G5G2.5 core‐shell tecto‐dendrimer specifically targets reactive glia in brain ischemia

Secondary neuronal death is a serious stroke complication. This process is facilitated by the conversion of glial cells to the reactive pro‐inflammatory phenotype that induces neurodegeneration. Therefore, regulation of glial activation is a compelling strategy to reduce brain damage after stroke. However, drugs have difficulties to access the CNS , and to specifically target glial cells. In the present work, we explored the use core‐shell polyamidoamine tecto‐dendrimer (G5G2.5 PAMAM ) and studied its ability to target distinct populations of stroke‐activated glial cells. We found that G5G2.5 tecto‐dendrimer is actively engulfed by primary glial cells in a time‐ and dose‐dependent manner showing high cellular selectivity and lysosomal localization. In addition, oxygen‐glucose deprivation or lipopolysaccharides exposure in vitro and brain ischemia in vivo increase glial G5G2.5 uptake; not being incorporated by neurons or other cell types. We conclude that G5G2.5 tecto‐dendrimer is a highly suitable carrier for targeted drug delivery to reactive glial cells in vitro and in vivo after brain ischemia.

     

Kinetic modeling of [18F]VAT,a novel radioligand for positron emission tomography imaging vesicular acetylcholine transporter in non‐human primate brain

Molecular imaging of vesicular acetylcholine transporter (VACh T) in the brain provides an important cholinergic biomarker for the pathophysiology and treatment of dementias including Alzheimer's disease. In this study, kinetics modeling methods were applied and compared for quantifying regional brain uptake of the VACh T‐specific positron emission tomography radiotracer, ((?)‐(1‐(‐8‐(2‐fluoroethoxy)‐3‐hydroxy‐1,2,3,4‐tetrahydronaphthalen‐2‐yl)piperidin‐4‐yl)(4‐fluorophenyl)‐methanone) ([¹⁸F]VAT ) in macaques. Total volume distribution (V _T ) estimates were compared for one‐tissue compartment model (1TCM ), two‐tissue compartment model (2TCM ), Logan graphic analysis (LoganAIF ) and multiple linear analysis (MA 1) with arterial blood input function using data from three macaques. Using the cerebellum‐hemispheres as the reference region with data from seven macaques, three additional models were compared: reference tissue model (RTM ), simplified RTM (SRTM ), and Logan graphic analysis (LoganREF ). Model selection criterion indicated that a) 2TCM and SRTM were the most appropriate kinetics models for [¹⁸F]VAT ; and b) SRTM was strongly correlated with 2TCM (Pearson's coefficients r  > 0.93, p  < 0.05). Test–retest studies demonstrated that [¹⁸F]VAT has good reproducibility and reliability (TRV < 10%, ICC > 0.72). These studies demonstrate [¹⁸F]VAT is a promising VACh T positron emission tomography tracer for quantitative assessment of VACh T levels in the brain of living subjects.

     

Neuroprotective effects of Argon are mediated via an ERK‐1/2 dependent regulation of heme‐oxygenase‐1 in retinal ganglion cells

Retinal ischemia and reperfusion injuries (R‐IRI) damage neuronal tissue permanently. Recently, we demonstrated that Argon exerts anti‐apoptotic and protective properties. The molecular mechanism remains unclear. We hypothesized that Argon inhalation exert neuroprotective effects in rats retinal ganglion cells (RGC) via an ERK‐1/2 dependent regulation of heat‐shock proteins. Inhalation of Argon (75 Vol%) was performed after R‐IRI on the rats′ left eyes for 1 h immediately or with delay. Retinal tissue was harvested after 24 h to analyze mRNA and protein expression of heat‐shock proteins ?70, ?90 and heme‐oxygenase‐1, mitogen‐activated protein kinases (p38, JNK, ERK‐1/2) and histological changes. To analyze ERK dependent effects, the ERK inhibitor PD98059 was applicated prior to Argon inhalation. RGC count was analyzed 7 days after injury. Statistics were performed using anova . Argon significantly reduced the R‐IRI‐affected heat‐shock protein expression (p < 0.05). While Argon significantly induced ERK‐1/2 expression (p < 0.001), inhibition of ERK‐1/2 before Argon inhalation resulted in significantly lower vital RGCs (p < 0.01) and increase in heme‐oxygenase‐1 (p < 0.05). R‐IRI‐induced RGC loss was reduced by Argon inhalation (p < 0.001). Immunohistochemistry suggested ERK‐1/2 activation in Müller cells. We conclude, that Argon treatment protects R‐IRI‐induced apoptotic loss of RGC via an ERK‐1/2 dependent regulation of heme‐oxygenase‐1.

     

Hexokinase 2‐dependent hyperglycolysis driving microglial activation contributes to ischemic brain injury

The cytoplasmic trafficking of docosahexaenoic acid (DHA ), a cognitively beneficial fatty acid, across the blood–brain barrier (BBB ) is governed by fatty acid‐binding protein 5 (FABP 5). Lower levels of brain DHA have been observed in Alzheimer's disease (AD ), which is associated with diminished BBB expression of FABP 5. Therefore, up‐regulating FABP 5 expression at the BBB may be a novel approach for enhancing BBB transport of DHA in AD . DHA supplementation has been shown to be beneficial in various mouse models of AD , and therefore, the aim of this study was to determine whether DHA has the potential to up‐regulate the BBB expression of FABP 5, thereby enhancing its own uptake into the brain. Treating human brain microvascular brain endothelial (hCMEC /D3) cells with the maximum tolerable concentration of DHA (12.5 μM) for 72 h resulted in a 1.4‐fold increase in FABP 5 protein expression. Associated with this was increased expression of fatty acid transport proteins 1 and 4. To study the impact of dietary DHA supplementation, 6‐ to 8‐week‐old C57BL /6 mice were fed with a control diet or a DHA ‐enriched diet for 21 days. Brain microvascular FABP 5 protein expression was up‐regulated 1.7‐fold in mice fed the DHA ‐enriched diet, and this was associated with increased brain DHA levels (1.3‐fold). Despite an increase in brain DHA levels, reduced BBB transport of ¹⁴C‐DHA was observed over a 1 min perfusion, possibly as a result of competitive binding to FABP 5 between dietary DHA and ¹⁴C‐DHA . This study has demonstrated that DHA can increase BBB expression of FABP 5, as well as fatty acid transporters, overall increasing brain DHA levels.

     

Girdin,an actin‐binding protein,is critical for migration,adhesion, and invasion of human glioblastoma cells

Girdin, an actin‐binding protein, possesses versatile functions in a multitude of cellular processes. Although several studies have shown that Girdin is involved in the cell DNA synthesis, actin cytoskeleton rearrangement, and cell motility, the molecular mechanisms of Girdin in tumor development and progression remain elusive. In this study, through over‐expression and siRNA experiments, we found that Girdin increased migration of LN229 human glioblastoma cells. On the other hand, reducing Girdin impaired F‐actin polymerization, which is essential for cell morphogenesis and motility. Matrix metalloproteinase 2, critical in human glioma migration and invasion, was down‐regulated upon Girdin reduction and led to decreased invasion in vitro and in vivo. In addition, silencing Girdin expression impaired the phosphorylation of two important adhesion molecules, integrin β1 and focal adhesion kinase, resulting in cell adhesion defects. Our immunohistochemical study on human gliomas tissue sections indicated that Girdin expression was positively related with glioma malignancy, supporting the in vitro and in vivo results from cell lines. Collectively, our findings suggest a critical role for Girdin in glioma infiltration.

     

Zinc transporter‐1: a novel NMDA receptor‐binding protein at the postsynaptic density

Zinc (Zn²⁺) is believed to play a relevant role in the physiology and pathophysiology of the brain. Hence, Zn²⁺ homeostasis is critical and involves different classes of molecules, including Zn²⁺ transporters. The ubiquitous Zn²⁺ transporter‐1 (ZNT‐1) is a transmembrane protein that pumps cytosolic Zn²⁺ to the extracellular space, but its function in the central nervous system is not fully understood. Here, we show that ZNT‐1 interacts with GluN2A‐containing NMDA receptors, suggesting a role for this transporter at the excitatory glutamatergic synapse. First, we found that ZNT‐1 is highly expressed at the hippocampal postsynaptic density (PSD) where NMDA receptors are enriched. Two‐hybrid screening, coimmunoprecipitation experiments and clustering assay in COS‐7 cells demonstrated that ZNT‐1 specifically binds the GluN2A subunit of the NMDA receptor. GluN2A deletion mutants and pull‐down assays indicated GluN2A(1390–1464) domain as necessary for the binding to ZNT‐1. Most importantly, ZNT‐1/GluN2A complex was proved to be dynamic, since it was regulated by induction of synaptic plasticity. Finally, modulation of ZNT‐1 expression in hippocampal neurons determined a significant change in dendritic spine morphology, PSD‐95 clusters and GluN2A surface levels, supporting the involvement of ZNT‐1 in the dynamics of excitatory PSD.

     

The APPswe/PS1A246E mutations in an astrocytic cell line leads to increased vulnerability to oxygen and glucose deprivation,Ca2+ dysregulation,and mitochondrial abnormalities

Growing evidence suggests a close relationship between Alzheimer′s Disease (AD ) and cerebral hypoxia. Astrocytes play a key role in brain homeostasis and disease states, while some of the earliest changes in AD occur in astrocytes. We have therefore investigated whether mutations associated with AD increase astrocyte vulnerability to ischemia. Two astroglioma cell lines derived from APP_SWE /PS 1A246E (APP , amyloid precursor protein; PS 1, presenilin 1) transgenic mice and controls from normal mice were subjected to oxygen and glucose deprivation (OGD ), an in vitro model of ischemia. Cell death was increased in the APP_SWE /PS 1A246E line compared to the control. Increasing extracellular calcium concentration ([Ca²⁺]) exacerbated cell death in the mutant but not in the control cells. In order to explore cellular Ca²⁺ homeostasis, the cells were challenged with ATP or thapsigargin and [Ca²⁺] was measured by fluorescence microscopy. Changes in cytosolic Ca²⁺ concentration ([Ca²⁺]_c) were potentiated in the APP_SWE /PS 1A246E transgenic line. Mitochondrial function was also altered in the APP_SWE /PS 1A246E astroglioma cells; mitochondrial membrane potential and production of reactive oxygen species were increased, while mitochondrial basal respiratory rate and ATP production were decreased compared to control astroglioma cells. These results suggest that AD mutations in astrocytes make them more sensitive to ischemia; Ca²⁺ dysregulation and mitochondrial dysfunction may contribute to this increased vulnerability. Our results also highlight the role of astrocyte dyshomeostasis in the pathophysiology of neurodegenerative brain disorders.

     

Insulin and IGF1 modulate turnover of polysialylated neural cell adhesion molecule (PSA–NCAM) in a process involving specific extracellular matrix components

Cellular interactions mediated by the neural cell adhesion molecule (NCAM) are critical in cell migration, differentiation and plasticity. Switching of the NCAM‐interaction mode, from adhesion to signalling, is determined by NCAM carrying a particular post‐translational modification, polysialic acid (PSA). Regulation of cell‐surface PSA‐NCAM is traditionally viewed as a direct consequence of polysialyltransferase activity. Taking advantage of the polysialyltransferase Ca²⁺‐dependent activity, we demonstrate in TE671 cells that downregulation of PSA‐NCAM synthesis constitutes a necessary but not sufficient condition to reduce cell‐surface PSA‐NCAM; instead, PSA‐NCAM turnover required internalization of the molecule into the cytosol. PSA‐NCAM internalization was specifically triggered by collagen in the extracellular matrix (ECM) and prevented by insulin‐like growth factor (IGF1) and insulin. Our results pose a novel role for IGF1 and insulin in controlling cell migration through modulation of PSA‐NCAM turnover at the cell surface.

     

Dimethyl fumarate treatment after traumatic brain injury prevents depletion of antioxidative brain glutathione and confers neuroprotection

Dimethyl fumarate (DMF) is an immunomodulatory compound to treat multiple sclerosis and psoriasis with neuroprotective potential. Its mechanism of action involves activation of the antioxidant pathway regulator Nuclear factor erythroid 2‐related factor 2 thereby increasing synthesis of the cellular antioxidant glutathione (GSH). The objective of this study was to investigate whether post‐traumatic DMF treatment is beneficial after experimental traumatic brain injury (TBI). Adult C57Bl/6 mice were subjected to controlled cortical impact followed by oral administration of DMF (80 mg/kg body weight) or vehicle at 3, 24, 48, and 72 h after the inflicted TBI. At 4 days after lesion (dal), DMF‐treated mice displayed less neurological deficits than vehicle‐treated mice and reduced histopathological brain damage. At the same time, the TBI‐evoked depletion of brain GSH was prevented by DMF treatment. However, nuclear factor erythroid 2‐related factor 2 target gene mRNA expression involved in antioxidant and detoxifying pathways was increased in both treatment groups at 4 dal. Blood brain barrier leakage, as assessed by immunoglobulin G extravasation, inflammatory marker mRNA expression, and CD45⁺ leukocyte infiltration into the perilesional brain tissue was induced by TBI but not significantly altered by DMF treatment. Collectively, our data demonstrate that post‐traumatic DMF treatment improves neurological outcome and reduces brain tissue loss in a clinically relevant model of TBI. Our findings suggest that DMF treatment confers neuroprotection after TBI via preservation of brain GSH levels rather than by modulating neuroinflammation.

     

Functional properties of dopamine transporter oligomers after copper linking

Although it is universally accepted that dopamine transporters (DAT s) exist in monomers, dimers and tetramers (i.e. dimers of dimers), it is not known whether the oligomeric organization of DAT is a prerequisite for its ability to take up dopamine (DA ), or whether each DAT protomer, the subunit of quaternary structure, functions independently in terms of DA translocation. In this study, copper phenanthroline (CuP) was used to selectively target surface DAT : increasing concentrations of CuP gradually cross‐linked natural DAT dimers in LLC ‐PK ₁ cells stably expressing hDAT and thereby reduced DA uptake functionality until all surface DAT s were inactivated. DAT s that were not cross‐linked by CuP showed normal DA uptake with DA K _m at ~ 0.5 μM and DA efflux with basal and amphetamine‐induced DA efflux as much as control values. The cocaine analog 2β‐carbomethoxy‐3β‐[4‐fluorophenyl]‐tropane (CFT ) was capable to bind to copper‐cross‐linked DAT s, albeit with an affinity more than fivefold decreased (K _d of CFT  = 109 nM after cross‐linking vs 19 nM before). A kinetic analysis is offered describing the changing amounts of dimers and monomers with increasing [CuP], allowing the estimation of dimer functional activity compared with a DAT monomer. Consonant with previous conclusions for serotonin transporter and NET that only one protomer of an oligomer is active at the time, the present data indicated a functional activity of the DAT dimer of 0.74 relative to a monomer.

     

A conserved sequence in calmodulin regulated spectrin‐associated protein 1 links its interaction with spectrin and calmodulin to neurite outgrowth

Calmodulin regulated spectrin‐associated protein 1 (CAMSAP1) is a vertebrate microtubule‐binding protein, and a representative of a family of cytoskeletal proteins that arose with animals. We reported previously that the central region of the protein, which contains no recognized functional domain, inhibited neurite outgrowth when over‐expressed in PC12 cells [Baines et al., Mol. Biol. Evol. 26 (2009), p. 2005]. The CKK domain (DUF1781) binds microtubules and defines the CAMSAP/ssp4 family of animal proteins (Baines et al. 2009). In the central region, three short well‐conserved regions are characteristic of CAMSAP‐family members. One of these, CAMSAP‐conserved region 1 (CC1), bound to both βIIΣ1‐spectrin and Ca²⁺/calmodulin in vitro. The binding of Ca²⁺/calmodulin inhibited spectrin binding. Transient expression of CC1 in PC12 cells inhibited neurite outgrowth. siRNA knockdown of CAMSAP1 inhibited neurite outgrowth in PC12 cells or primary cerebellar granule cells: this could be rescued in PC12 cells by wild‐type CAMSAP1‐enhanced green fluorescent protein, but not by a CC1 mutant. We conclude that CC1 represents a functional region of CAMSAP1, which links spectrin‐binding to neurite outgrowth.

     

Cyclophilin D regulates neuronal activity‐induced filopodiagenesis by fine‐tuning dendritic mitochondrial calcium dynamics

Recent studies have highlighted the role of mitochondria in dendritic protrusion growth and plasticity. However, the detailed mechanisms that mitochondria regulate dendritic filopodia morphogenesis remain elusive. Cyclophilin D (CypD, gene name: Ppif ) controls the opening of mitochondrial permeability transition pore. Although the pathological relevance of CypD has been intensively investigated, little is known about its physiological function in neurons. Here, we have found that genetic depletion of or pharmaceutical inhibition of CypD blunts the outgrowth of dendritic filopodia in response to KC l‐stimulated neuronal depolarization. Further cell biological studies suggest that such inhibitory effect of CypD loss‐of‐function is closely associated with compromised flexibility of dendritic mitochondrial calcium regulation during neuronal depolarization, as well as the resultant changes in intradendritic calcium homeostasis, calcium signaling activation, dendritic mitochondrial motility and redistribution. Interestingly, loss of CypD attenuates oxidative stress‐induced mitochondrial calcium perturbations and dendritic protrusion injury. Therefore, our study has revealed the physiological function of CypD in dendritic plasticity by acting as a fine‐tuner of mitochondrial calcium homeostasis. Moreover, CypD plays distinct roles in neuronal physiology and pathology.

Cover Image for this issue: doi: 10.1111/jnc.14189 .

     

Urban air pollutants reduce synaptic function of CA1 neurons via an NMDA/NȮ pathway in vitro

Airborne particulate matter (PM) from urban vehicular aerosols altered glutamate receptor functions and induced glial inflammatory responses in rodent models after chronic exposure. Potential neurotoxic mechanisms were analyzed in vitro. In hippocampal slices, 2 h exposure to aqueous nanosized PM (nPM) selectively altered post‐synaptic proteins in cornu ammonis area 1 (CA1) neurons: increased GluA1, GluN2A, and GluN2B, but not GluA2, GluN1, or mGlur5; increased post synaptic density 95 and spinophilin, but not synaptophysin, while dentate gyrus (DG) neurons were unresponsive. In hippocampal slices and neurons, MitoSOX red fluorescence was increased by nPM, implying free radical production. Specifically, N? production by slices was increased within 15 min of exposure to nPM with dose dependence, 1–10 μg/mL. Correspondingly, CA1 neurons exhibited increased nitrosylation of the GluN2A receptor and dephosphorylation of GluN2B (S1303) and of GluA1 (S831 & S845). Again, DG neurons were unresponsive to nPM. The induction of N? and nitrosylation were inhibited by AP5, an NMDA receptor antagonist, which also protects neurite outgrowth in vitro from inhibition by nPM. Membrane injury (EthidiumD‐1 uptake) showed parallel specificity. Finally, nPM decreased evoked excitatory post‐synaptic currents of CA1 neurons. These findings further document the selective impact of nPM on glutamatergic functions and identify novel responses of NMDA receptor‐stimulated N? production and nitrosylation reactions during nPM‐mediated neurotoxicity.

     

Growth hormone releasing hormone (GHRH) signaling modulates intermittent hypoxia‐induced oxidative stress and cognitive deficits in mouse

Intermittent hypoxia (IH) during sleep, such as occurs in obstructive sleep apnea (OSA), leads to degenerative changes in the hippocampus, and is associated with spatial learning deficits in adult mice. In both patients and murine models of OSA, the disease is associated with suppression of growth hormone (GH) secretion, which is actively involved in the growth, development, and function of the central nervous system (CNS). Recent work showed that exogenous GH therapy attenuated neurocognitive deficits elicited by IH during sleep in rats. Here, we show that administration of the Growth Hormone Releasing Hormone (GHRH) agonist JI‐34 attenuates IH‐induced neurocognitive deficits, anxiety, and depression in mice along with reduction in oxidative stress markers such as MDA and 8‐hydroxydeoxyguanosine, and increases in hypoxia inducible factor‐1α DNA binding and up‐regulation of insulin growth factor‐1 and erythropoietin expression. In contrast, treatment with a GHRH antagonist (MIA‐602) during intermittent hypoxia did not affect any of the IH‐induced deleterious effects in mice. Thus, exogenous GHRH administered as the formulation of a GHRH agonist may provide a viable therapeutic intervention to protect IH‐vulnerable brain regions from OSA‐associated neurocognitive dysfunction.

     

A dual role for Integrin α6β4 in modulating hereditary neuropathy with liability to pressure palsies

Peripheral myelin protein 22 (PMP 22) is a component of compact myelin in the peripheral nervous system. The amount of PMP 22 in myelin is tightly regulated, and PMP 22 over or under‐expression cause Charcot‐Marie‐Tooth 1A (CMT 1A) and Hereditary Neuropathy with Pressure Palsies (HNPP ). Despite the importance of PMP 22 , its function remains largely unknown. It was reported that PMP 22 interacts with the β4 subunit of the laminin receptor α6β4 integrin, suggesting that α6β4 integrin and laminins may contribute to the pathogenesis of CMT 1A or HNPP . Here we asked if the lack of α6β4 integrin in Schwann cells influences myelin stability in the HNPP mouse model. Our data indicate that PMP 22 and β4 integrin may not interact directly in myelinating Schwann cells, however, ablating β4 integrin delays the formation of tomacula, a characteristic feature of HNPP . In contrast, ablation of integrin β4 worsens nerve conduction velocities and non‐compact myelin organization in HNPP animals. This study demonstrates that indirect interactions between an extracellular matrix receptor and a myelin protein influence the stability and function of myelinated fibers.

     

Botulinum protease‐cleaved SNARE fragments induce cytotoxicity in neuroblastoma cells

Soluble N‐ethylmaleimide sensitive factor attachment protein receptors (SNAREs) are crucial for exocytosis, trafficking, and neurite outgrowth, where vesicular SNAREs are directed toward their partner target SNAREs: synaptosomal‐associated protein of 25 kDa and syntaxin. SNARE proteins are normally membrane bound, but can be cleaved and released by botulinum neurotoxins. We found that botulinum proteases types C and D can easily be transduced into endocrine cells using DNA‐transfection reagents. Following administration of the C and D proteases into normally refractory Neuro2A neuroblastoma cells, the SNARE proteins were cleaved with high efficiency within hours. Remarkably, botulinum protease exposures led to cytotoxicity evidenced by spectrophotometric assays and propidium iodide penetration into the nuclei. Direct delivery of SNARE fragments into the neuroblastoma cells reduced viability similar to botulinum proteases' application. We observed synergistic cytotoxic effects of the botulinum proteases, which may be explained by the release and interaction of soluble SNARE fragments. We show for the first time that previously observed cytotoxicity of botulinum neurotoxins/C in neurons could be achieved in cells of neuroendocrine origin with implications for medical uses of botulinum preparations.

     

A two‐hybrid screen identifies an unconventional role for the intermediate filament peripherin in regulating the subcellular distribution of the SNAP25‐interacting protein,SIP30

Peripherin is a type III intermediate filament protein, the expression of which is associated with the acquisition and maintenance of a terminally differentiated neuronal phenotype. Peripherin up‐regulation occurs during acute neuronal injury and in degenerating motor neurons of amyotrophic lateral sclerosis. The functional role(s) of peripherin during normal, injurious, and disease conditions remains unknown, but may be related to differential expression of spliced isoforms. To better understand peripherin function, we performed a yeast two‐hybrid screen on a mouse brain cDNA library using an assembly incompetent peripherin isoform, Per‐61, as bait. We identified new peripherin interactors with roles in vesicular trafficking, signal transduction, DNA/RNA processing, protein folding, and mitochondrial metabolism. We focused on the interaction of Per‐61 and the constitutive isoform, Per‐58, with SNAP25 interacting protein 30 (SIP30), a neuronal protein involved in SNAP receptor‐dependent exocytosis. We found that peripherin and SIP30 interacted through coiled‐coil domains and colocalized in cytoplasmic aggregates in SW13vim(?) cells. Interestingly, Per‐61 and Per‐58 differentially altered the subcellular distribution of SIP30 and SNAP25 in primary motor neurons. Our findings suggest a novel role of peripherin in vesicle trafficking.