Intrastriatal Administration Of LV-Shh Stimulates Neuroregeneration In Parkinson Disease Model Rats

Parkinson disease (PD) is a progressive neurodegenerative disorder in which the degeneration of dopaminergic neurons (DANs) projecting from the substantia nigra to the striatum is a key pathological feature. It is characterized clinically by motor dysfunctions including tremor, rigidity, bradykinesia, postural instability and disturbances in sleep, cognition and mood with anxiety and depression. The main current treatment for PD is drug therapies, which can alleviate motor deficits temporarily. Both levodopa and dopamine agonists can significantly improve motor symptoms, particularly in the early stages of the disease. However, long-term use of dopaminergic drug therapies produces an array of side effects with the development of disease progress such as levodopa-induced dyskinesias,"on-off"fluctuations, hallucinations, excessive sleepiness and addictive behaviors. In addition, non-pharmacological methods (i.e., deep brain stimulation, DBS) improve motor and daily living scores, but the precise mechanism of action remains uncertain.Therefore, in contrast to the above-described symptomatic treatments, cell replacement therapy (CRT) which is based on more complete understanding of PD pathogenesis has been being a major research interest in treatment of Parkinson disease. The aim of CRT is to replace degenerated and lost dopaminergic neuronal innervations of the striatum from the nigra by transplantation of different cell sources. However, effects of exogenous cell replacement therapy (exo-CRT) for PD are proved to be disappointed. Studies have failed to meet their primary endpoints, and transplantation of dopamine cells derived from the fetal mesencephalon is associated with a potentially disabling form of dyskinesia and even nondopaminergic pathology such as gait dysfunction, freezing, falling, and dementia. Besides, Lewy bodies which suggest dysfunction were detected in implanted DA neurons. In addition, other sources of stem cells (neural stem cells, bone marrow mesenchymal stem cells, embryonic stem cells) may not have the similar transplanted effects with FVM cells, coupled with poor sources, uncertain survival and functional efficacy, problems of transplanted safety and ethical problems. Thus, we should focus on endogenous cell replacement therapy and pay attention to refine microenvironment in lesion sites.Endogenous cell replacement therapy (endo-CRT) is a process that exogenous stimulus which affects host microenvironment triggers endogenous adult neural stem/progenitor cell proliferation, differentiation, migration and ultimate restoration toward altered structures. It provides an alternative and attractive cell source for neuroregeneration. Throughout the life span of rodents, adult neural stem cells reside in two main neurogenic niches, the subventricular zone (SVZ) and the dentate gyrus (DG) of the hippocampus. Progenitors produced by neural stem cells which resided in these structures migrate toward their ultimate location:the olfactory bulb and granular cell layer of the DG respectively. The SVZ along the lateral wall of the lateral ventricles harbors the largest pool of proliferating cells in the adult brain. In this study, we will focus on how a key factor in midbrain development, Shh, stimulates the neuroregeneration of impaired nigrostriatal system through certain mechanism called exo-CRT.Sonic hedgehog homolog (SHH) is one of three proteins in the mammalian signaling pathway family called hedgehog, the others being desert hedgehog (DHH) and Indian hedgehog (IHH). SHH belongs to a family of secreted polypeptides implicated in embryonic development. It plays important roles in a wide variety of regulatory functions during the development of vertebrate and invertebrate organisms, regulating morphogenesis of a variety of tissues and organs. SHH is produced by the notochord and floor plate and is responsible for inducing the specification of ventral neural cell types and regulating NSCs proliferation and development in a concentration-dependent manner. Besides, SHH displays inductive, proliferative, axon growth and guide, neurotrophic and neuroprotective activities on various neural cells and signals through a receptor complex associating Patched (Ptc) and Smoothened (Smo). There SHH undergoes autoprocessing to generate a20kDa N-terminal signaling domain (SHH-N) and a25kDa C-terminal domain without known roles in signaling. The cleavage is catalysed by a protease within the C-terminal domain. During the reaction, a cholesterol molecule is added to the C-terminus of SHH-N. Thus the C-terminal domain acts as an intein and a cholesterol transferase. Therefore, we focus on N-terminal product of Shh (SHH-N).In this experiment, we have cloned N-terminal product of sonic hedgehog gene (Shh-N) and constructed a Shh-N lentiviral vector, then injected the packed lentiviruses containing Shh-N into the striatum of Parkinson disease model rats. First of all, in vivo expressions of Shh-N at both mRNA and protein levels were detected by Nested RT-PCR and Western blot, respectively. Secondly, we injected Shh-lentivirus particles into the striatum so as to find a new treatment which could stimulate residuary neurons or gliocytes to proliferate and even to induce endogenous NSCs proliferation and differentiation. Lastly, we want to identify the most effective time point for the treatment of PD with Shh-lentivirus stereotactic injection.Objective:To observe in vivo overexpression of Shh-N and its therapeutic effects after stereotactic administration of Shh-lentivirus into the striatum of Parkinson disease model rats.Methods:1) Total RNA was extracted from the notochord of a Sprague-Dawley (SD) embryonic rat (E9). Nested RT-PCR was used to amplify Shh-N. Shh-N modified lentiviral vector (LV-Shh-N) was constructed. Virus titer was measured by real-time PCR.2) PD model rats underwent a unilateral stereotaxic injection of LV-Shh-N in group1and LV in group2into the striatum. Besides, contusion injury sham group (group3) and blank control group (group4) were also be added. To explore the effect of administration, functional analysis was performed at2,4and8weeks after the injection. The mRNA and protein expression of Shh-N in each group were detected by Nested RT-PCR and Western blot.3) The rats were given a single injection of fluoro-ruby (FR) into the bilateral striatum7days before functional analysis (2weeks,4weeks,8weeks). On2weeks、4weeks and8weeks, rats were challenged with apomorphine and ipsilateral rotations for40minutes were recorded. Afterward, functional analysis such as immunofluorescence, Western blot and fluoro-ruby tracing were taken to observe the effects of Shh-lentivirus administration for residuary dopaminergic neurons, other neurons and gliocytes or endogenous NSCs in PD model.Results:1) Shh-N-lentiviral vector (PCDH-Shh-T2A-copGFP-MSCV) has been successfully constructed the with a virus titer of2×107IU/ml.2) Shh-N was significantly over-expressed at2,4and8weeks after administration of Shh-LV at mRNA level by Nested RT-PCR. However, SHH-N expression in protein level by Western blot was significantly higher at8weeks than that at2or4weeks, and higher than that at all the time points in other groups (P<0.01)3) Administration of Shh-N-LV improved the behavior of PD model rats and partially recovered functional status of dopaminergic neurons. In apomorphine-induced rotation test, rotation rounds within40minutes were significantly reduced at8weeks compared with those at2or4weeks (P<0.01) Similarly, both TH positive fibers in the striatum and TH positive neurons in the substantia nigra were observably increased compared with those at2or4weeks (P<0.05).4) Administration of Shh-N-LV partially restored nigrostriatal pathway by fluoro-ruby (FR) tracing. The distribution range and mean optical density of FR labeled axons in the striatum and substantia nigra at8weeks were improved compared with those at2and4weeks (P<0.05)5) Intrastriatal administration of Shh-N-LV induced presence of new NSCs and neurogliocytes. At8weeks, GFAP-and Nestin-positive cells were found in the lesion area of the striatum in LV-Shh group and LV group, respectively. Besides, in the Western blot, GFAP and Nestin expressed in LV-Shh group and LV group, respectively.Conclusion:All the above results suggest that stereotaxical administration of Shh-N-LV into the striatum could significantly improve the behaviour of PD model rats by restoring partial functions of dopaminergic neurons and nigrostriatal pathway, and by inducing the generation of new NSCs and neurogliocytes either in situ or from other sources.