Kristin M. Bullock

Central Nervous System Delivery of Intranasal Insulin: Mechanisms of Uptake and Effects on Cognition

Intranasal insulin has shown efficacy in patients with Alzheimers disease (AD), but there are no preclinical studies determining whether or how it reaches the brain. Here, we showed that insulin applied at the level of the cribriform plate via the nasal route quickly distributed throughout the brain and reversed learning and memory deficits in an AD mouse model. Intranasal insulin entered the blood stream poorly and had no peripheral metabolic effects. Uptake into the brain from the cribriform plate was saturable, stimulated by PKC inhibition, and responded differently to cellular pathway inhibitors than did insulin transport at the blood-brain barrier. In summary, these results show intranasal delivery to be an effective way to deliver insulin to the brain.

CNS tau efflux via exosomes is likely increased in Parkinson's disease but not in Alzheimer's disease.

Alzheimers disease (AD) and Parkinsons disease (PD) involve tau pathology. Tau is detectable in blood, but its clearance from neuronal cells and the brain is poorly understood.

Sleep fragmentation and sepsis differentially impact blood-brain barrier integrity and transport of tumor necrosis factor-α in aging

The factors by which aging predisposes to critical illness are varied, complex, and not well understood. Sepsis is considered a quintessential disease of old age because the incidence and mortality of severe sepsis increases in old and the oldest old individuals. Aging is associated with dramatic changes in sleep quality and quantity and sleep increasingly becomes fragmented with age. In healthy adults, sleep disruption induces inflammation. Multiple aspects of aging and of sleep dysregulation interact via neuroimmune mechanisms. Tumor necrosis factor-α (TNF), a cytokine involved in sleep regulation and neuroimmune processes, exerts some of its effects on the CNS by crossing the blood-brain barrier (BBB). In this study we examined the impact of sepsis, sleep fragmentation, and aging on BBB disruption and TNF transport into brain. We used the cecal ligation and puncture (CLP) model of sepsis in young and aged mice that were either undisturbed or had their sleep disrupted. There was a dichotomous effect of sepsis and sleep disruption with age: sepsis disrupted the BBB and increased TNF transport in young mice but not in aged mice, whereas sleep fragmentation disrupted the BBB and increased TNF transport in aged mice, but not in young mice. Combining sleep fragmentation and CLP did not produce a greater effect on either of these BBB parameters than did either of these manipulations alone. These results suggest that the mechanisms by which sleep fragmentation and sepsis alter BBB functions are fundamentally different from one another and that a major change in the organisms responses to those insults occurs with aging.

Tau Proteins Cross the Blood-Brain Barrier.

Tauopathies are a hallmark of many neurodegenerative diseases, including Alzheimers disease and traumatic brain injuries. It has been demonstrated that amyloid-beta peptides, alpha-synuclein, and prion proteins cross the blood-brain barrier (BBB), contributing to their abilities to induce disease. Very little is known about whether tau proteins can cross the BBB. Here we systematically characterized several key forms of tau proteins to cross the BBB, including Tau-441 (2N4R), Tau-410 (2N3R), truncated tau 151-391 (0N4R), and truncated tau 121-227. All of these tau proteins crossed the BBB readily and bidirectonally; however, only Tau-410 had a saturable component to its influx. The tau proteins also entered the blood after their injection into the brain, with Tau 121-227 having the slowest exit from brain. The tau proteins varied in regards to their enzymatic stability in brain and blood and in their peripheral pharmacokinetics. These results show that blood-borne tau proteins could contribute to brain tauopathies. The result also suggest that the CNS can contribute to blood levels of tau, raising the possibility that, as suggested for other misfolded proteins, blood levels of tau proteins could be used as a biomarker of CNS disease.

Multiple lipopolysaccharide (LPS) injections alter interleukin 6 (IL-6), IL-7, IL-10 and IL-6 and IL-7 receptor mRNA in CNS and spleen

Neuroinflammation is proposed to be an important component in the development of several central nervous system (CNS) disorders including depression, Alzheimers disease, Parkinsons disease, and traumatic brain injury. However, exactly how neuroinflammation leads to, or contributes to, these central disorders is unclear. The objective of the study was to examine and compare the expression of mRNAs for interleukin-6 (IL-6), IL-7, IL-10 and the receptors for IL-6 (IL-6R) and IL-7 (IL-7R) using in situ hybridization in discrete brain regions and in the spleen after multiple injections of 3mg/kg lipopolysaccharide (LPS), a model of neuroinflammation. In the spleen, LPS significantly elevated IL-6 mRNA expression, then IL-10 mRNA, with no effect on IL-7 or IL-7R mRNA, while significantly decreasing IL-6R mRNA expression. In the CNS, LPS administration had the greatest effect on IL-6 and IL-6R mRNA. LPS increased IL-6 mRNA expression only in non-neuronal cells throughout the brain, but significantly elevated IL-6R mRNA in neuronal populations, where observed, except the cerebellum. LPS resulted in variable effects on IL-10 mRNA, and had no effect on IL-7 or IL-7R mRNA expression. These studies indicate that LPS-induced neuroinflammation has substantial but variable effects on the regional and cellular patterns of CNS IL-6, IL-7 and IL-10, and for IL-6R and IL-7R mRNA expression. It is apparent that administration of LPS can affect non-neuronal and neuronal cells in the brain. Further research is required to determine how CNS inflammatory changes associated with IL-6, IL-10 and IL-6R could in turn contribute to the development of CNS neurological disorders.

Intranasal delivery of N-terminal modified leptin-pluronic conjugate for treatment of obesity

ABSTRACT Leptin is an adipocyte‐secreted hormone that is delivered via a specific transport system across the blood‐brain barrier (BBB) to the brain where it acts on the hypothalamus receptors to control appetite and thermogenesis. Peripheral resistance to leptin due to its impaired brain delivery prevents therapeutic use of leptin in overweight and moderately obese patients. To address this problem, we modified the N‐terminal amine of leptin with Pluronic P85 (LepNP85) and administered this conjugate intranasally using the nose‐to‐brain (INB) route to bypass the BBB. We compared this conjugate with the native leptin, the N‐terminal leptin conjugate with poly(ethylene glycol) (LepNPEG5K), and two conjugates of leptin with Pluronic P85 attached randomly to the lysine amino groups of the hormone. Compared to the random conjugates of leptin with P85, LepNP85 has shown higher affinity upon binding with the leptin receptor, and similarly to native hormone activated hypothalamus receptors after direct injection into brain. After INB delivery, LepNP85 conjugate was transported to the brain and accumulated in the hypothalamus and hippocampus to a greater extent than the native leptin and LepNPEG5K and activated leptin receptors in hypothalamus at lower dose than native leptin. Our work suggests that LepNP85 can access the brain directly after INB delivery and confirms our hypothesis that the improvement in brain accumulation of this conjugate is due to its enhanced brain absorption. In conclusion, the LepNP85 with optimized conjugation chemistry is a promising candidate for treatment of obesity. Graphical abstract Pluronic P85 is selectively attached to the N‐terminal amine of leptin to reduce the steric hindrance to leptin receptor binding and enhance the direct nose‐to‐brain transport of leptin. Figure. No Caption available.

Transmission of α-synuclein-containing erythrocyte-derived extracellular vesicles across the blood-brain barrier via adsorptive mediated transcytosis: another mechanism for initiation and progression of Parkinson’s disease?

Parkinson’s disease (PD) pathophysiology develops in part from the formation, transmission, and aggregation of toxic species of the protein α-synuclein (α-syn). Recent evidence suggests that extracellular vesicles (EVs) may play a vital role in the transport of toxic α-syn between brain regions. Moreover, increasing evidence has highlighted the participation of peripheral molecules, particularly inflammatory species, which may influence or exacerbate the development of PD-related changes to the central nervous system (CNS), although detailed characterization of these species remains to be completed. Despite these findings, little attention has been devoted to erythrocytes, which contain α-syn concentrations ~1000-fold higher than the cerebrospinal fluid, as a source of potentially pathogenic α-syn. Here, we demonstrate that erythrocytes produce α-syn-rich EVs, which can cross the BBB, particularly under inflammatory conditions provoked by peripheral administration of lipopolysaccharide. This transport likely occurs via adsorptive-mediated transcytosis, with EVs that transit the BBB co-localizing with brain microglia. Examination of microglial reactivity upon exposure to α-syn-containing erythrocyte EVs in vitro and in vivo revealed that uptake provoked an increase in microglial inflammatory responses. EVs derived from the erythrocytes of PD patients elicited stronger responses than did those of control subjects, suggesting that inherent characteristics of EVs arising in the periphery might contribute to, or even initiate, CNS α-syn-related pathology. These results provide new insight into the mechanisms by which the brain and periphery communicate throughout the process of synucleinopathy pathogenesis.

Nanoformulation of Brain-Derived Neurotrophic Factor with Target Receptor-Triggered-Release in the Central Nervous System

Brain-derived neurotrophic factor (BDNF) is identified as a potent neuroprotective and neuroregenerative agent for many neurological diseases. Regrettably, its delivery to the brain is hampered by poor serum stability and rapid brain clearance. Here, a novel nanoformulation is reported composed of a bio-compatible polymer, poly(ethylene glycol)-b-poly(L-glutamic acid) (PEG-PLE), that hosts the BDNF molecule in a nanoscale complex, termed here Nano-BDNF. Upon simple mixture, Nano-BDNF spontaneously forms uniform spherical particles with a core-shell structure. Molecular dynamics simulations suggest that binding between BDNF and PEG-PLE is mediated through electrostatic coupling as well as transient hydrogen bonding. The formation of Nano-BDNF complex stabilizes BDNF and protects it from nonspecific binding with common proteins in the body fluid, while allowing it to associate with its receptors. Following intranasal administration, the nanoformulation improves BDNF delivery throughout the brain and displays a more preferable regional distribution pattern than the native protein. Furthermore, intranasally delivered Nano-BDNF results in superior neuroprotective effects in the mouse brain with lipopolysaccharides-induced inflammation, indicating promise for further evaluation of this agent for the therapy of neurologic diseases.

Blast exposure elicits blood-brain barrier disruption and repair mediated by tight junction integrity and nitric oxide dependent processes

Mild blast-induced traumatic brain injury (TBI) is associated with blood-brain barrier (BBB) disruption. However, the mechanisms whereby blast disrupts BBB integrity are not well understood. To address this issue BBB permeability to peripherally injected 14C-sucrose and 99mTc-albumin was quantified in ten brain regions at time points ranging from 0.25 to 72 hours. In mice, repetitive (2X) blast provoked BBB permeability to 14C-sucrose that persisted in specific brain regions from 0.25 to 72 hours. However, 99mTc-albumin revealed biphasic BBB disruption (open-closed-open) over the same interval, which was most pronounced in frontal cortex and hippocampus. This indicates that blast initiates interacting BBB disruption and reparative processes in specific brain regions. Further investigation of delayed (72 hour) BBB disruption revealed that claudin-5 (CLD5) expression was disrupted specifically in the hippocampus, but not in dorsal striatum, a brain region that showed no blast-induced BBB permeability to sucrose or albumin. In addition, we found that delayed BBB permeability and disrupted CLD5 expression were blocked by the nitric oxide synthase inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME). These data argue that latent nitric oxide-dependent signaling pathways initiate processes that result in delayed BBB disruption, which are manifested in a brain-region specific manner.

Effect of controlled cortical impact on the passage of pituitary adenylate cyclase activating polypeptide (PACAP) across the blood-brain barrier

HighlightsPACAP38 transport across the BBB is not dramatically altered following CCI.The ipsilateral cortex has increased PACAP38 levels 2 h and 24 h following CCI.There is no change in the rate of PACAP38 transport at these times.The cerebellum contains the greatest amount and fastest rate of PACAP38 transport. ABSTRACT Injuries to the central nervous system can affect the blood‐brain barrier (BBB), including disruption and influencing peptide transport across the BBB. Pituitary adenylate cyclase‐activating polypeptide 38 (PACAP38) is a potent neurotrophic and neuroprotective peptide currently being investigated for its therapeutic role following injury to the central nervous system and can cross the BBB in a saturable manner. The goal of the current study was to investigate for the first time PACAP38 uptake by the brain following traumatic brain injury (TBI). Using radioactively labeled PACAP38, we measured the levels of PACAP38 present in the injured, ipsilateral cortex in Sham‐treated mice compared to mice receiving a controlled cortical impact (CCI), a model of TBI. Experiments were conducted at 6 different time points (from 2 h up to 4 weeks) following CCI to determine temporal changes in PACAP38 transport. PACAP38 uptake was increased at 2 and 72 h post‐CCI compared to Sham. We did not detect changes in PACAP38 uptake in the contralateral cortex and cerebellum between Sham and CCI‐treatment. The rate of PACAP38 transport into the ipsilateral cortex following CCI was increased 3.6‐fold 72 h after compared to 2 h post‐CCI. In addition, the rate of transport into the cerebellum was greater than that of the cortices. The data presented here shows PACAP38 transport is temporally altered following CCI‐treatment and PACAP38 uptake is greater in the cerebellum compared to the cortices.