Hana Panet

Increased survival and migration of engrafted mesenchymal bone marrow stem cells in 6-hydroxydopamine-lesioned rodents.

Parkinsons disease is characterized by the loss of dopaminergic neurons in the substantia nigra. Attempted replacement of these neurons by stem cells has proved inconclusive. Bone marrow mesenchymal stem cells (MSC) are multipotent, differentiating into a variety of cells, including neuron-like cells. We used the 6-hydroxydopamine (6-OHDA) animal model of Parkinsons disease to assess migration and differentiation of transplanted MSC. We found in rodents that transplanted MSC survive better in the 6-OHDA-induced damaged hemisphere compared to the unlesioned side. Moreover, contralaterally engrafted MSC migrated through the corpus callosum to populate the striatum, thalamic nuclei and substantia nigra of the 6-OHDA-lesioned hemisphere. In conclusion, we demonstrate that 6-OHDA-induced damage increases the viability of transplanted MSC and attracts these cells from the opposite hemisphere.

Regenerative effect of neural-induced human mesenchymal stromal cells in rat models of Parkinson's disease

BACKGROUND Human bone marrow multipotent mesenchymal stromal cells (hMSC), because of their capacity of multipotency, may provide an unlimited cell source for cell replacement therapy. The purpose of this study was to assess the developmental potential of hMSC to replace the midbrain dopamine neurons selectively lost in Parkinsons disease. METHODS Cells were isolated and characterized, then induced to differentiate toward the neural lineage. In vitro analysis of neural differentiation was achieved using various methods to evaluate the expression of neural and dopaminergic genes and proteins. Neural-induced cells were then transplanted into the striata of hemi-Parkinsonian rats; animals were tested for rotational behavior and, after killing, immunohistochemistry was performed. RESULTS Following differentiation, cells displayed neuronal morphology and were found to express neural genes and proteins. Furthermore, some of the cells exhibited gene and protein profiles typical of dopaminergic precursors. Finally, transplantation of neural-induced cells into the striatum of hemi-Parkinsonian rats resulted in improvement of their behavioral deficits, as determined by apomorphine-induced rotational behavior. The transplanted induced cells proved to be of superior benefit compared with the transplantation of naive hMSC. Immunohistochemical analysis of grafted brains revealed that abundant induced cells survived the grafts and some displayed dopaminergic traits. DISCUSSION Our results demonstrate that induced neural hMSC may serve as a new cell source for the treatment of neurodegenerative diseases and have potential for broad application. These results encourage further developments of the possible use of hMSC in the treatment of Parkinsons disease.

Riluzole suppresses experimental autoimmune encephalomyelitis: implications for the treatment of multiple sclerosis

Recent studies suggest that glutamate neurotoxicity is involved in the pathogenesis of multiple sclerosis (MS), and that treatment with glutamate receptor (AMPA/kainate) antagonists inhibits experimental autoimmune encephalomyelitis (EAE), the conventional model of MS. Therefore, we examined whether riluzole, an inhibitor of glutamate transmission, affects the pathogenesis and clinical features of MS-like disease in myelin oligodendrocyte glycoprotein (MOG)-induced EAE in mice. Here we report that riluzole (10 mg/kgx2/day, i.p.), administered before and even after the appearance of clinical symptoms, dramatically reduced the clinical severity of MOG-induced EAE, while all the MOG-immunized control mice developed significant clinical manifestations. Moreover, the riluzole-treated mice demonstrated only mild focal inflammation, and less demyelination, compared to MOG-treated mice, using histological methods. Furthermore, riluzole markedly reduced axonal disruption, as assessed by Bielshoweskys silver staining and by antibodies against non-phosphorylated neurofilaments (SMI-32). No difference was detected in the immune system potency, as T-cell proliferative responses to MOG were similar in both groups. In conclusion, our study demonstrates, for the first time, that riluzole can reduce inflammation, demyelination and axonal damage in the CNS and attenuate the clinical severity of MOG-induced EAE. These results suggest that riluzole, a drug used in amyotrophic lateral sclerosis (ALS), might be beneficial for the treatment of MS.

Activation of nuclear transcription factor kappa B (NF-κB) is essential for dopamine-induced apoptosis in PC12 cells

The etiology of Parkinsons disease is still unknown, though current investigations support the notion of the pivotal involvement of oxidative stress in the process of neurodegeneration in the substantia nigra (SN). In the present study, we investigated the molecular mechanisms underlying cellular response to a challenge by dopamine, one of the local oxidative stressors in the SN. Based on studies showing that nuclear factor kappa B (NF‐κB) is activated by oxidative stress, we studied the involvement of NF‐κB in the toxicity of PC12 cells following dopamine exposure. We found that dopamine (0.1–0.5 m m) treatment increased the phosphorylation of the IκB protein, the inhibitory subunit of NF‐κB in the cytoplasm. Immunoblot analysis demonstrated the presence of NF‐κB‐p65 protein in the nuclear fraction and its disappearance from the cytoplasmic fraction after 2 h of dopamine exposure. Dopamine‐induced NF‐κB activation was also evidenced by electromobility shift assay using radioactive labeled NF‐κB consensus DNA sequence. Cell‐permeable NF‐κB inhibitor SN‐50 rescued the cells from dopamine‐induced apoptosis and showed the importance of NF‐κB activation to the induction of apoptosis. Furthermore, flow cytometry assay demonstrated a higher level of translocated NF‐κB‐p65 in the apoptotic nuclei than in the unaffected nuclei. In conclusion, our findings suggest that NF‐κB activation is essential to dopamine‐induced apoptosis in PC12 cells and it may be involved in nigral neurodegeneration in patients with Parkinsons disease.

Axonal damage is reduced following glatiramer acetate treatment in C57/bl mice with chronic-induced experimental autoimmune encephalomyelitis.

Glatiramer acetate (GA) is efficacious in reducing demyelinating-associated exacerbations in patients with relapsing-remitting multiple sclerosis (RRMS) and in several experimental autoimmune encephalomyelitis (EAE) models. Here we report that GA reduced the clinical and pathological signs of mice in chronic EAE induced by myelin oligodendrocyte glycoprotein (MOG). GA-treated mice demonstrated only mild focal inflammation, and less demyelination, compared with controls. Moreover, we also found minimal axonal disruption, as assessed by silver staining, antibodies against amyloid precursor protein (APP) and non-phosphorylated neurofilaments (SMI-32), in the GA-treated group. In conclusion, our study demonstrated for the first time that axonal damage is reduced following GA treatment in C57/bl mice with chronic MOG-induced EAE.

Dopamine-induced apoptosis is inhibited in PC12 cells expressing Bcl-2.

Abstract1. Degeneration of nigrostriatal dopaminergic neurons is the major pathogenic substrate of Parkinsons disease (PD). It is assumed that the lethal trigger is the accumulation of oxidative reactive species generated during metabolism of the natural neurotransmitter dopamine.2. We have recently shown that dopamine is capable of inducing programmed cell death (PCD) or apoptosis in cultured postmitotic chick sympathetic neurons and rat PC12 pheochromocytoma cells.3. The bcl-2 gene encodes a protein which blocks physiological PCD in many mammalian cells. In an attempt to elucidate further the mechanism of dopamine toxicity, we examined the potential protective effect of bcl-2 in PC12 cells which were transfected with the protooncogene.4. In our experiments, Bcl-2 producing cells showed a marked resistance to dopamine toxicity. The percentage of nuclear condensation and DNA fragmentation visualized by the end-labeling method following dopamine treatment was significantly lower in bcl-2 expressing cells. Bcl-2 did not protect PC12 cells against toxicity induced by exposure to dopamine-melanin. Extracts of PC12 cells containing Bcl-2 inhibited dopamine autooxidation and formation of dopamine-melanin. Furthermore, the presence of Bcl-2 protected cells from thiol imbalance and prevented thiol loss following exposure to dopamine.5. The protective effects of Bcl-2 against dopamine toxicity may be explained, in part, by its action as an antioxidant and by its interference in the production of toxic agents. The possible protection by Bcl-2 against neuronal degeneration caused by dopamine may play a role in the pathogenesis of PD andmay provide a new direction for the development of neuroprotective therapies.

A novel thiol antioxidant that crosses the blood brain barrier protects dopaminergic neurons in experimental models of Parkinson's disease.

It is believed that oxidative stress (OS) plays an important role in the loss of dopaminergic nigrostriatal neurons in Parkinsons disease (PD) and that treatment with antioxidants might be neuroprotective. However, most currently available antioxidants cannot readily penetrate the blood brain barrier after systemic administration. We now report that AD4, the novel low molecular weight thiol antioxidant and the N‐acytel cysteine (NAC) related compound, is capable of penetrating the brain and protects neurons in general and especially dopaminergic cells against various OS‐generating neurotoxins in tissue cultures. Moreover, we found that treatment with AD4 markedly decreased the damage of dopaminergic neurons in three experimental models of PD. AD4 suppressed amphetamine‐induced rotational behaviour in rats with unilateral 6‐OHDA‐induced nigral lesion. It attenuated the reduction in striatal dopamine levels in mice treated with 1‐methyl‐4‐phenyl‐1,2,3,6,‐tetrahydropyridine (MPTP). It also reduced the dopaminergic neuronal loss following chronic intrajugular administration of rotenone in rats. Our findings suggest that AD4 is a novel potential new neuroprotective drug that might be effective at slowing down nigral neuronal degeneration and illness progression in patients with PD.

Intrastriatal transplantation of mouse bone marrow-derived stem cells improves motor behavior in a mouse model of Parkinson’s disease

Strategies of cell therapy for the treatment of Parkinsons disease (PD) are focused on replacing damaged neurons with cells to restore or improve function that is impaired due to cell population damage. In our studies, we used mesenchymal stromal cells (MSCs) from mouse bone marrow. Following our novel neuronal differentiation method, we found that the basic cellular phenotype changed to cells with neural morphology that express specific markers including those characteristic for dopaminergic neurons, such as tyrosine hydroxylase (TH). Intrastriatal transplantation of the differentiated MSCs in 6-hydroxydopamine-lesioned mice led to marked reduction in the amphetamine-induced rotations. Immunohistological analysis of the mice brains four months post transplantation, demonstrated that most of the transplanted cells survived in the striatum and expressed TH. Some of the TH positive cells migrated toward the substantia nigra. In conclusion, transplantation of bone marrow derived stem cells differentiated to dopaminergic-like cells, successfully improved behavior in an animal model of PD suggesting an accessible source of cells that may be used for autotransplantation in patient with PD.

Mice overexpressing Bcl-2 in their neurons are resistant to myelin oligodendrocyte glycoprotein (MOG)-induced experimental autoimmune encephalomyelitis (EAE)

Multiple sclerosis (MS) is an inflammatory disease of the central nervous system (CNS) characterized by destruction of myelin. Recent studies have indicated that axonal damage is involved in the pathogenesis of the progressive disability of this disease. To study the role of axonal damage in the pathogenesis of MS-like disease induced by myelin oligodendrocyte glycoprotein (MOG), we compared experimental autoimmune encephalomyelitis (EAE) in wild-type (WT) and transgenic mice expressing the human bcl-2 gene exclusively in neurons under the control of the neuron-specific enolase (NSE) promoter. Our study shows that, following EAE induction with pMOG 35-55, the WT mice developed significant clinical manifestations with complete hind-limb paralysis. In contrast, most of the NSE-bcl-2 mice (16/27) were completely resistant, whereas the others showed only mild clinical signs. Histological examination of CNS tissue sections showed multifocal areas of perivascular lymphohistiocytic inflammation with loss of myelin and axons in the WT mice, whereas only focal inflammation and minimal axonal damage were demonstrated in NSE-bcl-2 mice. No difference could be detected in the immune potency as indicated by delayed-type hypersensitivity (DTH) and T-cell proliferative responses to MOG. We also demonstrated that purified synaptosomes from the NSE-bcl-2 mice produce significantly lower level of reactive oxygen species (ROS) following exposure to H202 and nitric oxide (NO) than WT mice. In conclusion, we demonstrated that the expression of the antiapoptotic gene, bcl-2, reduces axonal damage and attenuates the severity of MOG-induced EAE. Our results emphasize the importance of developing neuroprotective therapies, in addition to immune-specific approaches, for treatment of MS.

Photoconversion of DAPI following UV or violet excitation can cause DAPI to fluoresce with blue or cyan excitation

4’‐6‐Diamidino‐2‐phenylindole is a fluorescent dye commonly used to visualize deoxyribonucleic acid or cell nuclei in fixed cell preparations, and is often used together with fluorescein or green fluorescent protein, which can be excited without exciting 4’‐6‐Diamidino‐2‐phenylindole. It is assumed that when using typical fluorescein or green fluorescent protein filter cubes, 4’‐6‐Diamidino‐2‐phenylindole will not be observed. In this paper, we show that following observation of 4’‐6‐Diamidino‐2‐phenylindole using UV or violet excitation, it may become sensitive to the blue/cyan excitation used in fluorescein/green fluorescent protein filter cubes. This has serious implications for the use of 4’‐6‐Diamidino‐2‐phenylindole together with widely used green fluorophores in double labelling experiments.