RVG29 peptide was confirmed by NMR and FTIR. Their morphology was assessed by transmission electron microscopy and nanoparticles size, polydispersity and zeta potential were determined by dynamic light scattering. The in vitro validation tests were conducted in hCMEC/D3 cells, a human blood-brain barrier model and thioflavin T binding assay was conducted to assess the process of amyloid-beta peptide fibrillation typical of Alzheimer's disease. RVG29-nanoparticles displayed spherical morphology and size below 250 nm, which is compatible with brain applications. Zeta potential values were between -20 and -25 mV. Quercetin entrapment efficiency was generally higher than 80% and NLC nanoparticles were able to encapsulate up to 90%. The LDH assay showed that there is no cytotoxicity in hCMEC/D3 cell line and RVG29-nanoparticles clearly increased in 1.5-fold the permeability across the in vitro model of blood-brain barrier after 4 h of incubation compared with non-functionalized nanoparticles. Finally, this nanosystem was capable of inhibiting amyloid-beta aggregation in thioflavin T binding assay, suggesting its great potential for neuroprotection. RVG29-nanoparticles that simultaneously target the blood-brain barrier and induce neurons protection against amyloid-beta fibrillation proved to be an efficient way of quercetin delivery and a promising strategy for future approaches in Alzheimer's disease. Graphical Abstract.

PMID: 33417459 Mapped to Reference [6]

ID: 33417459 Title: Relevance of Electrostatics for the Interaction of Tyrosine Hydroxylase with Porous Silicon Nanoparticles. Abstract: Tyrosine hydroxylase (TH) is the enzyme catalyzing the rate-limiting step in the synthesis of dopamine in the brain. Developing enzyme replacement therapies using TH could therefore be beneficial to patient groups with dopamine deficiency, and the use of nanocarriers that cross the blood-brain barrier seems advantageous for this purpose. Nanocarriers may also help to maintain the structure and function of TH, which is complex and unstable. Understanding how TH may interact with a nanocarrier is therefore crucial for the investigation of such therapeutic applications. This work describes the interaction of TH with porous silicon nanoparticles (pSiNPs), chosen since they have been shown to deliver other macromolecular therapeutics successfully to the brain. Size distributions obtained by dynamic light scattering show a size increase of pSiNPs upon addition of TH and the changes observed at the surface of pSiNPs by transmission electron microscopy also indicated TH binding at pH 7. As pSiNPs are negatively charged, we also investigated the binding at pH 6, which makes TH less negatively charged than at pH 7. However, as seen by thioflavin-T fluorescence, TH aggregated at this more acidic pH. TH activity was unaffected by the binding to pSiNPs most probably because the active site stays available for catalysis, in agreement with calculations of the surface electrostatic potential pointing to the most positively charged regulatory domains in the tetramer as the interacting regions. These results reveal pSiNPs as a promising delivery device of enzymatically active TH to increase local dopamine synthesis.

PMID: 34544422 Mapped to Reference [24]

ID: 34544422 Title: Investigating receptor-mediated antibody transcytosis using blood-brain barrier organoid arrays. Abstract: The pathways that control protein transport across the blood-brain barrier (BBB) remain poorly characterized. Despite great advances in recapitulating the human BBB in vitro, current models are not suitable for systematic analysis of the molecular mechanisms of antibody transport. The gaps in our mechanistic understanding of antibody transcytosis hinder new therapeutic delivery strategy development. We applied a novel bioengineering approach to generate human BBB organoids by the self-assembly of astrocytes, pericytes and brain endothelial cells with unprecedented throughput and reproducibility using micro patterned hydrogels. We designed a semi-automated and scalable imaging assay to measure receptor-mediated transcytosis of antibodies. Finally, we developed a workflow to use CRISPR/Cas9 gene editing in BBB organoid arrays to knock out regulators of endocytosis specifically in brain endothelial cells in order to dissect the molecular mechanisms of receptor-mediated transcytosis. BBB organoid arrays allowed the simultaneous growth of more than 3000 homogenous organoids per individual experiment in a highly reproducible manner. BBB organoid arrays showed low permeability to macromolecules and prevented transport of human non-targeting antibodies. In contrast, a monovalent antibody targeting the human transferrin receptor underwent dose- and time-dependent transcytosis in organoids. Using CRISPR/Cas9 gene editing in BBB organoid arrays, we showed that clathrin, but not caveolin, is required for transferrin receptor-dependent transcytosis. Human BBB organoid arrays are a robust high-throughput platform that can be used to discover new mechanisms of receptor-mediated antibody transcytosis. The implementation of this platform during early stages of drug discovery can accelerate the development of new brain delivery technologies.

PMID: 34579098 Mapped to Reference [27]

ID: 34579098 Title: A Proton-Coupled Transport System for β-Hydroxy-β-Methylbutyrate (HMB) in Blood-Brain Barrier Endothelial Cell Line hCMEC/D3. Abstract: β-Hydroxy-β-methylbutyrate (HMB), a leucine metabolite, is used as a nutritional ingredient to improve skeletal muscle health. Preclinical studies indicate that this supplement also elicits significant benefits in the brain; it promotes neurite outgrowth and prevents age-related reductions in neuronal dendrites and cognitive performance. As orally administered HMB elicits these effects in the brain, we infer that HMB crosses the blood-brain barrier (BBB). However, there have been no reports detailing the transport mechanism for HMB in BBB. Here we show that HMB is taken up in the human BBB endothelial cell line hCMEC/D3 via H+-coupled monocarboxylate transporters that also transport lactate and β-hydroxybutyrate. MCT1 (monocarboxylate transporter 1) and MCT4 (monocarboxylate transporter 4) belonging to the solute carrier gene family SLC16 (solute carrier, gene family 16) are involved, but additional transporters also contribute to the process. HMB uptake in BBB endothelial cells results in intracellular acidification, demonstrating cotransport with H+. Since HMB is known to activate mTOR with potential to elicit transcriptomic changes, we examined the influence of HMB on the expression of selective transporters. We found no change in MCT1 and MCT4 expression. Interestingly, the expression of LAT1 (system L amino acid transporter 1), a high-affinity transporter for branched-chain amino acids relevant to neurological disorders such as autism, is induced. This effect is dependent on mTOR (mechanistic target of rapamycine) activation by HMB with no involvement of histone deacetylases. These studies show that HMB in systemic circulation can cross the BBB via carrier-mediated processes, and that it also has a positive influence on the expression of LAT1, an important amino acid transporter in the BBB.

PMID: 34668776 Mapped to Reference [28]

ID: 34668776 Title: The HIV-1 Matrix Protein p17 Does Cross the Blood-Brain Barrier. Abstract: Human immunodeficiency virus type 1 (HIV-1)-associated neurocognitive disorder (HAND) remains an important neurological manifestation in HIV-1-infected (HIV+) patients. Furthermore, detection of the HIV-1 matrix protein p17 (p17) in the central nervous system (CNS) and its ability to form toxic assemblies in the brain have been recently confirmed. Here, we show for the first time, using both an in vitro blood-brain barrier (BBB) model and in vivo biodistribution studies in healthy mice, that p17 can cross the BBB. There is rapid brain uptake with 0.35% ± 0.19% of injected activity per gram of tissue (IA/g) 2 min after administration, followed by brain accumulation with 0.28% ± 0.09% IA/g after 1 h. The interaction of p17 with chemokine receptor 2 (CXCR2) at the surface of brain endothelial cells triggers transcytosis. The present study supports the hypothesis of a direct role of free p17 in neuronal dysfunction in HAND by demonstrating its intrinsic ability to reach the CNS. IMPORTANCE The percentage of patients affected by HIV-1-associated neurocognitive disorder (HAND) ranges from 30% to 50% of HIV-infected (HIV+) patients. The mechanisms leading to HAND development need to be elucidated, but the roles of secreted viral proteins, chemokines, and proinflammatory molecules appear to be clear. In particular, the blood-brain barrier (BBB) represents a route for entry into the central nervous system (CNS) and thus plays an important role in HAND. Several findings suggest a key role for the HIV-1 matrix protein p17 (p17) as a microenvironmental factor capable of inducing neurocognitive disorders. Here, we show the ability of the p17 to cross the BBB and to reach the CNS, thus playing a crucial role in neuronal dysfunction in HAND.

PMID: 35002279 Mapped to Reference [19]

ID: 35002279 Title: Dysregulation of the Retromer Complex in Brain Endothelial Cells Results in Accumulation of Phosphorylated Tau. Abstract: Transport through endothelial cells of the blood-brain barrier (BBB) involves a complex group of structures of the endo-lysosome system such as early and late endosomes, and the retromer complex system. Studies show that neuronal dysregulation of the vacuolar protein sorting 35 (VPS35), the main component of the retromer complex recognition core, results in altered protein trafficking and degradation and is involved in neurodegeneration. Since the functional role of VPS35 in endothelial cells has not been fully investigated, in the present study we aimed at characterizing the effect of its downregulation on these pathways. Genetic silencing of VPS35 in human brain endothelial cells; measurement of retromer complex system proteins, autophagy and ubiquitin-proteasome systems. VPS35-downregulated endothelial cells had increased expression of LC3B2/1 and more ubiquitinated products, markers of autophagy flux and impaired proteasome activity, respectively. Additionally, compared with controls VPS35 downregulation resulted in significant accumulation of tau protein and its phosphorylated isoforms. Our findings demonstrate that in brain endothelial cells retromer complex dysfunction by influencing endosome-lysosome degradation pathways results in altered proteostasis. Restoration of the retromer complex system function should be considered a novel therapeutic approach to rescue endothelial protein transport.

PMID: 37461525 Mapped to Reference [1]

ID: 37461525 Title: β-hydroxybutyrate is a metabolic regulator of proteostasis in the aged and Alzheimer disease brain. Abstract: Loss of proteostasis is a hallmark of aging and Alzheimer disease (AD). Here, we identify β-hydroxybutyrate (βHB), a ketone body, as a regulator of protein solubility in the aging brain. βHB is a small molecule metabolite which primarily provides an oxidative substrate for ATP during hypoglycemic conditions, and also regulates other cellular processes through covalent and noncovalent protein interactions. We demonstrate βHB-induced protein insolubility across in vitro, ex vivo, and in vivo mouse systems. This activity is shared by select structurally similar metabolites, is not dependent on covalent protein modification, pH, or solute load, and is observable in mouse brain in vivo after delivery of a ketone ester. Furthermore, this phenotype is selective for pathological proteins such as amyloid-β, and exogenous βHB ameliorates pathology in nematode models of amyloid-β aggregation toxicity. We have generated a comprehensive atlas of the βHB-induced protein insolublome ex vivo and in vivo using mass spectrometry proteomics, and have identified common protein domains within βHB target sequences. Finally, we show enrichment of neurodegeneration-related proteins among βHB targets and the clearance of these targets from mouse brain, likely via βHB-induced autophagy. Overall, these data indicate a new metabolically regulated mechanism of proteostasis relevant to aging and AD.

PMID: 38666466 Mapped to Reference [14]

ID: 38666466 Title: Adaptive Metabolic Responses Facilitate Blood-Brain Barrier Repair in Ischemic Stroke via BHB-Mediated Epigenetic Modification of ZO-1 Expression. Abstract: Adaptive metabolic responses and innate metabolites hold promising therapeutic potential for stroke, while targeted interventions require a thorough understanding of underlying mechanisms. Adiposity is a noted modifiable metabolic risk factor for stroke, and recent research suggests that it benefits neurological rehabilitation. During the early phase of experimental stroke, the lipidomic results showed that fat depots underwent pronounced lipolysis and released fatty acids (FAs) that feed into consequent hepatic FA oxidation and ketogenesis. Systemic supplementation with the predominant ketone beta-hydroxybutyrate (BHB) is found to exert discernible effects on preserving blood-brain barrier (BBB) integrity and facilitating neuroinflammation resolution. Meanwhile, blocking FAO-ketogenesis processes by administration of CPT1α antagonist or shRNA targeting HMGCS2 exacerbated endothelial damage and aggravated stroke severity, whereas BHB supplementation blunted these injuries. Mechanistically, it is unveiled that BHB infusion is taken up by monocarboxylic acid transporter 1 (MCT1) specifically expressed in cerebral endothelium and upregulated the expression of tight junction protein ZO-1 by enhancing local β-hydroxybutyrylation of H3K9 at the promoter of TJP1 gene. Conclusively, an adaptive metabolic mechanism is elucidated by which acute lipolysis stimulates FAO-ketogenesis processes to restore BBB integrity after stroke. Ketogenesis functions as an early metabolic responder to restrain stroke progression, providing novel prospectives for clinical translation.

PMID: 39587264 Mapped to Reference [29]

ID: 39587264 Title: The tRNA methyltransferase Mettl1 governs ketogenesis through translational regulation and drives metabolic reprogramming in cardiomyocyte maturation. Abstract: After birth, the heart undergoes a shift in energy metabolism and cytoarchitecture to enhance efficient energy production and cardiac contraction, which is essential for postnatal development and growth. However, the precise mechanisms regulating this process remain elusive. Here we show that the RNA modification enzyme Mettl1 is a critical regulator of postnatal metabolic reprogramming and cardiomyocyte maturation in mice, primarily through its influence on the translation of the rate-limiting ketogenesis enzyme Hmgcs2. Our findings reveal that ketogenesis is vital for the postnatal transition of fuel from glucose to fatty acids in cardiomyocytes, achieved by modulating tricarboxylic acid cycle-related enzymatic activity via lysine β-hydroxybutyrylation protein modification. Loss of Mettl1 results in aberrant metabolic reprogramming and cardiomyocyte immaturity, leading to heart failure, although some clinical features can be rescued by β-hydroxybutyrate supplementation. Our study provides mechanistic insights into how Mettl1 regulates metabolic reprogramming in neonatal cardiomyocytes and highlights the importance of ketogenesis in cardiomyocyte maturation.

PMID: 39626664 Mapped to Reference [2]

ID: 39626664 Title: β-hydroxybutyrate is a metabolic regulator of proteostasis in the aged and Alzheimer disease brain. Abstract: Loss of proteostasis is a hallmark of aging and Alzheimer disease (AD). We identify β-hydroxybutyrate (βHB), a ketone body, as a regulator of protein solubility. βHB primarily provides ATP substrate during periods of reduced glucose availability, and regulates other cellular processes through protein interactions. We demonstrate βHB-induced protein insolubility is not dependent on covalent protein modification, pH, or solute load, and is observable in mouse brain in vivo after delivery of a ketone ester. This mechanism is selective for pathological proteins such as amyloid-β, and exogenous βHB ameliorates pathology in nematode models of amyloid-β aggregation toxicity. We generate libraries of the βHB-induced protein insolublome using mass spectrometry proteomics, and identify common protein domains and upstream regulators. We show enrichment of neurodegeneration-related proteins among βHB targets and the clearance of these targets from mouse brain. These data indicate a metabolically regulated mechanism of proteostasis relevant to aging and AD.

PMID: 41213123 Mapped to Reference [20]

ID: 41213123 Title: β-Hydroxybutyrylation Links Ketone Metabolism to Mitochondrial Remodeling in Diabetic Cardiomyopathy. Abstract: Diabetic cardiomyopathy (DbCM) is characterized by metabolic remodeling and energetic stress independent of coronary artery disease. Increased reliance on fatty acid and ketone body metabolism has been observed in DbCM, but the regulatory mechanisms linking altered substrate use to myocardial dysfunction remain poorly understood. In particular, lysine β-hydroxybutyrate (Kbhb), a ketone body-derived, posttranslational modification, has emerged as a potentially critical regulator but has not been fully investigated. We conducted a comprehensive multiomics study integrating metabolomics, transcriptomics, proteomics, and Kbhb-specific proteomics on myocardial tissues in a well-established mouse model of DbCM. Kbhb-modified proteins were systematically mapped and quantified, followed by motif, subcellular localization, and protein-protein interaction analyses. DbCM cardiac tissue exhibited coordinated upregulations of fatty acid β-oxidation, ketone metabolism, and tricarboxylic acid cycle activity at the transcriptomic, proteomic, and metabolomic levels. Kbhb profiling revealed extensive mitochondrial protein modification, with Atp5f1a-K239 identified as a key modification site strongly correlated with β-hydroxybutyrate and isocitric acid concentrations. This study identifies Kbhb as a potential metabolic-epigenetic modifier linking ketone body availability to the regulation of mitochondrial proteins in DbCM. Our findings provide novel insights into metabolic-epigenetic cross talk and identify potential therapeutic targets for interventions to restore mitochondrial function in alleviating diabetic heart disease. We performed a multiomics study to better understand dysfunctions in diabetic cardiomyopathy (DbCM) and specifically identify links between lysine β-hydroxybutyrylation (Kbhb), a ketone body-derived, posttranslational modification, and cardiac dysfunction. DbCM cardiac tissue exhibited coordinated upregulations of fatty acid β-oxidation, ketone metabolism, and tricarboxylic acid cycle activity at the transcriptomic, proteomic, and metabolomic levels. Mitochondrial proteins showed that high Kbhb modification and modification of the Atp5f1a-K239 site were strongly correlated with high β-hydroxybutyrate and isocitric acid concentrations. This study identifies Kbhb modification of mitochondrial proteins as a potential mechanism linking ketone body availability to mitochondrial function in DbCM.

PMID: 41828397 Mapped to Reference [22]

ID: 41828397 Title: Impact of the Combination of Epigallocatechin Gallate and Ellagic Acid Supplemented with Ketone Bodies on Energetic Restoration of Mitochondrial Dysfunction and Metabolic Inefficiencies in Patients with Multiple Sclerosis: A Review. Abstract: Multiple sclerosis (MS) is characterized by progressive mitochondrial dysfunction affecting complexes I, III, and IV of the electron transport chain, contributing to axonal energy failure and neurodegeneration. This review examines the potential of combining β-hydroxybutyrate (βHB), epigallocatechin-3-gallate (EGCG), and ellagic acid (EA) as a multi-target therapeutic strategy to restore mitochondrial function in patients with MS. Experimental and clinical studies demonstrate that each compound exerts complementary mechanisms. Ketone bodies provide an alternative energy substrate and restore complex I activity via sirtuin-dependent pathways. EGCG acts predominantly at the peripheral level by reducing systemic inflammation and oxidative stress. EA-derived urolithins effectively cross the blood-brain barrier to directly enhance mitochondrial biogenesis and respiratory chain function in the central nervous system. Clinical trials have reported improvements in fatigue, cognition, mood, and muscle function following supplementation with these compounds. The convergence of their actions on energy restoration, reactive oxygen species reduction, and epigenetic modulation of protective pathways suggests their synergistic potential. Optimized delivery strategies, including exogenous ketone salts, liposomal EGCG, and microencapsulated EA, may overcome bioavailability limitations and interindividual variability in the gut microbiota metabolism.

PMID: 41961384 Mapped to Reference [26]

ID: 41961384 Title: Spermidine Attenuates Neuroimmune Dysfunction in Gulf War Illness via Modulation of the Gut- Brain Axis. Abstract: Gulf War illness (GWI) affects nearly one-third of US veterans deployed during the 1990-1991 Gulf War (GW) and is characterized by chronic fatigue, neuroinflammation, and gut dysbiosis. Through comprehensive fecal metabolomics sequencing, our lab previously reported the depletion of beneficial metabolites including spermidine in the preclinical GWI mouse model. Spermidine is an endogenously synthesized polyamine known for its anti-inflammatory and mucosal barrier protective effects in various pathological diseases. Given its established role in mitigating intestinal inflammation and maintaining homeostasis, this study investigated the therapeutic potential of spermidine in a persistent (22 weeks) GWI mouse model, with a specific focus on gut-brain axis regulation. Our results demonstrated that spermidine effectively restored both microbial richness and diversity by selectively enriching beneficial bacterial taxa and suppressing growth of opportunistic pathogens, which are otherwise dysregulated following exposure to GW chemicals. Spermidine treatment also improved gut epithelial barrier integrity and reduced epithelial release of high-mobility group box 1 (HMGB1) into systemic circulation. Recent studies on GWI have implicated a critical role of gut-derived damage-associated molecular patterns (DAMPs), particularly HMGB1 in mediating neuroinflammation. Our findings indicate that systemic levels of HMGB1 critically influence the extent of blood-brain barrier (BBB) disruption and subsequent microglial activation. Mechanistically, spermidine activated intestinal aryl hydrocarbon receptor (AhR)/nuclear factor erythroid 2-related factor 2 (Nrf2)/heme oxygenase-1 (HO-1) signaling, which played a role in limiting intestinal HMGB1 release and suppressing downstream receptor for advanced glycation end-product (RAGE)-mediated microglial activation in the brain. In vitro results indicate spermidine promoted AhR/Nrf2 nuclear translocation which reduced LPS-induced HMGB1 release from primary intestinal epithelial cells (IECs), effects abrogated by AhR inhibition. Additionally, we observed that HMGB1 directly induces microglial activation via RAGE receptors in immortalized microglial (IMG) cell lines in a dose-dependent manner. These results demonstrate that spermidine decreases neuroinflammation by modulating gut-brain axis pathophysiology associated with GWI. Together, this study demonstrates the therapeutic role of spermidine in ameliorating systemic and neurological disturbances in GWI.

PMID: 42012729 Mapped to Reference [23]

ID: 42012729 Title: Spermidine in the aging brain: mechanisms, preclinical evidence, and clinical perspectives. Abstract: Spermidine, a naturally occurring polyamine, has emerged as a candidate modulator of brain aging because it regulates autophagy, mitochondrial function, oxidative balance, and neuroinflammatory signaling. This review summarizes current knowledge on spermidine biology, including its synthesis, catabolism, transport, and cell-specific handling in neurons and glia, with emphasis on ATP13A2/ATP13A4-dependent trafficking and EP300-linked autophagy. Preclinical studies consistently show that spermidine enhances proteostasis, reduces soluble amyloid-beta, improves microglial phagocytic activity, preserves mitochondrial fitness, and attenuates inflammatory responses in models of aging and neurodegeneration. Human evidence is encouraging but still limited: observational studies associate higher dietary spermidine intake with better cognitive outcomes, whereas interventional trials suggest possible benefits in subjective cognitive decline and mild dementia, with variable dose-dependent effects. Overall, spermidine is a biologically plausible nutraceutical for healthy brain aging, but larger, well-designed clinical trials are needed to define efficacy, bioavailability, safety, and optimal therapeutic use across prodromal, early-stage, and disease-specific patient populations.

PMID: 42498022 Mapped to Reference [21]

ID: 42498022 Title: Genome-resolved characterization of candidate thermotolerance traits and predicted protein conformational behavior in Calditerricola during hyperthermophilic composting of organic wastes. Abstract: Hyperthermophilic composting (HC) can generate temperatures above 80 °C without external heating, thereby accelerating organic-waste stabilization; however, how dominant heat-adapted microorganisms maintain cellular function under such extreme conditions remains unclear. Here, we integrated metagenomics, metagenome-assembled genome reconstruction, Calditerricola-resolved functional profiling, partial least squares path modeling, and molecular dynamics simulations to investigate candidate thermotolerance-related traits associated with Calditerricola enrichment during HC. The pile temperature reached 82.6 °C on day 2 and peaked at 86.6 °C on day 4, accompanied by progressive humification, with humic substances increasing from 40.45 to 51.28 mg/g and HA/FA reaching 3.45. Microbial communities differed significantly among composting phases (R2 = 0.975, P = 0.004), and Calditerricola increased from 0.02% in the initial phase to 6.1% in the thermophilic phase before declining to 0.7% in maturation. Community-level profiles showed comparatively modest variation in selected thermotolerance-related pathways, whereas the independently normalized Calditerricola profile displayed clearer phase-associated increases in functions linked to polyamine metabolism, membrane/envelope homeostasis, proteostasis, and DNA maintenance. Path modeling revealed consistent positive associations between Calditerricola enrichment and polyamine synthesis, membrane stability, and proteostasis. qPCR further revealed phase-associated increases in the community-level copy numbers of representative target genes, particularly polA and speE. During 100-ns simulations at 360 K, the predicted apo structures of PolA, AtpD, SpeE, and FabH retained their overall folds and comparatively stable catalytic-residue geometries. Together, these results define an association-based multi-module framework of candidate traits linked to Calditerricola persistence during HC, providing a genome-resolved basis for prioritizing testable thermotolerance targets in engineered high-temperature waste-treatment systems.

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Run1 Eval1 Synthesis

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