The many "Neurofaces" of Prohibitins 1 and 2: Crucial for the healthy brain, dysregulated in numerous brain disorders.

Prohibitin 1 (PHB1) and prohibitin 2 (PHB2) are proteins that are nearly ubiquitously expressed. They are localized in mitochondria, cytosol and cell nuclei. In the healthy CNS, they occur in neurons and non-neuronal cells (oligodendrocytes, astrocytes, microglia, and endothelial cells) and fulfill pivotal functions in brain development and aging, the regulation of brain metabolism, maintenance of structural integrity, synapse formation, aminoacidergic neurotransmission and, probably, regulation of brain action of certain hypothalamic-pituitary hormones.With regard to the diseased brain there is increasing evidence that prohibitins are prominently involved in numerous major diseases of the CNS, which are summarized and discussed in the present review (brain tumors, neurotropic viruses, Alzheimer disease, Down syndrome, Fronto-temporal and vascular dementia, dementia with Lewy bodies, Parkinson disease, Huntington disease, Multiple sclerosis, Amyotrophic lateral sclerosis, stroke, alcohol use disorder, schizophrenia and autism). Unfortunately, there is no PHB-targeted therapy available for any of these diseases.

The many facets of CD26/dipeptidyl peptidase 4 and its inhibitors in disorders of the CNS - a critical overview.

Dipeptidyl peptidase 4 is a serine protease that cleaves X-proline or X-alanine in the penultimate position. Natural substrates of the enzyme are glucagon-like peptide-1, glucagon inhibiting peptide, glucagon, neuropeptide Y, secretin, substance P, pituitary adenylate cyclase-activating polypeptide, endorphins, endomorphins, brain natriuretic peptide, beta-melanocyte stimulating hormone and amyloid peptides as well as some cytokines and chemokines. The enzyme is involved in the maintenance of blood glucose homeostasis and regulation of the immune system. It is expressed in many organs including the brain. DPP4 activity may be effectively depressed by DPP4 inhibitors. Apart from enzyme activity, DPP4 acts as a cell surface (co)receptor, associates with adeosine deaminase, interacts with extracellular matrix, and controls cell migration and differentiation. This review aims at revealing the impact of DPP4 and DPP4 inhibitors for several brain diseases (virus infections affecting the brain, tumours of the CNS, neurological and psychiatric disorders). Special emphasis is given to a possible involvement of DPP4 expressed in the brain.While prominent contributions of extracerebral DPP4 are evident for a majority of diseases discussed herein; a possible role of "brain" DPP4 is restricted to brain cancers and Alzheimer disease. For a number of diseases (Covid-19 infection, type 2 diabetes, Alzheimer disease, vascular dementia, Parkinson disease, Huntington disease, multiple sclerosis, stroke, and epilepsy), use of DPP4 inhibitors has been shown to have a disease-mitigating effect. However, these beneficial effects should mostly be attributed to the depression of "peripheral" DPP4, since currently used DPP4 inhibitors are not able to pass through the intact blood-brain barrier.

From putative brain tumor marker to high cognitive abilities: Emerging roles of a disintegrin and metalloprotease (ADAM) 12 in the brain.

ADAM (a disintergin and metalloprotease) 12 is a member of the large family of multidomain metalloprotease-disintegrins, which possess cell-binding and metalloprotease properties. The enzyme is responsible for the shedding of a number of membrane-bound proteins (heparin-binding-EGF, insulin-like growth factor 2-binding proteins 3 and 5, oxytocinase, glycoprotein non-metastatic melanoma protein B and basigin). In rat and human CNS, ADAM12 is predominantly localized in white and gray matter oligodendrocytes. In addition it can be detected in astrocytes, neurons and endothelial cells. Its function in healthy brain is not well established yet, but prominent roles in CNS development, myelination and high cognitive abilities are discussed. There is increasing evidence that ADAM12 is involved in numerous major diseases of the CNS, which are summarized in the present review (brain tumors, multiple sclerosis, amyotrophic lateral sclerosis, Alzheimer´s disease, stroke, schizophrenia, autism and bipolar disorder).

Binding varicella zoster virus: an underestimated facet of insulin-degrading enzyme´s implication for Alzheimer´s disease pathology?

We shortly discuss a possible contribution of insulin-degrading enzyme to Alzheimer´s disease pathology by binding varicella zoster virus glycoprotein E, which increases the infectivity and cell-cell spread of the virus.

Relative Resilience of Cerebellar Purkinje Cells in a Cardiac Arrest/Resuscitation Rat Model.

BACKGROUND: In studies on cardiac arrest (CA)/resuscitation (R) injury, Purkinje cell degeneration was described, however, with inconsistent data concerning severity and time point of manifestation. Moreover, CA/R studies paid only limited attention to inhibitory stellate interneurons. To this aim, the hypothesis that cerebellar could be relatively resilient toward CA/R because of diverse cellular defense mechanisms including interaction with stellate cells was tested. METHODS: We examined rats with survival times of 6, 24, and 48 h, and 7 and 21 days in comparison with sham- and nonoperated animals. Thereby, we focused on the immunohistochemical expression of cfos, MnSOD, Bcl2, caspase 3, parvalbumin, calbindin D28 k, MAP2, IBA1, and GFAP, especially in the particular sensitivity to CA/R cerebellar lobule IX. Hippocampal CA1 degeneration was demonstrated by expression patterns of MAP2 and NeuN in combination with IBA1 and GFAP. RESULTS/CONCLUSIONS: Comparative analysis of hippocampal CA1 pyramidal cells and cerebellar Purkinje cells confirmed a relative resil-ience of Purkinje cells to CA/R. We found only a notable degeneration of Purkinje cell neuronal fiber network, which, however, not necessarily led to neuronal cell death. To induce significant Purkinje cell loss, a stronger ischemic trigger seems to be needed. As possible Purkinje cell-protecting mechanisms, we would propose: (1) activation of inhibitory stellate cells, shown by cfos, MnSOD, and Bcl2 expression, balancing out ischemia-induced excitation and inhibition of Purkinje cells; (2) translocation of the calcium-buffering system, shown by parvalbumin and calbindin D28 k expression, protecting Purkinje cells from detrimental calcium overload; (3) activation of the neuron-astrocyte cross talk, protecting Purkinje cells from over-excitation by removing potassium and neurotransmitters from the extracellular space; (4) activation of the effective and long-lasting MnSOD defense system; and (5) of the anti-apoptotic protein Bcl2 in Purkinje cells itself. Moreover, the results emphasize the limited comparability of animal CA/R studies because of the heterogeneity of the used experimental regimes.

The Differentiation of Spinal Cord Motor Neurons is Associated with Changes of the Mitochondrial Phospholipid Cardiolipin.

Motor neuron damage caused by diseases, traumatic insults or de-afferentation of the spinal cord is often incurable due to the poor intrinsic regenerative capacity. Moreover, regenerated peripheral nerves often do not reach normal functionality. Here, we investigated cardiolipin in the process of neuro-differentiation, since cardiolipin is closely linked to the mitochondrial energy supply in cells. The NSC-34 hybrid cell line, produced by fusing neuroblastoma cells with primary spinal cord motor neurons, was used, since it shares several morphological and physiological characteristics with mature primary motor neurons. Their neuro-differentiation was supported by switching from normal to differentiation medium or by fatty acid supplementation. Differentiation was evaluated by measuring neurite-sprouting parameters and PPARα expression. Cellular fatty acid distribution was analyzed to indicate changes in lipid metabolism during differentiation. Cardiolipin was characterized by acyl-chain composition and the distribution of molecular cardiolipin species. Both, the switch from normal to differentiation medium as well as the administration of palmitic and oleic acid promoted neuro-differentiation. Stimulated differentiation was accompanied by changes in cardiolipin content and composition. The positive correlation between neuro-differentiation and concentration of those molecular cardiolipin species containing palmitic and oleic acid implied a link between differentiation of NSC-34 cells and cardiolipin metabolism. We further demonstrated the impact of cellular lipid metabolism, and particularly cardiolipin metabolism, during and NSC-34 neuritogenesis. Thus, cardiolipin may represent a new therapeutic target for axon regeneration after peripheral nerve injuries or when axon sprouting is required to compensate for motor neuron loss in response to aging and/or disease.

The expression of the MSC-marker CD73 and of NF2/Merlin are correlated in meningiomas.

Mesenchymal stem cells (MSC) have been found in various cancers and were discussed to influence tumor biology. Cells fulfilling the complete MSC criteria, including surface marker expression (CD73, CD90, CD105) and tri-lineage differentiation, have been isolated solely from a low percentage of high-grade meningiomas. In contrast, pure co-expression of the surface-markers was relatively frequent, raising the question for an additional role of these membrane proteins in meningiomas. Therefore, here we analyzed the expression of CD73, CD90 and CD105 in a series of meningiomas of all grades. Although no significant association of any marker with meningeal tumor growth per se or with tumor-grade was observed, we detected a positive Pearson correlation (r = 0.55, p ≤ 0.05) in low-grade tumors between CD73 and the most relevant tumor suppressor NF2/Merlin, supported by a tendency of lower CD73 expression in cases with allelic losses at the NF2-locus, which express significantly lower NF2/Merlin-mRNA (p ≤ 0.05). In two pairs of syngenous meningeal or meningioma cell lines with or without shRNA-mediated knockdown of NF2/Merlin a nearly complete loss of CD73 mRNA expression was observed after the knockdown (p ≤ 0.001). This suggested that the correlation observed in tumors may result from a direct functional link between Merlin and CD73. Since CD73 is a 5'-exonucleotidase (termed NT5E), we discuss a potential role of NT5E-mediated purinergic signaling to modulate actin-cytoskeleton and cell contacts, which may be a functional link to NF2/Merlin.

Dipeptidyl peptidase IV, which probably plays important roles in Alzheimer disease (AD) pathology, is upregulated in AD brain neurons and associates with amyloid plaques.

There is evidence from in vitro experiments that dipeptidyl peptidase IV (DPP IV) might play role(s) in amyloid formation. However, nothing is known about the localization of the enzyme in brains of individuals with Alzheimer's disease. We herein show that in comparison to non-demented controls DPP IV is upregulated in AD brain neurons and occurs in multiple amyloid plaques.

Insulin-regulated aminopeptidase immunoreactivity is abundantly present in human hypothalamus and posterior pituitary gland, with reduced expression in paraventricular and suprachiasmatic neurons in chronic schizophrenia.

The vasopressin- and oxytocin-degrading enzyme insulin-regulated aminopeptidase (IRAP) is expressed in various organs including the brain. However, knowledge about its presence in human hypothalamus is fragmentary. Functionally, for a number of reasons (genetic linkage, hydrolysis of oxytocin and vasopressin, its role as angiotensin IV receptor in learning and memory and others) IRAP might play a role in schizophrenia. We studied the regional and cellular localization of IRAP in normal human brain with special emphasis on the hypothalamus and determined numerical densities of IRAP-expressing cells in the paraventricular, supraoptic and suprachiasmatic nuclei in schizophrenia patients and controls. By using immunohistochemistry and Western blot analysis, IRAP was immunolocalized in postmortem human brains. Cell countings were performed to estimate numbers and numerical densities of IRAP immunoreactive hypothalamic neurons in schizophrenia patients and control cases. Shape, size and regional distribution of IRAP-expressing cells, as well the lack of co-localization with the glia marker glutamine synthetase, show that IRAP is expressed in neurons. IRAP immunoreactive cells were observed in the hippocampal formation, cerebral cortex, thalamus, amygdala and, abundantly, hypothalamus. Double labeling experiments (IRAP and oxytocin/neurophysin 1, IRAP with vasopressin/neurophysin 2) revealed that IRAP is present in oxytocinergic and in vasopressinergic neurons. In schizophrenia patients, the numerical density of IRAP-expressing neurons in the paraventricular and the suprachiasmatic nuclei is significantly reduced, which might be associated with the reduction in neurophysin-containing neurons in these nuclei in schizophrenia. The pathophysiological role of lowered hypothalamic IRAP expression in schizophrenia remains to be established.

Selected gene profiles of stressed NSC-34 cells and rat spinal cord following peripheral nerve reconstruction and minocycline treatment.

The present study was conducted to investigate the effects of minocycline on the expression of selected transcriptional and translational profiles in the rat spinal cord following sciatic nerve (SNR) transection and microsurgical coaptation. The mRNA and protein expression levels of B cell lymphoma-2 (Bcl-2), Bcl-2-associated X protein (Bax), caspase-3, major histocompatibility complex I (MHC I), tumor necrosis factor-α (TNF-α), activating transcription factor 3 (ATF3), vascular endothelial growth factor (VEGF), matrix metalloproteinase 9 (MMP9), and growth associated protein-43 (GAP-43) were monitored in the rat lumbar spinal cord following microsurgical reconstruction of the sciatic nerves and minocycline treatment. The present study used semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemistry. As a PCR analysis of spinal cord tissue enabled the examination of the expression patterns of all cell types including glia, the motorneuron-like NSC-34 cell line was used to investigate expression level changes in motorneurons. As stressors, oxygen glucose deprivation (OGD) and lipopolysaccharide (LPS) treatment were performed. SNR did not induce significant degeneration of ventral horn motorneurons, whereas microglia activation and synaptic terminal retraction were detectable. All genes were constitutively expressed at the mRNA and protein levels in untreated spinal cord and control cells. SNR significantly increased the mRNA expression levels of all genes, albeit only temporarily. In all genes except MMP9 and GAP-43, the induction was seen ipsilaterally and contralaterally. The effects of minocycline were moderate. The expression levels of MMP9, TNF-α, MHC I, VEGF, and GAP-43 were reduced, whereas those of Bax and Bcl-2 were unaffected. OGD, but not LPS, was toxic for NSC-34 cells. No changes in the expression levels of Bax, caspase-3, MHC I or ATF3 were observed. These results indicated that motorneurons were not preferentially or solely responsible for SNR-mediated upregulation of these genes. MMP9, TNF-α, VEGF and Bcl-2 were stress-activated. These results suggest that a substantial participation of motorneurons in gene expression levels in vivo. Minocycline was also shown to have inhibitory effects. The nuclear factor-κB signalling pathway may be a possible target of minocycline.

Growth factor choice is critical for successful functionalization of nanoparticles.

Nanoparticles (NPs) show new characteristics compared to the corresponding bulk material. These nanoscale properties make them interesting for various applications in biomedicine and life sciences. One field of application is the use of magnetic NPs to support regeneration in the nervous system. Drug delivery requires a functionalization of NPs with bio-functional molecules. In our study, we functionalized self-made PEI-coated iron oxide NPs with nerve growth factor (NGF) and glial cell-line derived neurotrophic factor (GDNF). Next, we tested the bio-functionality of NGF in a rat pheochromocytoma cell line (PC12) and the bio-functionality of GDNF in an organotypic spinal cord culture. Covalent binding of NGF to PEI-NPs impaired bio-functionality of NGF, but non-covalent approach differentiated PC12 cells reliably. Non-covalent binding of GDNF showed a satisfying bio-functionality of GDNF:PEI-NPs, but turned out to be unstable in conjugation to the PEI-NPs. Taken together, our study showed the importance of assessing bio-functionality and binding stability of functionalized growth factors using proper biological models. It also shows that successful functionalization of magnetic NPs with growth factors is dependent on the used binding chemistry and that it is hardly predictable. For use as therapeutics, functionalization strategies have to be reproducible and future studies are needed.

Evaluation of In-Situ Magnetic Signals from Iron Oxide Nanoparticle-Labeled PC12 Cells by Atomic Force Microscopy.

The magnetic signals from magnetite nanoparticle-labeled PC12 cells were assessed by magnetic force microscopy by deploying a localized external magnetic field to magnetize the nanoparticles and the magnetic tip simultaneously so that the interaction between the tip and PC12 cell-associated Fe3O4 nanoparticles could be detected at lift heights (the distance between the tip and the sample) larger than 100 nm. The use of large lift heights during the raster scanning of the probe eliminates the non-magnetic interference from the complex and rugged cell surface and yet maintains the sufficient sensitivity for magnetic detection. The magnetic signals of the cell-bound nanoparticles were semi-quantified by analyzing cell surface roughness upon three-dimensional reconstruction generated by the phase shift of the cantilever oscillation. The obtained data can be used for the evaluation of the overall cellular magnetization as well as the maximum magnetic forces from magnetic nanoparticle-labeled cells which is crucial for the biomedical application of these nanomaterials.

Effects of nitric oxide synthase deficiency on a disintegrin and metalloproteinase domain-containing protein 12 expression in mouse brain samples.

A disintegrin and metalloproteinase domain-containing protein 12 (ADAM12) belongs to the ADAM family of transmembrane proteins. Via proteolysis, cell adhesion, cell-cell fusion, cell-matrix interaction and membrane protein shedding, ADAM proteins are involved in normal brain development, and also in cancer genesis and progression, and in inflammation. Therefore, neurobiological research focusing on this protein is increasing. Nitric oxide (NO), which is endogenously produced by NO synthases (NOS), is associated with glial tumors. However, knock-out of NOS produces only limited antitumor effects. The present study analyzed the expression of ADAM12 in the cortex and hippocampus of C57/BL6 wild-type mice, and endothelial NOS-, neuronal NOS-(nNOS) or inducible NOS (iNOS)-deficient (-/-) mice, at different stages of development. Expression of ADAM12 was quantified using immunoblot analysis of cortical and hippocampal tissue samples from fetal, neonatal (5 days postnatal), adult (12 weeks old) or >1 year old mice. Using reverse transcription-quantitative polymerase chain reaction, ADAM12 expression was analyzed in cultured N9, OLN93, C6 and PC12 cells, representing the four main cell types in the brain, following NOS inhibition. ADAM12 expression was low in all mouse genotypes and regions of the brain, and in fetal and neonatal mice, an increase in expression was observed with increasing age. The highest levels of expression were observed in the cortex of adult mice, iNOS(-/-) mice of >1 year and wild-type mice, and in the hippocampus of adult and iNOS(-/-) mice of >1 year. By contrast, ADAM12 expression was lowest in adult nNOS(-/-) mice. Inhibition of NOS using N(ω)-Nitro-L-arginine methyl ester hydrochloride, induced ADAM12 mRNA expression in N9 and PC12 cell lines. Inhibition of NOS using L-N(6)-(1-Iminoethyl)lysine dihydrochloride, induced ADAM12 mRNA expression in N9 and C6 cell lines. No change in ADAM12 expression was observed in OLN93 cells following NOS inhibition. ADAM12 expression in mouse hippocampus and cortex samples demonstrated considerable variation during development, with a marked increase observed in adult and >1 year old mice, compared with that in fetal and neonatal mice.

Downregulation of neuregulin 1-ErbB4 signaling and antidepressant properties of ketamine: ErbB4 expressing pyramidal neurons may play a role.

We comment on a recent paper of Wang et al. (J Mol Neurosci, 2014), who have shown that ketamine reduces neuregulin1-ErbB4 signaling in interneurons.

Effects of cerebrolysin on motor-neuron-like NSC-34 cells.

Although the peripheral nervous system is capable of regeneration, this capability is limited. As a potential means of augmenting nerve regeneration, the effects of cerebrolysin (CL)--a proteolytic peptide fraction--were tested in vitro on the motor-neuron-like NSC-34 cell line and organotypic spinal cord cultures. Therefore, NSC-34 cells were subjected to mechanical stress by changing media and metabolic stress by oxygen glucose deprivation. Afterwards, cell survival/proliferation using MTT and BrdU-labeling (FACS) and neurite sprouting using ImageJ analysis were evaluated. Calpain-1, Src and α-spectrin protein expression were analyzed by Western blot. In organotypic cultures, the effect of CL on motor neuron survival and neurite sprouting was tested by immunohistochemistry. CL had a temporary anti-proliferative but initially neuroprotective effect on OGD-stressed NSC-34 cells. High-dosed or repeatedly applied CL was deleterious for cell survival. CL amplified neurite reconstruction to limited extent, affected calpain-1 protein expression and influenced calpain-mediated spectrin cleavage as a function of Src expression. In organotypic spinal cord slice cultures, CL was not able to support motor neuron survival/neurite sprouting. Moreover, it hampered astroglia and microglia activities. The data suggest that CL may have only isolated positive effects on injured spinal motor neurons. High-dosed or accumulated CL seemed to have adverse effects in treatment of spinal cord injury. Further experiments are required to optimize the conditions for a safe clinical administration of CL in spinal cord injuries.

Time-course of neuropathic pain in mice deficient in neuronal or inducible nitric oxide synthase.

Nitric oxide which is synthesised by nitric oxide synthase (NOS) is involved in processes related to regeneration after nerve injury and neuropathic pain. Here we investigated functional aspects of the nociceptive system. For that purpose, the chronic constriction injury (CCI) model induced by loose ligation of the sciatic nerve was employed in C57Bl/6J wild-type (WT), nNOS and iNOS knock-out (-/-) mice. Their thermal and mechanical pain thresholds were then measured over a period of six weeks. In addition, (3)H-DAMGO, (3)H-CP 55.940, and (3)H-l-glutamate binding, and neuronal (NeuN-immunostained) and astroglial (GFAP-immunostained) cell composition were studied. There were no significant differences in cell composition between the three strains used. Significant differences between CCI and sham-operated animals were found in nNOS-/- after day 6, in WT mice after day 10, and in iNOS-/- after day 17 post surgery. The mechanical pain threshold was normalised after day 45 post surgery in WT mice only. There were no changes in DAMGO and glutamate binding. However, we found significant differences in CP 55.940 binding in the spinal cord. It was concluded that NOS-cannabinoid interaction contributes to differences in nociceptive behaviour.

ADAM 12: a putative marker of oligodendrogliomas?

ADAM 12 (meltrin alpha) belongs to a large family of molecules, consisting of members with both disintegrin and metalloproteinase properties. ADAMs have been implicated in several cell physiological processes including cell adhesion, cell fusion, proteolysis and signalling. ADAM 12 is widely expressed, including skeletal muscle, testis, bone, intestine, heart and kidney. In addition, a variety of tumours show elevated expression of ADAM12; among them being breast-, colon-, gastric- and lung-carcinoma. As to the brain, ADAM 12 has been shown previously to be expressed in rat and human oligodendrocytes. However, little is known about the expression of this protease in brain tumours. This study demonstrates the presence of ADAM 12 in non-neoplastic oligodendroglial cells of normal human brain as well as in neoplastic oligodendroglia and minigemistocytes arising from four pure oligodendrogliomas and three mixed oligoastrocytomas. Double stainings revealed a notable preference of ADAM 12 for the oligodendroglial over astroglial components. The results of immunohistochemistry are in accordance with the results obtained from the RT-PCR, which further demonstrated a mild difference concerning the mRNA concentration of ADAM 12 between similar grades of eight astrocytomas and eight oligodendrogliomas (namely four astrocytomas grade II versus four oligodendrogliomas grade II and four astrocytomas grade III versus four oligodendrogliomas grade III). Both cellular immunostaining for ADAM 12 and ADAM 12 mRNA content decrease with higher histologic grade of the tumour. Surprisingly, the latter parameter (ADAM12 mRNA) showed a significant opposite correlation to the degree of histologic tumour malignancy. From our data showing that ADAM 12 is highly expressed in, but not restricted to, oligodendrogliomas, we conclude that ADAM 12 immunohistochemistry may be a helpful tool in the diagnosis of brain tumours.

Neuronal cells loaded with PEI-coated Fe3O4 nanoparticles for magnetically guided nerve regeneration.

We report a one-step synthesis protocol for obtaining polymer-coated magnetic nanoparticles (MNPs) engineered for uploading neural cells. Polyethyleneimine-coated Fe3O4 nanoparticles (PEI-MNPs) with sizes of 25 ± 5 nm were prepared by oxidation of Fe(OH)2 by nitrate in basic aqueous media and adding PEI in situ during synthesis. The obtained PEI-MNP cores displayed a neat octahedral morphology and high crystallinity. The resulting nanoparticles were coated with a thin polymer layer of about 0.7-0.9 nm, and displayed a saturation magnetization value MS = 58 A m2 kg-1 at 250 K (64 A m2 kg-1 for T = 10 K). Cell uptake experiments on a neuroblastoma-derived SH-SY5Y cell line were undertaken over a wide time and MNP concentration range. The results showed a small decrease in cell viability for 24 h incubation (down to 70% viability for 100 µg ml-1), increasing the toxic effects with incubation time (30% cell survival at 100 µg ml-1 for 7 days of incubation). On the other hand, primary neuronal cells displayed higher sensitivity to PEI-MNPs, with a cell viability reduction of 44% of the control cells after 3 days of incubation with 50 µg ml-1. The amount of PEI-MNPs uploaded by SH-SY5Y cells was found to have a linear dependence on concentration. The intracellular distribution of the PEI-MNPs analyzed at the single-cell level by the dual-beam (FIB/SEM) technique revealed the coexistence of both fully incorporated PEI-MNPs and partially internalized PEI-MNP-clusters crossing the cell membrane. The resulting MNP-cluster distributions open the possibility of using these PEI-MNPs for magnetically driven axonal re-growth in neural cells.

Poly-l-lysine-coated magnetic nanoparticles as intracellular actuators for neural guidance.

PURPOSE: It has been proposed in the literature that Fe(3)O(4) magnetic nanoparticles (MNPs) could be exploited to enhance or accelerate nerve regeneration and to provide guidance for regenerating axons. MNPs could create mechanical tension that stimulates the growth and elongation of axons. Particles suitable for this purpose should possess (1) high saturation magnetization, (2) a negligible cytotoxic profile, and (3) a high capacity to magnetize mammalian cells. Unfortunately, the materials currently available on the market do not satisfy these criteria; therefore, this work attempts to overcome these deficiencies. METHODS: Magnetite particles were synthesized by an oxidative hydrolysis method and characterized based on their external morphology and size distribution (high-resolution transmission electron microscopy [HR-TEM]) as well as their colloidal (Z potential) and magnetic properties (Superconducting QUantum Interference Devices [SQUID]). Cell viability was assessed via Trypan blue dye exclusion assay, cell doubling time, and MTT cell proliferation assay and reactive oxygen species production. Particle uptake was monitored via Prussian blue staining, intracellular iron content quantification via a ferrozine-based assay, and direct visualization by dual-beam (focused ion beam/scanning electron microscopy [FIB/SEM]) analysis. Experiments were performed on human neuroblastoma SH-SY5Y cell line and primary Schwann cell cultures of the peripheral nervous system. RESULTS: This paper reports on the synthesis and characterization of polymer-coated magnetic Fe(3)O(4) nanoparticles with an average diameter of 73 ± 6 nm that are designed as magnetic actuators for neural guidance. The cells were able to incorporate quantities of iron up to 2 pg/cell. The intracellular distribution of MNPs obtained by optical and electronic microscopy showed large structures of MNPs crossing the cell membrane into the cytoplasm, thus rendering them suitable for magnetic manipulation by external magnetic fields. Specifically, migration experiments under external magnetic fields confirmed that these MNPs can effectively actuate the cells, thus inducing measurable migration towards predefined directions more effectively than commercial nanoparticles (fluidMAG-ARA supplied by Chemicell). There were no observable toxic effects from MNPs on cell viability for working concentrations of 10 µg/mL (EC(25) of 20.8 µg/mL, compared to 12 µg/mL in fluidMAG-ARA). Cell proliferation assays performed with primary cell cultures of the peripheral nervous system confirmed moderate cytotoxicity (EC(25) of 10.35 µg/mL). CONCLUSION: These results indicate that loading neural cells with the proposed MNPs is likely to be an effective strategy for promoting non-invasive neural regeneration through cell magnetic actuation.

Vav deficiency impedes peripheral nerve regeneration in mice.

PURPOSE: The regeneration of adult peripheral nerves is a complex, multi-step process that is often incomplete, resulting in pain and/or loss of muscle innervation. Success is based on a fine-tuned interplay of neurons, Schwann cells, fibrocytes and macrophages realizing Wallerian degeneration, fiber regrowth and revascularization. Following trauma, the nerves distal to the injury site undergo Wallerian degeneration, an event that includes the phagocytosis of debris and the formation of Schwann cell scaffolds that guide the sprouting nerve fibers. The actin cytoskeleton is critical to all of these processes; therefore, activators of the cytoskeleton such as Rho GTPases and RhoGEFS such as Vav2 and Vav3 represent attractive targets for therapeutic intervention. METHODS: Sciatic nerve segments were surgically resected and reconstructed, and the degenerative/regenerative outcomes were compared in wild-type and Vav2/3 double knockout mice. RESULTS: Vav2/3 knockout nerves showed delayed Wallerian degeneration and revascularization, a broadly control-like morphometry of the regenerated nerves including remyelination, and contradictory motor function recovery, whereby impaired toe spreading was accompanied by enhanced muscle weight recreation. CONCLUSIONS: The data suggest that Vav2 and Vav3 are required for normal peripheral nerve degeneration/regeneration, revascularization and functional recovery. Functional redundancy, compensatory mechanisms, and muscle (pseudo)hypertrophy, however, impede the understanding of and intervention in Vav-mediated processes.