This study demonstrates proof of concept for delivery and expression of compacted plasmid DNA in the central nervous system. Plasmid DNA was compacted with polyethylene glycol substituted lysine 30-mer peptides, forming rod-like nanoparticles with diameters between 8 and 11 nm. Here we show that an intracerebral injection of compacted DNA can transfect both neurons and glia, and can produce transgene expression in the striatum for up to 8 weeks, which was at least 100-fold greater than intracerebral injections of naked DNA plasmids. Bioluminescent imaging (BLI) of injected animals at the 11th postinjection week revealed significantly higher transgene activity in animals receiving compacted DNA plasmids when compared to animals receiving naked DNA. There was minimal evidence of brain inflammation. Intrastriatal injections of a compacted plasmid encoding for glial cell line-derived neurotrophic factor (pGDNF) resulted in a significant overexpression of GDNF protein in the striatum 1-3 weeks after injection.
Epilepsy is an important and common worldwide public health problem that affects people of all ages. A significant number of individuals with epilepsy will be intractable to medication. These individuals experience an elevated mortality rate and negative psychosocial consequences of recurrent seizures. Surgery of epilepsy is highly effective to stop seizures in well-selected individuals, and seizure freedom is the most desirable result of epilepsy treatment due to the positive improvements in psychosocial function and the elimination of excess mortality associated with intractable epilepsy. Globally, there is inadequate data to fully assess epilepsy-related quality of life and stigma, although the preponderance of information we have points to a significant negative impact on people with epilepsy (PWE) and families of PWE. This review of the psychosocial impact of epilepsy focuses on regions of Asia and Sub-Saharan Africa that have been analyzed with population study approaches to determine the prevalence of epilepsy, treatment gaps, as well as factors impacting psychosocial function of PWE and their families. This review additionally identifies models of care for medically intractable epilepsy that have potential to significantly improve psychosocial function.
A goal of our studies is to develop a potential therapeutic for Parkinson’s disease (PD) by a human GDNF (hGDNF) expression plasmid administered to the rat striatum as a compacted DNA nanoparticle (DNP) and which will generate long-term hGDNF expression at biologically active levels. In the present study we used a DNA plasmid encoding for hGDNF and a polyubiquitin C (UbC) promoter that was previously shown to have activity in both neurons and glia, but primarily in glia. A two-fold improvement was observed at the highest plasmid dose when using hGDNF DNA incorporating sequences found in RNA splice variant 1 compared to splice variant 2; of note, the splice variant 2 sequence is used in most preclinical studies. This optimized expression cassette design includes flanking scaffold matrix attachment elements (S/MARs) as well as a CpG-depleted prokaryotic domain and, where possible, eukaryotic elements. Stable long-term GDNF activity at levels 300–400% higher than baseline was observed following a single intracerebral injection. In a previous study DNPs plasmids encoding for reporter genes had been successful in generating long-term reporter transgene activity in the striatum (>365 days) and in this study produced sustained GDNF activity at the longest assessed time point (6 months).
In this study, we used bioluminescence imaging (BLI) to track long-term transgene activity following the transfection of brain cells using a nonviral gene therapy technique. Formulations of deoxyribonucleic acid (DNA) combined with 30-mer lysine polymers (substituted with 10 kDa polyethylene glycol) form nanoparticles that transfect brain cells in vivo and produce transgene activity. Here we show that a single intracerebral injection of these DNA nanoparticles (DNPs) into the rat cortex, striatum, or substantia nigra results in long-term and persistent luciferase transgene activity over an 8- to 11-week period as evaluated by in vivo BLI analysis, and single injections of DNPs into the mouse striatum showed stable luciferase transgene activity for 1 year. Compacted DNPs produced in vivo signals 7- to 34-fold higher than DNA alone. In contrast, ex vivo BLI analysis, which is subject to less signal quenching from surrounding tissues, demonstrated a DNP to DNA alone ratio of 76- to 280-fold. Moreover, the ex vivo BLI analysis confirmed that signals originated from the targeted brain structures. In summary, BLI permits serial analysis of luciferase transgene activity at multiple brain locations following gene transfer with DNPs. Ex vivo analysis may permit more accurate determination of relative activities of gene transfer vectors.
Calcitriol, the active metabolite of vitamin D, has been shown to have significant effects on the brain. These actions include reducing the severity of some central nervous system lesions, possibly by upregulating trophic factors such as glial cell line-derived neurotrophic factor (GDNF). GDNF has substantial effects on the nigrostriatal dopamine (DA) system of young adult, aged and lesioned animals. Thus, the administration of calcitriol may lead to significant effects on nigrostriatal DA neuron functioning. The present experiments were designed to examine the ability of calcitriol to alter striatal DA release, and striatal and nigral tissue levels of DA. Male Fischer-344 rats were administered vehicle or calcitriol (0.3, 1.0, or 3.0 μg/kg, s.c.) once daily for 8 consecutive days. Three weeks later in vivo microdialysis experiments were conducted to measure basal and stimulus evoked overflow of DA from the striatum. Basal levels of extracellular DA were not significantly affected by the calcitriol treatments. However, the 1.0 and 3.0 μg/kg doses of calcitriol led to increases in both potassium and amphetamine evoked overflow of striatal DA. Although post-mortem tissue levels of striatal DA were not altered by the calcitriol injections, nigral tissue levels of DA and its main metabolites were increased by both the 1.0 and 3.0 μg/kg doses of calcitriol. In a separate group of animals GDNF levels were augmented in the striatum and substantia nigra after eight consecutive daily injections of calcitriol. These results suggest that systemically administered calcitriol can upregulate dopaminergic release processes in the striatum and DA levels in the substantia nigra. Increases in levels of endogenous GDNF following calcitriol treatment may in part be responsible for these changes. The ability of calcitriol to lead to augmented DA release in the striatum suggests that calcitriol may be beneficial in disease processes involving dopaminergic dysfunction.
Different experimental and clinical strategies have been used to promote survival of transplanted embryonic ventral mesencephalic (VM) neurons. However, few studies have focused on the long-distance growth of dopaminergic axons from VM transplants. The aim of this study is to identify some of the growth and guidance factors that support directed long-distance growth of dopaminergic axons from VM transplants. Lentivirus encoding either glial cell line-derived neurotrophic factor (GDNF) or netrin-1, or a combination of lenti-GDNF with either lenti-GDNF family receptor α1 (GFRα-1) or lenti-netrin-1 was injected to form a gradient along the corpus callosum. Two weeks later, a piece of embryonic day 14 VM tissue was transplanted into the corpus callosum adjacent to the low end of the gradient. Results showed that tyrosine hydroxylase (TH+) axons grew a very short distance from the VM transplants in control groups, with few axons reaching the midline. In GDNF or Netrin-1 expressing groups, more TH+ axons grew out of transplants and reached the midline. Pathways co-expressing GDNF with either GFRα-1 or netrin-1 showed significantly increased axonal outgrowth. Interestingly, only the GDNF/netrin-1 combination resulted in the majority of axons reaching the distal target (80%), whereas along the GDNF/GFRα-1 pathway only 20% of the axons leaving the transplant reached the distal target. This technique of long-distance axon guidance may prove to be a useful strategy in reconstructing damaged neuronal circuits, such as the nigrostriatal pathway in Parkinson’s disease.
Current therapies for Parkinson's disease (PD) offer symptomatic relief but do not provide a cure or slow the disease process. Treatments that could halt progression of the disease or help restore function to damaged neurons would be of substantial benefit. Calcitriol, the active metabolite of vitamin D, has been shown to have significant effects on the brain. These effects include upregulating trophic factor levels, and reducing the severity of some central nervous system lesions. While previous studies have shown that calcitriol can be neuroprotective in 6-hydroxydopamine (6-OHDA) rodent models of PD, the present experiments were designed to examine the ability of calcitriol to promote restoration of extracellular DA levels and tissue content of DA in animals previously lesioned with 6-OHDA. Male Fischer-344 rats were given a single injection of 12 µg 6-OHDA into the right striatum. Four weeks later the animals were administered vehicle or calcitriol (0.3 or 1.0 µg/kg, s.c.) once a day for eight consecutive days. Three weeks after the calcitriol treatments in vivo microdialysis experiments were conducted to measure potassium and amphetamine evoked overflow of DA from both the left and right striata. In control animals treated with 6-OHDA and vehicle there were significant reductions in both potassium and amphetamine evoked overflow of DA on the lesioned side of the brain compared to the contralateral side. In animals treated with 6-OHDA followed by calcitriol there was significantly greater potassium and amphetamine evoked overflow of DA from the lesioned striatum compared to that from the control animals. The calcitriol treatments also led to increases in postmortem tissue levels of DA in the striatum and substantia nigra. These results suggest that calcitriol may help promote recovery of dopaminergic functioning in injured nigrostriatal neurons.
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