Pirh2-dependent DNA damage in neurons induced by the G-quadruplex ligand pyridostatin.

Noncanonical base pairing between four guanines (G) within single-stranded G-rich sequences leads to formation of а G-quartet. Self-stacking of G-quartets results in a columnar four-stranded DNA structure known as the G-quadruplex (G4 or G4-DNA). In cancer cells, G4-DNA regulates multiple DNA-dependent processes, including transcription, replication, and telomere function. How G4s function in neurons is poorly understood. Here, we performed a genome-wide gene expression analysis (RNA-Seq) to identify genes modulated by a G4-DNA ligand, pyridostatin (PDS), in primary cultured neurons. PDS promotes stabilization of G4 structures, thus allowing us to define genes directly or indirectly responsive to G4 regulation. We found that 901 genes were differentially expressed in neurons treated with PDS out of a total of 18,745 genes with measured expression. Of these, 505 genes were downregulated and 396 genes were upregulated and included gene networks regulating p53 signaling, the immune response, learning and memory, and cellular senescence. Within the p53 network, the E3 ubiquitin ligase Pirh2 (Rchy1), a modulator of DNA damage responses, was upregulated by PDS. Ectopically overexpressing Pirh2 promoted the formation of DNA double-strand breaks, suggesting a new DNA damage mechanism in neurons that is regulated by G4 stabilization. Pirh2 downregulated DDX21, an RNA helicase that unfolds G4-RNA and R-loops. Finally, we demonstrated that Pirh2 increased G4-DNA levels in the neuronal nucleolus. Our data reveal the genes that are responsive to PDS treatment and suggest similar transcriptional regulation by endogenous G4-DNA ligands. They also connect G4-dependent regulation of transcription and DNA damage mechanisms in neuronal cells.

Preventive and therapeutic reduction of amyloid deposition and behavioral impairments in a model of Alzheimer's disease by whole blood exchange.

Alzheimer's disease (AD) is the major form of dementia in the elderly population. The main neuropathological changes in AD patients are neuronal death, synaptic alterations, brain inflammation, and the presence of cerebral protein aggregates in the form of amyloid plaques and neurofibrillary tangles. Compelling evidence suggests that the misfolding, aggregation, and cerebral deposition of amyloid-beta (Aß) plays a central role in the disease. Thus, prevention and removal of misfolded protein aggregates is considered a promising strategy to treat AD. In the present study, we describe that the development of cerebral amyloid plaques in a transgenic mice model of AD (Tg2576) was significantly reduced by 40-80% through exchanging whole blood with normal blood from wild type mice having the same genetic background. Importantly, such reduction resulted in improvement in spatial memory performance in aged Tg2576 mice. The exact mechanism by which blood exchange reduces amyloid pathology and improves memory is presently unknown, but measurements of Aß in plasma soon after blood exchange suggest that mobilization of Aß from the brain to blood may be implicated. Our results suggest that a target for AD therapy may exist in the peripheral circulation, which could open a novel disease-modifying intervention for AD.

Activation of neuronal Ras-related C3 botulinum toxin substrate 1 (Rac1) improves post-stroke recovery and axonal plasticity in mice.

Long-term disability after stroke is common but the mechanisms of post-stroke recovery remain unclear. Cerebral Ras-related C3 botulinum toxin substrate (Rac) 1 contributes to functional recovery after ischemic stroke in mice. As Rac1 plays divergent roles in individual cell types after central neural system injury, we herein examined the specific role of neuronal Rac1 in post-stroke recovery and axonal regeneration. Young male mice were subjected to 60-min of middle cerebral artery occlusion (MCAO). Inducible deletion of neuronal Rac1 by daily intraperitoneal injection of tamoxifen (2 mg/40 g) into Thy1-creER/Rac1-floxed mice day 7-11 after MCAO worsened cognitive (assayed by novel object recognition test) and sensorimotor (assayed by adhesive removal and pellet reaching tests) recovery day 14-28 accompanied with the reduction of neurofilament-L (NFL) and myelin basic protein (MBP) and the elevation of glial fibrillary acidic protein (GFAP) in the peri-infarct zone assessed by immunostaining. Whereas the brain tissue loss was not altered assayed by cresyl violet staining. In another approach, delayed overexpression of neuronal Rac1 by injection of lentivirus encoding Rac1 with neuronal promotor into both the cortex and striatum (total 4 µl at 1 × 109 transducing units/mL) of stroke side in C57BL/6J mice day 7 promoted stroke outcome, NFL and MBP regrowth and alleviated GFAP invasion. Furthermore, neuronal Rac1 over-expression led to the activation of p21 activating kinases (PAK) 1, mitogen-activated protein kinase kinase (MEK) 1/2 and extracellular signal-regulated kinase (ERK) 1/2, and the elevation of brain-derived neurotrophic factor (BDNF) day 14 after stroke. Finally, we observed higher counts of neuronal Rac1 in the peri-infarct zone of subacute/old ischemic stroke subjects. This work identified a neuronal Rac1 signaling in improving functional recovery and axonal regeneration after stroke, suggesting a potential therapeutic target in the recovery stage of stroke.

Dysregulated Gut Homeostasis Observed Prior to the Accumulation of the Brain Amyloid-ß in Tg2576 Mice.

Amyloid plaques in Alzheimer's disease (AD) are associated with inflammation. Recent studies demonstrated the involvement of the gut in cerebral amyloid-beta (Aß) pathogenesis; however, the mechanisms are still not well understood. We hypothesize that the gut bears the Aß burden prior to brain, highlighting gut-brain axis (GBA) interaction in neurodegenerative disorders. We used pre-symptomatic (6-months) and symptomatic (15-months) Tg2576 mouse model of AD compared to their age-matched littermate WT control. We identified that dysfunction of intestinal epithelial barrier (IEB), dysregulation of absorption, and vascular Aß deposition in the IEB occur before cerebral Aß aggregation is detectible. These changes in the GBA were associated with elevated inflammatory plasma cytokines including IL-9, VEGF and IP-10. In association with reduced cerebral myelin tight junction proteins, we identified reduced levels of systemic vitamin B12 and decrease cubilin, an intestinal B12 transporter, after the development of cerebral Aß pathology. Lastly, we report Aß deposition in the intestinal autopsy from AD patients with confirmed cerebral Aß pathology that is not present in intestine from non-AD controls. Our data provide evidence that gut dysfunction occurs in AD and may contribute to its etiology. Future therapeutic strategies to reverse AD pathology may involve the early manipulation of gut physiology and its microbiota.

Efficient prion disease transmission through common environmental materials.

Prion diseases are a group of fatal neurodegenerative diseases associated with a protein-based infectious agent, termed prion. Compelling evidence suggests that natural transmission of prion diseases is mediated by environmental contamination with infectious prions. We hypothesized that several natural and man-made materials, commonly found in the environments of wild and captive animals, can bind prions and may act as vectors for disease transmission. To test our hypothesis, we exposed surfaces composed of various common environmental materials (i.e. wood, rocks, plastic, glass, cement, stainless steel, aluminum, and brass) to hamster-adapted 263K scrapie prions and studied their attachment and retention of infectivity in vitro and in vivo Our results indicated that these surfaces, with the sole exception of brass, efficiently bind, retain, and release prions. Prion replication was studied in vitro using the protein misfolding cyclic amplification technology, and infectivity of surface-bound prions was analyzed by intracerebrally challenging hamsters with contaminated implants. Our results revealed that virtually all prion-contaminated materials transmitted the disease at high rates. To investigate a more natural form of exposure to environmental contamination, we simply housed animals with large contaminated spheres made of the different materials under study. Strikingly, most of the hamsters developed classical clinical signs of prion disease and typical disease-associated brain changes. Our findings suggest that prion contamination of surfaces commonly present in the environment can be a source of disease transmission, thus expanding our understanding of the mechanisms for prion spreading in nature.

Activation of cerebral Ras-related C3 botulinum toxin substrate (Rac) 1 promotes post-ischemic stroke functional recovery in aged mice.

Brain functional impairment after stroke is common; however, the molecular mechanisms of post-stroke recovery remain unclear. It is well-recognized that age is the most important independent predictor of poor outcomes after stroke as older patients show poorer functional outcomes following stroke. Mounting evidence suggests that axonal regeneration and angiogenesis, the major forms of brain plasticity responsible for post-stroke recovery, diminished with advanced age. Previous studies suggest that Ras-related C3 botulinum toxin substrate (Rac) 1 enhances stroke recovery as activation of Rac1 improved behavior recovery in a young mice stroke model. Here, we investigated the role of Rac1 signaling in long-term functional recovery and brain plasticity in an aged (male, 18 to 22 months old C57BL/6J) brain after ischemic stroke. We found that as mice aged, Rac1 expression declined in the brain. Delayed overexpression of Rac1, using lentivirus encoding Rac1 injected day 1 after ischemic stroke, promoted cognitive (assessed using novel object recognition test) and sensorimotor (assessed using adhesive removal tests) recovery on days 14-28. This was accompanied by the increase of neurite and proliferative endothelial cells in the peri-infarct zone assessed by immunostaining. In a reverse approach, pharmacological inhibition of Rac1 by intraperitoneal injection of Rac1 inhibitor NSC23766 for 14 successive days after ischemic stroke worsened the outcome with the reduction of neurite and proliferative endothelial cells. Furthermore, Rac1 inhibition reduced the activation of p21-activated kinase 1, the protein level of brain-derived neurotrophic factor, and increased the protein level of glial fibrillary acidic protein in the ischemic brain on day 28 after stroke. Our work provided insight into the mechanisms behind the diminished plasticity after cerebral ischemia in aged brains and identified Rac1 as a potential therapeutic target for improving functional recovery in the older adults after stroke.

Iron overload induces cerebral endothelial senescence in aged mice and in primary culture in a sex-dependent manner.

Iron imbalance in the brain negatively affects brain function. With aging, iron levels increase in the brain and contribute to brain damage and neurological disorders. Changes in the cerebral vasculature with aging may enhance iron entry into the brain parenchyma, leading to iron overload and its deleterious consequences. Endothelial senescence has emerged as an important contributor to age-related changes in the cerebral vasculature. Evidence indicates that iron overload may induce senescence in cultured cell lines. Importantly, cells derived from female human and mice generally show enhanced senescence-associated phenotype, compared with males. Thus, we hypothesize that cerebral endothelial cells (CEC) derived from aged female mice are more susceptible to iron-induced senescence, compared with CEC from aged males. We found that aged female mice, but not males, showed cognitive deficits when chronically treated with ferric citrate (FC), and their brains and the brain vasculature showed senescence-associated phenotype. We also found that primary culture of CEC derived from aged female mice, but not male-derived CEC, exhibited senescence-associated phenotype when treated with FC. We identified that the transmembrane receptor Robo4 was downregulated in the brain vasculature and in cultured primary CEC derived from aged female mice, compared with those from male mice. We discovered that Robo4 downregulation contributed to enhanced vulnerability to FC-induced senescence. Thus, our study identifies Robo4 downregulation as a driver of senescence induced by iron overload in primary culture of CEC and a potential risk factor of brain vasculature impairment and brain dysfunction.

Argininosuccinate lyase deficiency causes blood-brain barrier disruption via nitric oxide-mediated dysregulation of claudin expression.

Nitric oxide (NO) is a critical signaling molecule that has been implicated in the pathogenesis of neurocognitive diseases. Both excessive and insufficient NO production have been linked to pathology. Previously, we have shown that argininosuccinate lyase deficiency (ASLD) is a novel model system to investigate cell-autonomous, nitric oxide synthase-dependent NO deficiency. Humans with ASLD are at increased risk for developing hyperammonemia due to a block in ureagenesis. However, natural history studies have shown that individuals with ASLD have multisystem disease including neurocognitive deficits that can be independent of ammonia. Here, using ASLD as a model of NO deficiency, we investigated the effects of NO on brain endothelial cells in vitro and the blood-brain barrier (BBB) in vivo. Knockdown of ASL in human brain microvascular endothelial cells (HBMECs) led to decreased transendothelial electrical resistance, indicative of increased cell permeability. Mechanistically, treatment with an NO donor or inhibition of Claudin-1 improved barrier integrity in ASL-deficient HBMECs. Furthermore, in vivo assessment of a hypomorphic mouse model of ASLD showed increased BBB leakage, which was partially rescued by NO supplementation. Our results suggest that ASL-mediated NO synthesis is required for proper maintenance of brain microvascular endothelial cell functions as well as BBB integrity.

Initial fate of prions upon peripheral infection: half-life, distribution, clearance, and tissue uptake.

Prion diseases are infectious neurodegenerative disorders associated with the misfolded prion protein (PrP(Sc)), which appears to be the sole component of the infectious agent (termed prion). To produce disease, prions have to be absorbed into the body and reach sufficient quantities in the brain. Very little is known about the biological mechanisms controlling the initial fate of prions. Here, we studied the systemic pharmacokinetics and biodistribution of PrP(Sc) in vivo. After an intravenous injection of highly purified radiolabeled or native unlabeled PrP(Sc), the protein was eliminated rapidly from the serum (half-life of 3.24 h), mostly through tissue uptake. The quantity of intact PrP(Sc) reaching the brain was ∼ 0.2% of the injected dose per gram of brain tissue (ID/g). The highest levels were found in liver (∼ 20% ID/g), spleen (∼ 13% ID/g), and kidney (∼ 7.4% ID/g). Cell surface PrP(C) does not appear to play a role in PrP(Sc) pharmacokinetics, since the infectious protein distributed similarly in wild-type and PrP-null mice. To measure tissue uptake kinetics and biodistribution accurately, vascular space in tissues was measured with radioactively labeled albumin coinjected with radioactively labeled PrP(Sc). Our results provide a fundamental pharmacokinetic characterization of PrP(Sc) in vivo, which may be relevant to estimate tissue risks and mechanisms of prion neuroinvasion and to identify novel therapeutic strategies.

Mucopolysaccharidoses and the blood-brain barrier.

Mucopolysaccharidoses comprise a set of genetic diseases marked by an enzymatic dysfunction in the degradation of glycosaminoglycans in lysosomes. There are eight clinically distinct types of mucopolysaccharidosis, some with various subtypes, based on which lysosomal enzyme is deficient and symptom severity. Patients with mucopolysaccharidosis can present with a variety of symptoms, including cognitive dysfunction, hepatosplenomegaly, skeletal abnormalities, and cardiopulmonary issues. Additionally, the onset and severity of symptoms can vary depending on the specific disorder, with symptoms typically arising during early childhood. While there is currently no cure for mucopolysaccharidosis, there are clinically approved therapies for the management of clinical symptoms, such as enzyme replacement therapy. Enzyme replacement therapy is typically administered intravenously, which allows for the systemic delivery of the deficient enzymes to peripheral organ sites. However, crossing the blood-brain barrier (BBB) to ameliorate the neurological symptoms of mucopolysaccharidosis continues to remain a challenge for these large macromolecules. In this review, we discuss the transport mechanisms for the delivery of lysosomal enzymes across the BBB. Additionally, we discuss the several therapeutic approaches, both preclinical and clinical, for the treatment of mucopolysaccharidoses.

Cerebral Amyloid Angiopathy and Blood-Brain Barrier Dysfunction.

Cerebral hemorrhage, a devastating subtype of stroke, is often caused by hypertension and cerebral amyloid angiopathy (CAA). Pathological evidence of CAA is detected in approximately half of all individuals over the age of 70 and is associated with cortical microinfarcts and cognitive impairment. The underlying pathophysiology of CAA is characterized by accumulation of pathogenic amyloid ß (Aß) fragments of amyloid precursor protein in the cerebral vasculature. Vascular deposition of Aß damages the vessel wall, results in blood-brain barrier (BBB) leakiness, vessel occlusion or rupture, and leads to hemorrhages and decreased cerebral blood flow that negatively affects vessel integrity and cognitive function. Currently, the main hypothesis surrounding the mechanism of CAA pathogenesis is that there is an impaired clearance of Aß peptides, which includes compromised perivascular drainage as well as dysfunction of BBB transport. Also, the immune response in CAA pathogenesis plays an important role. Therefore, the mechanism by which Aß vascular deposition occurs is crucial for our understanding of CAA pathogenesis and for the development of potential therapeutic options.

Sex differences in a murine model of Cerebral Amyloid Angiopathy.

Cerebral amyloid angiopathy (CAA) is one of the common causes of lobar intracerebral hemorrhage and vascular cognitive impairment (VCI) in the aging population. Increased amyloid plaque deposition within cerebral blood vessels, specifically the smooth muscle layer, is linked to increased cerebral microbleeds (CMBs) and impaired cognition in CAA. Studies in Alzheimer's disease (AD) have shown that amyloid plaque pathology is more prevalent in the brains of elderly women (2/3rd of the dementia population) compared with men, however, there is a paucity of studies on sex differences in CAA. The objective of this study was to discern the sexual dichotomies in CAA. We utilized male and female Tg-SwDI mice (mouse model of CAA) at 12-14 months of age for this study. We evaluated sex differences in CMBs, cognitive function and inflammation. Cognition was assessed using Y-maze (spatial working memory) and Fear Conditioning (contextual memory). CMBs were quantified by ex vivo brain MRI scans. Inflammatory cytokines in brain were quantified using ELISA. Our results demonstrated that aging Tg-SwDI female mice had a significantly higher burden of CMBs on MRI as compared to males. Interestingly, these aging Tg-SwDI female mice also had significantly impaired spatial and contextual memory on Y maze and Fear Conditioning respectively. Furthermore, female mice had significantly lower circulating inflammatory cytokines, IL-1α, IL-2, IL-9, and IFN-γ, as compared to males. Our results demonstrate that aging female Tg-SwDI mice are more cognitively impaired and have higher number of CMBs, as compared to males at 12-14 months of age. This may be secondary to reduced levels of neural repair cytokines (IL-1α, IL-2, IL-9 and IFN-γ) involved in sex specific inflammatory signaling in CAA.

G-quadruplexes Stabilization Upregulates CCN1 and Accelerates Aging in Cultured Cerebral Endothelial Cells.

Senescence in the cerebral endothelium has been proposed as a mechanism that can drive dysfunction of the cerebral vasculature, which precedes vascular dementia. Cysteine-rich angiogenic inducer 61 (Cyr61/CCN1) is a matricellular protein secreted by cerebral endothelial cells (CEC). CCN1 induces senescence in fibroblasts. However, whether CCN1 contributes to senescence in CEC and how this is regulated requires further study. Aging has been associated with the formation of four-stranded Guanine-quadruplexes (G4s) in G-rich motifs of DNA and RNA. Stabilization of the G4 structures regulates transcription and translation either by upregulation or downregulation depending on the gene target. Previously, we showed that aged mice treated with a G4-stabilizing compound had enhanced senescence-associated (SA) phenotypes in their brains, and these mice exhibited enhanced cognitive deficits. A sequence in the 3'-UTR of the human CCN1 mRNA has the ability to fold into G4s in vitro. We hypothesize that G4 stabilization regulates CCN1 in cultured primary CEC and induces endothelial senescence. We used cerebral microvessel fractions and cultured primary CEC from young (4-months old, m/o) and aged (18-m/o) mice to determine CCN1 levels. SA phenotypes were determined by high-resolution fluorescence microscopy in cultured primary CEC, and we used Thioflavin T to recognize RNA-G4s for fluorescence spectra. We found that cultured CEC from aged mice exhibited enhanced levels of SA phenotypes, and higher levels of CCN1 and G4 stabilization. In cultured CEC, CCN1 induced SA phenotypes, such as SA ß-galactosidase activity, and double-strand DNA damage. Furthermore, CCN1 levels were upregulated by a G4 ligand, and a G-rich motif in the 3'-UTR of the Ccn1 mRNA was folded into a G4. In conclusion, we demonstrate that CCN1 can induce senescence in cultured primary CEC, and we provide evidence that G4 stabilization is a novel mechanism regulating the SASP component CCN1.

The Role of Basement Membranes in Cerebral Amyloid Angiopathy.

Dementia is a neuropsychiatric syndrome characterized by cognitive decline in multiple domains, often leading to functional impairment in activities of daily living, disability, and death. The most common causes of age-related progressive dementia include Alzheimer's disease (AD) and vascular cognitive impairment (VCI), however, mixed disease pathologies commonly occur, as epitomized by a type of small vessel pathology called cerebral amyloid angiopathy (CAA). In CAA patients, the small vessels of the brain become hardened and vulnerable to rupture, leading to impaired neurovascular coupling, multiple microhemorrhage, microinfarction, neurological emergencies, and cognitive decline across multiple functional domains. While the pathogenesis of CAA is not well understood, it has long been thought to be initiated in thickened basement membrane (BM) segments, which contain abnormal protein deposits and amyloid-ß (Aß). Recent advances in our understanding of CAA pathogenesis link BM remodeling to functional impairment of perivascular transport pathways that are key to removing Aß from the brain. Dysregulation of this process may drive CAA pathogenesis and provides an important link between vascular risk factors and disease phenotype. The present review summarizes how the structure and composition of the BM allows for perivascular transport pathways to operate in the healthy brain, and then outlines multiple mechanisms by which specific dementia risk factors may promote dysfunction of perivascular transport pathways and increase Aß deposition during CAA pathogenesis. A better understanding of how BM remodeling alters perivascular transport could lead to novel diagnostic and therapeutic strategies for CAA patients.

Infusion of blood from mice displaying cerebral amyloidosis accelerates amyloid pathology in animal models of Alzheimer's disease.

Previous studies showed that injection of tissue extracts containing amyloid-ß (Aß) aggregates accelerate amyloid deposition in the brain of mouse models of Alzheimer's disease (AD) through prion-like mechanisms. In this study, we evaluated whether brain amyloidosis could be accelerated by blood infusions, procedures that have been shown to transmit prion diseases in animals and humans. Young transgenic mice infused with whole blood or plasma from old animals with extensive Aß deposition in their brains developed significantly higher levels brain amyloidosis and neuroinflammation compared to untreated animals or mice infused with wild type blood. Similarly, intra-venous injection of purified Aß aggregates accelerated amyloid pathology, supporting the concept that Aß seeds present in blood can reach the brain to promote neuropathological alterations in the brain of treated animals. However, an amyloid-enhancing effect of other factors present in the blood of donors cannot be discarded. Our results may help to understand the role of peripheral (amyloid-dependent or -independent) factors implicated in the development of AD and uncover new strategies for disease intervention.

Regulation of autophagy by DNA G-quadruplexes.

Guanine-rich DNA strands can form secondary structures known as G-quadruplexes (G4-DNA or G4s). G4-DNA is important for the regulation of replication and transcription. We recently showed that the expression of Atg7, a gene that is critical for macroautophagy/autophagy, is controlled by G4-DNA in neurons. We demonstrated that the transcription factor SUB1/PC4 and the G4-DNA-specific antibody HF2 bind to a putative G4-DNA motif located in the Atg7 gene. Stabilizing G4-DNA with the G4-ligand pyridostatin (PDS) downregulates Atg7 expression in neurons. Here, we further investigated how G4-DNA in the Atg7 gene is stabilized by PDS. We show that PDS can form 1:1 and 2:1 complexes with the Atg7's G4. We also demonstrate that PDS downregulates the ATG7 protein and the expression of Atg7 in astrocytes as well as in neurons. Together with our previous findings, these data establish a novel G4-DNA-associated mechanism of autophagy regulation at a transcriptional level in neurons and astrocytes.

Small-molecule G-quadruplex stabilizers reveal a novel pathway of autophagy regulation in neurons.

Guanine-rich DNA sequences can fold into four-stranded G-quadruplex (G4-DNA) structures. G4-DNA regulates replication and transcription, at least in cancer cells. Here, we demonstrate that, in neurons, pharmacologically stabilizing G4-DNA with G4 ligands strongly downregulates the Atg7 gene. Atg7 is a critical gene for the initiation of autophagy that exhibits decreased transcription with aging. Using an in vitro assay, we show that a putative G-quadruplex-forming sequence (PQFS) in the first intron of the Atg7 gene folds into a G4. An antibody specific to G4-DNA and the G4-DNA-binding protein PC4 bind to the Atg7 PQFS. Mice treated with a G4 stabilizer develop memory deficits. Brain samples from aged mice contain G4-DNA structures that are absent in brain samples from young mice. Overexpressing the G4-DNA helicase Pif1 in neurons exposed to the G4 stabilizer improves phenotypes associated with G4-DNA stabilization. Our findings indicate that G4-DNA is a novel pathway for regulating autophagy in neurons.

Mannose 6-phosphate receptor-mediated transport of sulfamidase across the blood-brain barrier in the newborn mouse.

Mucopolysaccharidosis type IIIA (MPS IIIA), which is a lysosomal storage disorder (LSD) caused by inherited deficiency of sulfamidase, is characterized by severe, progressive central nervous system (CNS) dysfunction. Enzyme replacement therapy (ERT) to treat CNS storage is challenging, because the access of enzymes to the brain is restricted by the blood-brain barrier (BBB). In a prior study, we found that phosphorylated beta-glucuronidase (P-GUS) could be transcytosed across the BBB in newborn mice by the mannose 6-phosphate (M6P) receptor. In order to determine whether sulfamidase can utilize this pathway, we examined brain influx and the specificity of uptake of sulfamidase after intravenous (i.v.) injection in 2-day-old and 8-week-old mice. [(131)I]Sulfamidase was transported across the BBB in neonates at rates higher than that of simultaneously injected [(125)I]albumin. In contrast, the transport of [(131)I]sulfamidase was negligible in 8-week-old mice, thereby showing that the BBB transport mechanism is developmentally downregulated. Capillary depletion revealed that 83.7% of the [(131)I]sulfamidase taken up by the brain was in the parenchyma, demonstrating transfer across the capillary wall. The uptake of [(131)I]sulfamidase into the brain was significantly reduced by co-injections of M6P and P-GUS. That is, the transport of sulfamidase into the brain parenchyma in early postnatal life is mediated by the M6P receptor, which is shared with P-GUS and is likely accessible to other M6P-containing lysosomal enzymes.

Activation of endothelial ras-related C3 botulinum toxin substrate 1 (Rac1) improves post-stroke recovery and angiogenesis via activating Pak1 in mice.

BACKGROUND AND PURPOSE: Long-term disability after stroke is common yet the mechanisms of post-stroke recovery are far from clear. It has been suggested that Ras-related C3 botulinum toxin substrate 1 (Rac1) contributes to functional recovery after ischemic stroke in mice. As Rac1 activation plays diverse roles in multiple cell types after central nervous system (CNS) injury, we herein examined the functional role of endothelial Rac1 in post-stroke recovery and angiogenesis. METHODS: Transient middle cerebral artery occlusion (MCAO) in mice and oxygen-glucose deprivation (OGD) in human brain endothelial cell line-5i (HEBC 5i) were performed to mimic ischemic stroke. Lentivirus vectors encoding Rac1 with GFP and endothelial promotor ENG were injected into the animal's brain after stroke to overexpress Rac1. After injection, stroke recovery was tested by multiple behavioral tests including novel object recognition, adhesive removal and single pellet reaching tests. Endothelial regeneration in the peri-infarct zone was detected by immunohistochemistry (IHC). In the vitro model, the effect of Rac1 and Pak1 inhibitors to cell proliferation and migration was examined by CCK-8 and wound healing assays after OGD. The cellular protein level of brain-derived neurotrophic factor (BDNF), phosphorylated cAMP response element-binding protein (CREB), extracellular signal-regulated kinase (ERK) 1/2 and mitogen-activated protein kinase kinase (MEK) 1/2 were detected by western blots. RESULTS: Delayed overexpression of endothelial Rac1 after MCAO improved cognitive and sensorimotor recovery from day 14 to 21 after stroke, increased vascular density and the protein level of pericytes in the peri-infarct zone without altering tissue loss in mice. Consistently, inhibition of Rac1 prevented endothelial proliferation and migration after OGD. Pak1 inhibition exerted a similar effect on endothelial cells. However, co-incubation of Rac1 and Pak1 inhibitors with cells did not lead to additive effects when compared with either inhibitor alone. Moreover, individual inhibition of Rac1 or Pak1 suppressed OGD-induced activation of pro-regenerative molecules, including CREB, MEK1/2 and ERK1/2, as well as the production of BDNF in vitro. The level of these proteins did not further decrease if both Rac1 and Pak1 were simultaneously inhibited. CONCLUSIONS: We conclude that activation of endothelial Rac1 improves functional recovery and angiogenesis after stroke, and this process is mediated by Pak1 signaling. This study provides novel insight for Rac1 in the mechanism of long-term stroke recovery.

Transforming growth factor-ß promotes basement membrane fibrosis, alters perivascular cerebrospinal fluid distribution, and worsens neurological recovery in the aged brain after stroke.

Aging and stroke alter the composition of the basement membrane and reduce the perivascular distribution of cerebrospinal fluid and solutes, which may contribute to poor functional recovery in elderly patients. Following stroke, TGF-ß induces astrocyte activation and subsequent glial scar development. This is dysregulated with aging and could lead to chronic, detrimental changes within the basement membrane. We hypothesized that TGF-ß induces basement membrane fibrosis after stroke, leading to impaired perivascular CSF distribution and poor functional recovery in aged animals. We found that CSF entered the aged brain along perivascular tracts; this process was reduced by experimental stroke and was rescued by TGF-ß receptor inhibition. Brain fibronectin levels increased with experimental stroke, which was reversed with inhibitor treatment. Exogenous TGF-ß stimulation increased fibronectin expression, both in vivo and in primary cultured astrocytes. Oxygen-glucose deprivation of cultured astrocytes induced multiple changes in genes related to astrocyte activation and extracellular matrix production. Finally, in stroke patients, we found that serum TGF-ß levels correlated with poorer functional outcomes, suggesting that serum levels may act as a biomarker for functional recovery. These results support a potential new treatment strategy to enhance recovery in elderly stroke patients.