From regenerative strategies to pharmacological approaches:can we fine-tune treatment for Parkinson's disease?

Parkinson's disease is the second most prevalent neurodegenerative disorder worldwide.Clinically,it is characterized by severe motor complications caused by progressive degeneration of dopaminergic neurons.Current treatment is focused on mitigating the symptoms through the administration of levodopa,rather than on preventing dopaminergic neuronal damage.Therefore,the use and development of neuroprotective/disease-modifying strategies is an absolute need that can lead to promising gains on translational research of Parkinson's disease.For instance,N-acetylcysteine,a natural compound with strong antioxidant effects,has been shown to modulate oxidative stress,preventing dopamine-induced cell death.Despite the evidence of neuroprotective and modulatory effects of this drug,as far as we know,it does not induce per se any regenerative process.Therefore,it would be of interest to combine the latter with innovative therapies that induce dopaminergic neurons repair or even differentiation,as stem cell-based strategies.Stem cells secretome has been proposed as a promising therapeutic approach for Parkinson's disease,given its ability to modulate cell viability/preservation of dopaminergic neurons.Such approach represents a shift in the paradigm,showing that cell-transplantation free therapies based on the use of stem cells secretome may represent a potential alternative for regenerative medicine of Parkinson's disease.Thus,in this review,we address the current understanding of the potential combination of stem cell free-based strategies and neuroprotective/disease-modifying strategies as a new paradigm for the treatment of central nervous system neurodegenerative diseases,like Parkinson's disease.     

Engineering synucleinopathy‐resistant human dopaminergic neurons by CRISPR‐mediated deletion of the SNCA gene

An emerging treatment for Parkinson's disease (PD) is cell replacement therapy. Authentic midbrain dopaminergic (mDA) neuronal precursors can be differentiated from human embryonic stem cells (hESCs) and human induced pluripotent stem cells (iPSCs). These laboratory‐generated mDA cells have been demonstrated to mature into functional dopaminergic neurons upon transplantation into preclinical models of PD. However, clinical trials with human fetal mesenchephalic cells have shown that cell replacement grafts in PD are susceptible to Lewy body formation suggesting host‐to‐graft transfer of α‐synuclein pathology. Here, we have used CRISPR/Cas9n technology to delete the endogenous SNCA gene, encoding for α‐synuclein, in a clinical‐grade hESC line to generate SNCA^+/? and SNCA^?/? cell lines. These hESC lines were first differentiated into mDA neurons, and then challenged with recombinant α‐synuclein preformed fibrils (PFFs) to seed the formation for Lewy‐like pathology as measured by phosphorylation of serine‐129 of α‐synuclein (pS129‐αSyn). Wild‐type neurons were fully susceptible to the formation of protein aggregates positive for pS129‐αSyn, while SNCA^+/? and SNCA^?/? neurons exhibited significant resistance to the formation of this pathological mark. This work demonstrates that reducing or completely removing SNCA alleles by CRISPR/Cas9n‐mediated gene editing confers a measure of resistance to Lewy pathology.     

Dopamine neuron precursors within the developing human mesencephalon show radial glial characteristics

Specification and differentiation of neural precursors into dopaminergic neurons within the ventral mesencephalon has been subject to much attention due to the implication of dopaminergic neurons in Parkinson's disease and the perspective of generating sources of therapeutically active cells to be used for cell replacement therapy for the disease. However, despite intensive research efforts, little is known about the characteristics of the dopamine neuron progenitors in human. We show that the dopamine neuron determinant LMX1a is expressed in the diencephalic and mesencephalic dopaminergic neuron domains during human development. Within the mesencephalon, LMX1a is expressed in the dopaminergic neurons and their progenitors located in the ventricular zone of the floor plate region. Furthermore, the neural progenitors in the developing human ventral mesencephalon have a radial morphology and express the radial glial markers Vimentin and BLBP. These radial glia are mitotic and act as precursors for the dopaminergic neurons. Finally, we show that progenitors isolated from the human ventral mesencephalon maintain their radial glial characteristics and neurogenic capacity after expansion in vitro, making them a promising future source of cells to be used in cell replacement therapy for Parkinson's disease. © 2009 Wiley‐Liss, Inc.     

Stem cell‐based cell therapy in neurological diseases: A review

Human neurological disorders such as Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis (ALS), Alzheimer's disease, multiple sclerosis (MS), stroke, and spinal cord injury are caused by a loss of neurons and glial cells in the brain or spinal cord. Cell replacement therapy and gene transfer to the diseased or injured brain have provided the basis for the development of potentially powerful new therapeutic strategies for a broad spectrum of human neurological diseases. However, the paucity of suitable cell types for cell replacement therapy in patients suffering from neurological disorders has hampered the development of this promising therapeutic approach. In recent years, neurons and glial cells have successfully been generated from stem cells such as embryonic stem cells, mesenchymal stem cells, and neural stem cells, and extensive efforts by investigators to develop stem cell‐based brain transplantation therapies have been carried out. We review here notable experimental and preclinical studies previously published involving stem cell‐based cell and gene therapies for Parkinson's disease, Huntington's disease, ALS, Alzheimer's disease, MS, stroke, spinal cord injury, brain tumor, and lysosomal storage diseases and discuss the future prospects for stem cell therapy of neurological disorders in the clinical setting. There are still many obstacles to be overcome before clinical application of cell therapy in neurological disease patients is adopted: 1) it is still uncertain what kind of stem cells would be an ideal source for cellular grafts, and 2) the mechanism by which transplantation of stem cells leads to an enhanced functional recovery and structural reorganization must to be better understood. Steady and solid progress in stem cell research in both basic and preclinical settings should support the hope for development of stem cell‐based cell therapies for neurological diseases. © 2009 Wiley‐Liss, Inc.     

Panax notoginseng saponins influence on transplantation of neural stem cell-derived dopaminergic neurons in a rat model of Parkinson’s disease *☆

BACKGROUND： Dopaminergic neurons differentiated from neural stem cells have been successfully used in the treatment of rat models of Parkinson＇s disease; however, the survival rate of transplanted cells has been low. Most cells die by apoptosis as a result of overloaded intracellular calcium and the formation of oxygen free radicals.
OBJECTIVE： To observe whether survival of transplanted cells, transplantation efficacy, and dopaminergic differentiation from neural stem cells is altered by Panax notoginseng saponins （PNS） in a rat model of Parkinson＇s disease.
DESIGN, TIME AND SETTING： Cellular and molecular biology experiments with randomized group design. The experiment was performed at the Animal Experimental Center, First Hospital of Sun Yat-sen University from April to October 2007.
MATERIALS： Thirty-two adult, healthy, male Sprague Dawley rats, and four healthy Sprague Dawley rat embryos at gestational days 14-15 were selected. The right ventral mesencephalon was injected with 6-hydroxydopamine to establish a model of Parkinson＇s disease. 6-hydroxydopamine and apomorphine were purchased from Sigma, USA.
METHODS： Neural stem cells derived from the mesencephalon of embryonic rats were cultivated and passaged in serum-free culture medium. Lesioned animals were randomly divided into four groups （n = 8）： dopaminergic neuron, dopaminergic neuron ＋ PNS, PNS, and control. The dopaminergic neuron group was injected with 3 μL cell suspension containing dopaminergic neurons differentiated from neural stem cells. The dopaminergic neurons ＋ PNS group received 3 μ L dopaminergic cell suspension combined with PNS （250 mg/L）. The PNS group received 3 μL PNS （250 mg/L）, and the control group received 3 μL DMEM/F12 culture medium.
MAIN OUTCOME MEASURES： The rats were transcardially perfused with 4% paraformaldehyde at 60 days post-grafting for immunohistochemistry. The rats were intraperitoneally injected with apomorphine （0.5 mg/kg） to induce rotational behavior. RESU     

Fibroblast growth factor 2 regulates adequate nigrostriatal pathway formation in mice

Fibroblast growth factor 2 (FGF‐2) is an important neurotrophic factor that promotes survival of adult mesencephalic dopaminergic (mDA) neurons and regulates their adequate development. Since mDA neurons degenerate in Parkinson's disease, a comprehensive understanding of their development and maintenance might contribute to the development of causative therapeutic approaches. The current analysis addressed the role of FGF‐2 in mDA axonal outgrowth, pathway formation, and innervation of respective forebrain targets using organotypic explant cocultures of ventral midbrain (VM) and forebrain (FB). An enhanced green fluorescent protein (EGFP) transgenic mouse strain was used for the VM explants, which allowed combining and distinguishing of individual VM and FB tissue from wildtype and FGF‐2‐deficient embryonic day (E)14.5 embryos, respectively. These cocultures provided a suitable model to study the role of target‐derived FB and intrinsic VM‐derived FGF‐2. In fact, we show that loss of FGF‐2 in both FB and VM results in significantly increased mDA fiber outgrowth compared to wildtype cocultures, proving a regulatory role of FGF‐2 during nigrostriatal wiring. Further, we found in heterogeneous cocultures deficient for FGF‐2 in FB and VM, respectively, similar phenotypes with wider fiber tracts compared to wildtype cocultures and shorter fiber outgrowth distance than cocultures completely deficient for FGF‐2. Additionally, the loss of target‐derived FGF‐2 in FB explants resulted in decreased caudorostral glial migration. Together these findings imply an intricate interplay of target‐derived and VM‐derived FGF signaling, which assures an adequate nigrostriatal pathway formation and target innervation. J. Comp. Neurol. 520:3949–3961, 2012. © 2012 Wiley Periodicals, Inc.     

IL6 Protects MN9D Cells and Midbrain Dopaminergic Neurons from MPP+-Induced Neurodegeneration

The degeneration of midbrain dopaminergic (mDA) neurons is the hallmark of Parkinson??s disease (PD), and several in vivo and in vitro models have been established to resemble the processes occurring during disease progression. One of the most commonly used disease models for PD is the toxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), which selectively kills mDA neurons when applied systemically. In vivo, MPTP intoxication is accompanied by a strong microglia response which is characterised by the release of inflammatory molecules such as tumour necrosis factor alpha (TNF-alpha) and interleukin-6 (IL6) that are believed to further drive inflammation-mediated degeneration of mDA neurons. Here, we addressed the question whether primary ventral mDA neurons and MN9D cells release cytokines in vitro and how these cytokine profiles change after treatment with MPP⁺. Our results demonstrate that both culture models show different cytokine profiles under control conditions indicating that comparisons between both models should be made very carefully. Moreover, MN9D cells released high levels of IL6 and IP10/CXCL10, both of which were down regulated after treatment with MPP⁺. MN9D-derived IL6 seems to be important for MN9D survival since neutralisation of endogenous IL6 resulted in degeneration of MN9D cells. Moreover, recombinant IL6 was able to rescue MN9D cells and primary mDA neuron cultures from MPP⁺-induced neurotoxicity, underlining the neuroprotective properties of IL6.     

Primary tissue for cellular brain repair in Parkinson's disease: Promise,problems and the potential of biomaterials

The dopamine precursor, levodopa, remains the “gold standard” treatment for Parkinson's disease, and, although it provides superlative efficacy in the early stages of the disease, its long‐term use is limited by the development of severe motor side effects and a significant abating of therapeutic efficacy. Therefore, there remains a major unmet clinical need for the development of effective neuroprotective, neurorestorative or neuroreparatory therapies for this condition. The relatively selective loss of dopaminergic neurons from the nigrostriatal pathway makes Parkinson's disease an ideal candidate for reparative cell therapies, wherein the dopaminergic neurons that are lost in the condition are replaced through direct cell transplantation into the brain. To date, this approach has been developed, validated and clinically assessed using dopamine neuron‐rich foetal ventral mesencephalon grafts which have been shown to survive and reinnervate the denervated brain after transplantation, and to restore motor function. However, despite long‐term symptomatic relief in some patients, significant limitations, including poor graft survival and the impact this has on the number of foetal donors required, have prevented this therapy being more widely adopted as a restorative approach for Parkinson's disease. Injectable biomaterial scaffolds have the potential to improve the delivery, engraftment and survival of these grafts in the brain through provision of a supportive microenvironment for cell adhesion, growth and immune shielding. This article will briefly review the development of primary cell therapies for brain repair in Parkinson's disease and will consider the emerging literature which highlights the potential of using injectable biomaterial hydrogels in this context.     

Development of stem cell-based therapies for Parkinson's disease

Parkinson's disease (PD) is a common neurodegenerative disorder characterized by the progressive loss of dopaminergic neurons of the substantia nigra pars compacta in the brain with an unknown cause. Current pharmacological treatments for PD are only symptomatic and there is still no cure for this disease nowadays. In fact, transplantation of human fetal ventral midbrain cells into PD brains has provided a proof of concept that cell replacement therapy can be used for some PD patients, beneficial for improving their symptoms. However, the ethical and practical issues of human fetal tissue will inevitably limit its widespread clinical use. Therefore, it is essential to find alternative cell sources for the future cell transplantation for PD patients. With recent development in stem cell technology, here, we review the different types of stem cells and their main properties currently explored, which could be developed as a possible cell therapy for PD treatment.     

Estradiol promotes proliferation of dopaminergic precursors resulting in a higher proportion of dopamine neurons derived from mouse embryonic stem cells

Estradiol protects dopamine neurons of the substantia nigra from toxic insults. Such neurons succumb in Parkinson's disease; one strategy for restoring dopamine deficiency is cell therapy with neurons differentiated from embryonic stem cells. We investigated the effects of 17β-estradiol on dopaminergic induction of embryonic stem cells using the 5-stage protocol. Cells were incubated with different steroid concentrations during the proliferation (stage 4) or differentiation (stage 5) phases. Estradiol added at nM concentrations only during stage 4 increases the proliferation of dopaminergic precursors expressing Lmx1a, inducing a higher proportion of dopamine neurons at stage 5. These actions were mediated by activation of estrogen receptors, because co-incubation of cells with estradiol and ICI 182,780 completely abolished the positive effect on both proliferation of committed precursors, and subsequent differentiation to dopaminergic neurons. Our results suggest that estradiol should be useful in producing higher proportions of dopamine neurons from embryonic stem cells aimed for treating Parkinson's disease.     

Neural stem cell‐based treatment for neurodegenerative diseases

Human neurodegenrative diseases such as Parkinson's disease (PD), Huntington's disease (HD), amyotrophic lateral sclerosis (ALS) and Alzheimer's disease (AD) are caused by a loss of neurons and glia in the brain or spinal cord. Neurons and glial cells have successfully been generated from stem cells such as embryonic stem cells (ESCs), mesenchymal stem cells (MSCs) and neural stem cells (NSCs), and stem cell‐based cell therapies for neurodegenerative diseases have been developed. A recent advance in generatioin of a new class of pluripotent stem cells, induced pluripotent stem cells (iPSCs), derived from patients' own skin fibroblasts, opens doors for a totally new field of personalized medicine. Transplantation of NSCs, neurons or glia generated from stem cells in animal models of neurodegenrative diseases, including PD, HD, ALS and AD, demonstrates clinical improvement and also life extension of these animals. Additional therapeutic benefits in these animals can be provided by stem cell‐mediated gene transfer of therapeutic genes such as neurotrophic factors and enzymes. Although further research is still needed, cell and gene therapy based on stem cells, particularly using neurons and glia derived from iPSCs, ESCs or NSCs, will become a routine treatment for patients suffering from neurodegenerative diseases and also stroke and spinal cord injury.     

Subthalamic nucleus neuronal firing rate increases with Parkinson's disease progression

Parkinson's disease is a neurodegenerative disorder characterized by progressive loss of dopaminergic cells in the central nervous system, in particular the substantia nigra, resulting in an unrelenting loss of motor and nonmotor function. Animal models of Parkinson's disease reveal hyperactive neurons in the subthalamic nucleus that have increased firing rates and bursting activity compared with controls. Although subthalamic nucleus activity has been characterized in patients with advanced‐stage Parkinson's disease, it has not been described in patients with early‐stage Parkinson's disease. Here we present the results of subthalamic nucleus neuronal recordings from patients with early‐stage Parkinson's disease (Hoehn and Yahr stage II) enrolled in an ongoing clinical trial compared with recordings from age‐ and sex‐matched patients with advanced Parkinson's disease. Subthalamic nucleus neurons had a significantly lower firing rate in early versus advanced Parkinson's disease (28.7 vs 36.3 Hz; P < .01). The overall activity of the subthalamic nucleus was also significantly lower in early versus late Parkinson's disease, as measured by background neuronal noise (12.4 vs 14.0 mV; P < .05). No significant difference was identified between groups in the bursting or variability of neuronal firing in the subthalamic nucleus, as measured by a burst index or the interspike interval coefficient of variability. The results suggest that neuronal firing in the subthalamic nucleus increases with Parkinson's disease progression. © 2011 Movement Disorder Society     

Peptide hormone exendin-4 stimulates subventricular zone neurogenesis in the adult rodent brain and induces recovery in an animal model of Parkinson's disease

We investigated the effects of exendin-4 on neural stem/progenitor cells in the subventricular zone of the adult rodent brain and its functional effects in an animal model of Parkinson's disease. Our results showed expression of GLP-1 receptor mRNA or protein in the subventricular zone and cultured neural stem/progenitor cells isolated from this region. In vitro, exendin-4 increased the number of neural stem/progenitor cells, and the number of cells expressing the neuronal markers microtubule-associated protein 2, β-III-tubulin, and neuron-specific enolase. When exendin-4 was given intraperitoneally to naïve rodents together with bromodeoxyuridine, a marker for DNA synthesis, both the number of bromodeoxyuridine-positive cells and the number of neuronal precursor cells expressing doublecortin were increased. Exendin-4 was tested in the 6-hydroxydopamine model of Parkinson's disease to investigate its possible functional effects in an animal model with neuronal loss. After unilateral lesion and a 5-week stabilization period, the rats were treated for 3 weeks with exendin-4. We found a reduction of amphetamine-induced rotations in animals receiving exendin-4 that persisted for several weeks after drug administration had been terminated. Histological analysis showed that exendin-4 significantly increased the number of both tyrosine hydroxylase- and vesicular monoamine transporter 2-positive neurons in the substantia nigra. In conclusion, our results show that exendin-4 is able to promote adult neurogenesis in vitro and in vivo, normalize dopamine imbalance, and increase the number of cells positive for markers of dopaminergic neurons in the substantia nigra in a model of Parkinson's disease. © 2007 Wiley-Liss, Inc.     

Comparison of GDF5 and GDNF as neuroprotective factors for postnatal dopamine neurons in ventral mesencephalic cultures

Loss of dopamine neurons is associated with the motor deficits that occur in Parkinson's disease. Although many drugs have proven to be useful in the treatment of the symptoms of this disease, none has been shown to have a significant impact on the development of the disease. However, we believe that several neurotrophic factors have the potential to reduce its progression. Glial cell line‐derived neurotrophic factor (GDNF), a member of the transforming growth factor‐β superfamily of neurotrophic factors, has been extensively studied in this regard. Less attention has been paid to growth/differentiation factor 5 (GDF5), another member of the same superfamily. This study compares GDNF and GDF5 in dissociated cultures prepared from ventral mesencephalon and in organotypic co‐cultures containing substantia nigra, striatum, and neocortex. We report that both GDNF (10–500 ng/ml) and GDF5 (100–500 ng/ml) promoted the survival of dopamine neurons from the substantia nigra of postnatal rats, although GDNF was considerably more potent than GDF5. In contrast, neither factor had any significant effect on the survival of dopamine neurons from the rat ventral tegmental area. Using organotypic co‐cultures, we also compared GDF5 with GDNF as chemoattractants for the innervation of the striatum and the neocortex by dopamine neurons from the substantia nigra. The addition of either GDF5 or GDNF (100–500 ng/ml) caused innervation by dopamine neurons into the cortex as well as the striatum, which did not occur in untreated cultures. Our results are consistent with similar findings suggesting that GDF5, like GDNF, deserves attention as a possible therapeutic intervention for Parkinson's disease. © 2014 Wiley Periodicals, Inc.     

Zhichan decoction induces differentiation of dopaminergic neurons in Parkinson’s disease rats after neural stem cell transplantation

The goal of this study was to increase the dopamine content and reduce dopaminergic metabolites in the brain of Parkinson’s disease rats. Using high-performance liquid chromatography, we found that dopamine and dopaminergic metabolite（dihydroxyphenylacetic acid and homovanillic acid） content in the midbrain of Parkinson’s disease rats was increased after neural stem cell transplantation ＋ Zhichan decoction, compared with neural stem cell transplantation alone. Our genetic algorithm results show that dihydroxyphenylacetic acid and homovanillic acid levels achieve global optimization. Neural stem cell transplantation ＋ Zhichan decoction increased dihydroxyphenylacetic acid levels up to 10-fold, while transplantation alone resulted in a 3-fold increment. Homovanillic acid levels showed no apparent change. Our experimental findings show that after neural stem cell transplantation in Parkinson’s disease rats, Zhichan decoction can promote differentiation of neural stem cells into dopaminergic neurons.     

Review: The future of cell therapies and brain repair: Parkinson's disease leads the way

During the past 40 years brain tissue grafting techniques have been used both to study fundamental neurobiological questions and to treat neurological diseases. Motor symptoms of Parkinson's disease are largely due to degeneration of midbrain dopamine neurones. Because the nigrostriatal pathology is relatively focused anatomically, Parkinson's disease is considered the ideal candidate for brain repair by neural grafting and dopamine neurone transplantation for it has led the way in the neural transplantation research field. In this mini‐review, we briefly highlight four important areas of development. First, we describe marked functional benefits up to 18 years after transplantation surgery in patients with Parkinson's disease. This is proof‐of‐principle that, using optimal techniques and patient selection, grafted dopamine neurones can work in humans and the duration of the benefit exceeds placebo effects associated with surgery. Second, we describe that eventually protein aggregates containing α‐synuclein, identical to Lewy bodies, develop inside foetal dopamine neurones transplanted to patients with Parkinson's disease. This gives clues about pathogenetic mechanisms operating in Parkinson's disease, and also raises the question whether neural graft function will eventually decline as the result of the disease process. Third, we describe new emerging sources of transplantable dopamine neurones derived from pluripotent stem cells or reprogrammed adult somatic cells. Fourth, we highlight an important European Union‐funded multicentre clinical trial involving transplantation of foetal dopamine neurones in Parkinson's disease. We describe the design of this ongoing trial and how it can impact on the overall future of cell therapy in Parkinson's disease.     

Tyrosine hydroxylase-immunoreactive neurons are decreased in number in the cerebral cortex of Parkinson's disease

Tyrosine hydroxylase (TH)-immunoreactive (IR) neurons and their relationship with Lewy bodies were investigated in Parkinson's disease. Using anti-TH and/or anti-ubiquitin antibodies, we evaluated the cerebral cortices included superior frontal gyrus, precentral gyrus, postcentral gyrus, inferior temporal gyrus, cuneus, cingulate gyrus, short and long gyri of insula, and parahippocampal gyrus from 18 autopsy cases of Parkinson's disease and 16 controls. The appearance of TH-IR neurons in cerebral cortices was suggestive of non-pyramidal interneurons. The mean number of TH-IR neurons and the density of TH-IR fibers in Parkinson's disease were decreased in comparison with the controls. The cognitive impairment in Parkinson's disease has been accounted for by the lesions of the basal nucleus of Meynert, the locus ceruleus, and the cortical Lewy bodies in the cerebral cortex. In addition to these lesions, the global loss of non-pyramidal TH-IR cortical neurons and TH-IR fibers would induce the dysfunction of higher-order control of the neocortex and the limbic system in Parkinson's disease. Double-immunostaining with anti-TH and anti-ubiquitin antibodies did not show the TH-IR neuron with cortical Lewy body in the cerebral cortices of Parkinson's disease. In the cerebral cortices of Parkinson's disease, TH-IR non-pyramidal neurons in which cortical Lewy body is not formed decreased in number.     

Cell‐based therapy for Parkinson's disease: A journey through decades toward the light side of the Force

This review describes the history, development, and evolution of cell‐based replacement therapy for Parkinson's disease (PD), from the first pioneering trials with fetal ventral midbrain progenitors to future trials using stem cells as well as reprogrammed cells. In the spirit of Tom Isaacs, the review takes parallels to the storyline of Star Wars, including the temptations from the dark side and the continuous fight for the light side of the Force. It is subdivided into headings based on the original movies, spanning from A New Hope to the Last Jedi.