Periurethral and Intravenous Injections of Adipose-Derived Stem Cells to Promote Local Tissue Recovery in a Rat Model of Stress Urinary Incontinence.

OBJECTIVE: To compare the effects of periurethral and intravenous injection of adipose-derived stem cells (ADSCs) on voiding function and tissue recovery in a stress urinary incontinence (SUI) rat model. METHODS: Sixty-four postpartum rats were randomly allocated to a normal group and the SUI model was established in 48 rats by vagina balloon dilation and bilateral ovariectomy. The SUI rats were randomized into 3 groups and received urethral injection of PBS (SUI group), periurethral injection of ADSCs (PU group), and intravenous injection of ADSCs (IV group) in 10 days after the ovariectomy. After 1, 7, and 14 days, ADSCs were tracked in urethra specimen. The urinary function of the remaining rats was analyzed at day 28, and urethral tissues were harvested for Western blotting and histochemical analyses. RESULTS: Alpha smooth muscle actin, myosin heavy chain, vascular endothelial growth factor, and neurofilament protein expression was increased in the IV and PU groups. Voiding function was also improved, with no significant differences between the IV and PU groups. The cell retention rate in rat urethral tissues was higher in the PU group than that in the IV group. Compared with the IV group, myosin heavy chain, vascular endothelial growth factor, neurofilament and transforming growth factor-ß1 (TGF-ß1)/Smad pathway protein expression levels were significantly higher in the PU group, while alpha smooth muscle actin expression was significantly lower (P < .05). CONCLUSION: Periurethral and intravenous injection of ADSCs induces different degrees of recovery of the urethral sphincter, cytokine secretion levels and cell retention rates in the urethral tissues in SUI rats, however, there was no significant difference in 2 methods.

Comparing effects of L-carnitine and sildenafil citrate on histopathologic recovery from sciatic nerve crush injury in female albino rats.

BACKGROUND: The sciatic nerve is a peripheral nerve and is more vulnerable to compression with subsequent short- or long-term neuronal dysfunction. The current study was designed to elucidate the possible ameliorative effect of L-carnitine and sildenafil (SIL) on sciatic nerve crush injury. We sought to determine the effects of L-carnitine, a neuroprotective and a neuro-modulatory agent, and SIL citrate, a selective peripheral phosphodiesterases inhibitor, on modulating neuro-degenerative changes due to sciatic nerve compression. MATERIALS AND METHODS: The comparative effect of L-carnitine (at an oral dose of 20 mg/kg/day) or SIL citrate (20 mg/kg/day orally) administration for 21 days was studied in a rat model of sciatic nerve compression. Sciatic nerve sections were subjected to biochemical, histological, ultrastructure, and immunohistochemical studies to observe the effects of these treatments on neurofilament protein. RESULTS: The sciatic nerve crush injury group (group II) showed a significant decrease in tissue catalase (CAT), superoxide dismutase (SOD) and increase in malondialdehyde (MDA) as compared to control group (p < 0.01). Histological changes in the form of degenerated and vacuolated axoplasm with areas of nerve fibre loss and pyknotic nuclei were reported. The blood vessels were dilated, congested with areas of haemorrhage and mononuclear cell infiltration. Histo-morphometrically, a statistically significant reduction in the nerve fibres' number, mean axon cross-sectional area, myelin sheath thickness and a significant increase in collagen fibres' percentage (p < 0.05) as compared to control group. Immunohistochemically, neurofilament protein was significantly downregulated as proved by a significant reduction in mean area per cent of neurofilament expression. L-carnitine ameliorated the studied parameters through its neuroprotective effect while SIL, a selective peripheral phosphodiesterases (PDE-5) inhibitor, improved crush injury parameters but with less extent than L-carnitine. CONCLUSIONS: These findings indicate the valuable effects of L-carnitine administration compared to that of SIL citrate in alleviating the serious debilitating effects of sciatic nerve crush injury. Our results provide a new insight into the scope of neuroprotective and neuro-regenerative effects of L-carnitine in a sciatic nerve compression model.

Lipid Nanoparticles Vectorized with NFL-TBS.40-63 Peptide Target Oligodendrocytes and Promote Neurotrophin-3 Effects After Demyelination In Vitro.

Promoting remyelination in multiple sclerosis is important to prevent axon degeneration, given the lack of curative treatment. Although some growth factors improve this repair, unspecific delivery to cells and potential side effects limit their therapeutic use. Thus, NFL-TBS.40-63 peptide (NFL)-known to enter specifically myelinating oligodendrocytes (OL)-was used to vectorize 100 nm diameter lipid nanoparticles (LNC), and the ability of NFL-LNC to specifically target OL from newborn rat brain was assessed in vitro. Specific uptake of DiD-labeled NFL-LNC by OL characterized by CNP and myelin basic protein was observed by confocal microscopy, as well as DiD colocalization with NFL and with Rab5-a marker of early endosomes. Unvectorized LNC did not significantly penetrate OL and there was no uptake of NFL-LNC by astrocytes. Canonical maturation of OL which extended compacted myelin-like membranes was observed by transmission electron microscopy in cells grown up to 9 days with NFL-LNC. Endocytosis of NFL-LNC appeared to depend on several pathways, as demonstrated by inhibitors. In addition, vectorized NFL-LNC adsorbed on neurotrophin-3 (NT-3) potentiated the proremyelinating effects of NT-3 after demyelination by lysophosphatidyl choline, allowing noticeably decreasing NT-3 concentration. Our results if they were confirmed in vivo suggest that NFL-vectorized LNC appear safe and could be considered as putative carriers for specific drug delivery to OL in order to increase remyelination.

The neurofilament derived-peptide NFL-TBS.40-63 enters in-vitro in human neural stem cells and increases their differentiation.

Regenerative medicine is a promising approach to treat neurodegenerative diseases by replacing degenerating cells like neurons or oligodendrocytes. Targeting human neural stem cells directly in the brain is a big challenge in such a strategy. The neurofilament derived NFL-TBS.40-63 peptide has recently been introduced as a novel tool to target neural stem cells. Previous studies showed that this peptide can be internalized by rat neural stem cells in vitro and in vivo, which coincided with lower proliferation and self-renewal capacity and increase of differentiation. In this study, we analyzed the uptake and potential effects of the NFL-TBS.40-63 peptide on human neural stem cells isolated from human fetuses. We showed that the peptide inhibits proliferation and the ability to produce neurospheres in vitro, which is consistent with an increase in cell adhesion and differentiation. These results confirm that the peptide could be a promising molecule to target and manipulate human neural stem cells and thus could serve as a strategic tool for regenerative medicine.

The Neurofilament-Derived Peptide NFL-TBS.40-63 Targets Neural Stem Cells and Affects Their Properties.

UNLABELLED: Targeting neural stem cells (NSCs) in the adult brain represents a promising approach for developing new regenerative strategies, because these cells can proliferate, self-renew, and differentiate into new neurons, astrocytes, and oligodendrocytes. Previous work showed that the NFL-TBS.40-63 peptide, corresponding to the sequence of a tubulin-binding site on neurofilaments, can target glioblastoma cells, where it disrupts their microtubules and inhibits their proliferation. We show that this peptide targets NSCs in vitro and in vivo when injected into the cerebrospinal fluid. Although neurosphere formation was not altered by the peptide, the NSC self-renewal capacity and proliferation were reduced and were associated with increased adhesion and differentiation. These results indicate that the NFL-TBS.40-63 peptide represents a new molecular tool to target NSCs to develop new strategies for regenerative medicine and the treatment of brain tumors. SIGNIFICANCE: In the present study, the NFL-TBS.40-63 peptide targeted neural stem cells in vitro when isolated from the subventricular zone and in vivo when injected into the cerebrospinal fluid present in the lateral ventricle. The in vitro formation of neurospheres was not altered by the peptide; however, at a high concentration of the peptide, the neural stem cell (NSC) self-renewal capacity and proliferation were reduced and associated with increased adhesion and differentiation. These results indicate that the NFL-TBS.40-63 peptide represents a new molecular tool to target NSCs to develop new strategies for regenerative medicine and the treatment of brain tumors.

The NFL-TBS.40-63 anti-glioblastoma peptide disrupts microtubule and mitochondrial networks in the T98G glioma cell line.

Despite aggressive therapies, including combinations of surgery, radiotherapy and chemotherapy, glioblastoma remains a highly aggressive brain cancer with the worst prognosis of any central nervous system disease. We have previously identified a neurofilament-derived cell-penetrating peptide, NFL-TBS.40-63, that specifically enters by endocytosis in glioblastoma cells, where it induces microtubule destruction and inhibits cell proliferation. Here, we explore the impact of NFL-TBS.40-63 peptide on the mitochondrial network and its functions by using global cell respiration, quantitative PCR analysis of the main actors directing mitochondrial biogenesis, western blot analysis of the oxidative phosphorylation (OXPHOS) subunits and confocal microscopy. We show that the internalized peptide disturbs mitochondrial and microtubule networks, interferes with mitochondrial dynamics and induces a rapid depletion of global cell respiration. This effect may be related to reduced expression of the NRF-1 transcription factor and of specific miRNAs, which may impact mitochondrial biogenesis, in regard to default mitochondrial mobility.

Neurofilament-tubulin binding site peptide NFL-TBS.40-63 increases the differentiation of oligodendrocytes in vitro and partially prevents them from lysophosphatidyl choline toxiciy.

During multiple sclerosis (MS), the main axon cystoskeleton proteins, neurofilaments (NF), are altered, and their release into the cerebrospinal fluid correlates with disease severity. The role of NF in the extraaxonal location is unknown. Therefore, we tested whether synthetic peptides corresponding to the tubulin-binding site (TBS) sequence identified on light NF chain (NFL-TBS.40-63) and keratin (KER-TBS.1-24), which could be released during MS, modulate remyelination in vitro. Biotinylated NFL-TBS.40-63, NFL-Scramble2, and KER-TBS.1-54 (1-100 µM, 24 hr) were added to rat oligodendrocyte (OL) and astrocyte (AS) cultures, grown in chemically defined medium. Proliferation and differentiation were characterized by using specific antibodies (A2B5, CNP, MBP, GFAP) and compared with untreated cultures. Lysophosphatidyl choline (LPC; 2 × 10(-5) M) was used to induce OL death and to test the effects of TBS peptides under these conditions. NFL-TBS.40-63 significantly increased OL differentiation and maturation, with more CNP(+) and MBP(+) cells characterized by numerous ramified processes, along with myelin balls. When OL were challenged with LPC, concomitant treatment with NFL-TBS.40-63 rescued more than 50% of OL compared with cultures treated with LPC only. Proliferation of OL progenitors was not affected, nor were AS proliferation and differentiation. NFL-TBS.40-63 peptide induces specific effects in vitro, increasing OL differentiation and maturation without altering AS fate. In addition, it partially protects OL from demyelinating injury. Thus release of NFL-TBS.40-63 caused by axonal damage in vivo could improve repair through increased OL differentiation, which is a prerequisite for remyelination.

The NFL-TBS.40-63 anti-glioblastoma peptide enters selectively in glioma cells by endocytosis.

Glioblastoma are the most frequent and aggressive tumour of the nervous system despite surgical resection associated with chemotherapy and radiotherapy. Recently, we showed that the NFL-TBS.40-63 peptide corresponding to the sequence of a tubulin-binding site of neurofilaments, enters selectively in glioblastoma cells where it blocks microtubule polymerization, inhibits their proliferation, and reduces tumour development in rats bearing glioblastoma (Bocquet et al., 2009; Berges et al., 2012a). Here, we characterized the molecular mechanism responsible for the uptake of NFL-TBS.40-63 peptide by glioblastoma cells. Unlike other cell penetrating peptides (CPPs), which use a balance between endocytosis and direct translocation, the NFL-TBS.40-63 peptide is unable to translocate directly through the membrane when incubated with giant plasma membrane vesicles. Then, using a panel of markers and inhibitors, flow cytometry and confocal microscopy investigations showed that the uptake occurs mainly through endocytosis. Moreover, glycosaminoglycans and αVß3 integrins are not involved in the NFL-TBS.40-63 peptide recognition and internalization by glioblastoma cells. Finally, the signalling of tyrosine kinase receptors is involved in the peptide uptake, especially via EGFR overexpressed in tumour cells, indicating that the uptake of NFL-TBS.40-63 peptide by glioblastoma cells is related to their abnormally high proliferative activity.

The effect of functionalizing lipid nanocapsules with NFL-TBS.40-63 peptide on their uptake by glioblastoma cells.

We previously described a neurofilament derived cell-penetrating peptide, NFL-TBS.40-63, that specifically enters in glioblastoma cells where it disturbs the microtubule network both in vitro and in vivo. The aim of this study is to test whether this peptide can increase the targeted uptake by glioblastoma cells of lipid nanocapsules filled with Paclitaxel, and thus can increase their anti-proliferation in vitro and in vivo. Here, using the drop tensiometry we show that approximately 60 NFL-TBS.40-63 peptides can bind to one 50 nm lipid nanocapsule. When nanocapsules are filled with a far-red fluorochrome (DiD) and Paclitaxel, the presence of the NFL-TBS.40-63 peptide increases their uptake by glioblastoma cells in culture as evaluated by FACS analysis, and thus reduces their proliferation. Finally, when such nanocapsules were injected in mice bearing a glioma tumour, they are preferentially targeted to the tumour and reduce its progression. These results show that nanocapsules functionalized with the NFL-TBS.40-63 peptide represent a powerful drug-carrier system for glioma targeted treatment.

A tubulin binding peptide targets glioma cells disrupting their microtubules, blocking migration, and inducing apoptosis.

Despite aggressive treatment regimes, glioma remains a largely fatal disease. Current treatment limitations are attributed to the precarious locations within the brain where such tumors grow, their highly infiltrative nature precluding complete resection and lack of specificity among agents capable of attenuating their growth. Here, we show that in vitro, glioma cells of diverse origins internalize a peptide encompassing a tubulin-binding site (TBS) on the neurofilament light protein. The internalized peptide disrupts the microtubule network, inhibits migration and proliferation, and leads to apoptosis. Using an intracerebral transplant model, we show that most, if not all, of these responses to peptide exposure also occur in vivo. Notably, a single intratumor injection significantly attenuates tumor growth, while neither peptide uptake nor downstream consequences are observed elsewhere in the host nervous system. Such preferential uptake suggests that the peptide may have potential as a primary or supplementary glioblastoma treatment modality by exploiting its autonomous microtubule-disrupting activity or engaging its capacity to selectively target glioma cells with other cell-disrupting cargos.

Axon cytoskeleton proteins specifically modulate oligodendrocyte growth and differentiation in vitro.

In multiple sclerosis (MS) remyelination by oligodendrocytes (OL) is incomplete, and it is associated with a decrease in axonal neurofilaments (NF) and tubulin (TUB). To determine whether these proteins could participate directly in MS remyelination failure, or indirectly through proteins that are co-associated, we have analysed their effects in pure OL cultures. Rat brain NF fractions, recovered by successive centrifugations increase either OL progenitor (OLP) proliferation (2nd pellet, P2), or only their maturation (P5), whereas albumin, liver and skin proteins, as well as recombinant GFAP or purified actin were ineffective. NF (P2) copurify mainly with TUB, as well as with other proteins, like MAPs, Tau, spectrin ß2, and synapsin 2. These purified, or recombinant, proteins increased OLP proliferation without delaying their maturation, and appeared responsible for the proliferation observed with P2 fractions. Among putative signaling pathways mediating these effects Fyn kinase was not involved. Whereas NF did not alter the growth of cultured astrocytes, the NF associated proteins enhanced their proliferation. This suggests that NF and their associated proteins exert specific effects on OL development, broadening the field of axon-oligodendrocyte interactions. In case of axon damage in vivo, extracellular release of such axonal proteins could regulate remyelination and astrocytic gliosis.

Immortalized CNS pericytes are quiescent smooth muscle actin-negative and pluripotent.

Despite their identification more than 100 years ago by the French scientist Charles-Marie Benjamin Rouget, microvascular pericytes have proven difficult to functionally characterize, due in part to their relatively low numbers and the lack of specific cell markers. However, recent progress is beginning to shed light on the diverse biological functions of these cells. Pericytes are thought to be involved in regulating vascular homeostasis and hemostasis as well as serving as a local source of adult stem cells. To further define the properties of these intriguing cells, we have isolated pericytes from transgenic mice (Immortomouse®) harboring a temperature-sensitive mutant of the SV40 virus target T-gene. This Immortopericyte (IMP) conditional cell line is stable for long periods of time and, at 33°C in the presence of interferon gamma, does not differentiate. Under these conditions IMPs are alpha muscle actin-negative and exhibit a pluripotent phenotype, but can be induced to differentiate along both mesenchymal and neuronal lineages at 37°C. Alternatively, differentiation of wild type pericytes and IMPs can be induced directly from capillaries in culture. Finally, the addition of endothelial cells to purified IMP cultures augments their rate of self-renewal and differentiation, possibly in a cell-to-cell contact dependent manner.

[Transcription of cytoskeleton protein genes in differentiation of neurons from mouse embryonic stem cells induced by small molecules].

OBJECTIVE: To investigate the transcription of cytoskeleton protein genes in differentiation of neurons from mouse embryonic stem (ES) cells induced by all-trans retinoic acid (RA), and to explore the possibility of setting up a method to screen small molecules with promoting or inhibiting effect. METHODS: The hanging drop method was employed for embryonic body formation to mimic embryo development in vivo. Reverse transcriptase PCR (RT-PCR) was performed to investigate mRNA expression of the neuron-specific cytoskeleton proteins including Mtap2, Nefm and beta-tubulin III which were regarded as the inducing effect indexes of RA. Morphological evaluation and immunocytochemistry staining were conducted to identify the neural derivatives. Moreover, the inducing effects of six synthetic molecules were further evaluated. RESULT: RA up-regulated the mRNA expression of Mtap2 and Nefm, especially Mtap2 increased by 1.27 times, which was consistent with the morphological alteration. However, there was no significant changes of beta-tubulin III expression. With addition of the six synthetic molecules, the transcription of Mtap2 was inhibited, while the Nefm mRNA expression was up-regulated in some degree, especially for molecule 1 and 3 that was increased by 1.4 and 1.2 times, which, however, was not parallel to the morphological changes. CONCLUSION: The transcriptional levels of Mtap2 and Nefm are both up-regulated in the RA-induced differentiation of ES cells towards neurons. The up-regulation of Mtap2 is consistent with the morphological alteration, which might be the key landmark in the RA-induced differentiation of ES cells into neurons.

Peripherin-mediated death of motor neurons rescued by overexpression of neurofilament NF-H proteins.

In previous studies, we showed that overexpression of peripherin, a neuronal intermediate filament (IF) protein, in mice deficient for neurofilament light (NF-L) subunits induced a progressive adult-onset degeneration of spinal motor neurons characterized by the presence of IF inclusion bodies reminiscent of axonal spheroids found in amyotrophic lateral sclerosis (ALS). In contrast, the overexpression of human neurofilament heavy (NF-H) proteins provoked the formation of massive perikaryal IF protein accumulations with no loss of motor neurons. To further investigate the toxic properties of IF protein inclusions, we generated NF-L null mice that co-express both peripherin and NF-H transgenes. The axonal count in L5 ventral roots from 6 and 8-month-old transgenic mice showed that NF-H overexpression rescued the peripherin-mediated degeneration of motor neurons. Our analysis suggests that the protective effect of extra NF-H proteins is related to the sequestration of peripherin into the perikaryon of motor neurons, thereby abolishing the development of axonal IF inclusions that might block transport. These findings illustrate the importance of IF protein stoichiometry in formation, localization and toxicity of neuronal inclusion bodies.

Untranslated element in neurofilament mRNA has neuropathic effect on motor neurons of transgenic mice.

Studies of experimental motor neuron degeneration attributable to expression of neurofilament light chain (NF-L) transgenes have raised the possibility that the neuropathic effects result from overexpression of NF-L mRNA, independent of NF-L protein effects (Cañete-Soler et al., 1999). The present study was undertaken to test for an RNA-mediated pathogenesis. Transgenic mice were derived using either an enhanced green fluorescent protein reporter construct or modified chimeric constructs that differ only in their 3' untranslated regions (UTRs). Motor function and spinal cord histology were normal in mice expressing the unmodified reporter transgene. In mice expressing a chimeric transgene in which sequence of NF-L 3' UTR was inserted into the 3' UTR of the reporter transgene, we observed growth retardation and reduced kinetic activity during postnatal development. Older mice developed impairment of motor function and atrophy of nerve fibers in the ventral roots. A similar but more severe phenotype was observed when the chimeric transgene contained a 36 bp c-myc insert in an mRNA destabilizing element of the NF-L sequence. Our results suggest that neuropathic effects of overexpressing NF-L can occur at the level of transgene RNA and are mediated by sequences in the NF-L 3' UTR.

Overexpression of neurofilament H disrupts normal cell structure and function.

Studying exogenously expressed tagged proteins in live cells has become a standard technique for evaluating protein distribution and function. Typically, expression levels of experimentally introduced proteins are not regulated, and high levels are often preferred to facilitate detection. However, overexpression of many proteins leads to mislocalization and pathologies. Therefore, for normative studies, moderate levels of expression may be more suitable. To understand better the dynamics of intermediate filament formation, transport, and stability in a healthy, living cell, we inserted neurofilament heavy chain (NFH)-green fluorescent protein (GFP) fusion constructs in adenoviral vectors with tetracycline (tet)-regulated promoters. This system allows for turning on or off the synthesis of NFH-GFP at a selected time, for a defined period, in a dose-dependent manner. We used this inducible system for live cell imaging of changes in filament structure and cell shape, motility, and transport associated with increasing NFH-GFP expression. Cells with low to intermediate levels of NFH-GFP were structurally and functionally similar to neighboring, nonexpressing cells. In contrast, overexpression led to pathological alterations in both filament organization and cell function.