Targeted viral vector-mediated gene transfer to specific population of neurons in the central nervous system (CNS) is a relatively novel, but fast developing approach to study gene function in a number of neurodegenerative diseases (reviewed in Korecka, Verhaagen, & Hol, 2007; Manfredsson & Mandel, 2010). Moreover, several early phase clinical trials based on viral vector-mediated therapeutic gene transfer have been completed or are underway for neurological disorders (Kaplitt et al., 2007; reviewed in Korecka et al., 2007; Marks, Jr. et al., 2010; Muramatsu et al., 2010; Tuszynski et al., 2005). Gene therapy is especially attractive for diseases where neuronal degeneration is largely restricted to a single neuronal population in a specific anatomical area. Parkinson disease (PD) is a neurodegenerative disease mainly characterized by a progressive degeneration of dopaminergic (DAergic) neurons in the Substantia Nigra (SN) (Dauer & Przedborski, 2003). It would be desirable to direct transgene expression to the dopaminergic neurons in animal models for neurodegenerative diseases, allowing for a range of investigations into the function of that gene in normal, adult DAergic neurons or following neurotoxic insult. Lentiviral vectors (LV) and adeno-associated viral vectors (AAV) are increasingly regarded as the two most useful gene therapy vectors for the CNS. Both vectors have been successfully used to express a foreign gene in a variety of brain regions and neuronal cell types (Lim, Airavaara, & Harvey, 2010; Manfredsson & Mandel, 2010; Papale, Cerovic, & Brambilla, 2009; Schneider, Zufferey, & Aebischer, 2008). LV vectors have been shown to direct long-lasting expression of a number of transgenes in the brain (Lundberg et al., 2008) including in neurons in the rat SN (Deglon et al., 2000). AAV vectors are considered to be the most appealing vectors for transgene expression in the CNS, due to their efficient neuronal transduction, their capacity to direct long-term transgene expression and their safety profile (Kaplitt et al., 2007; Mandel et al., 2006; McCown, 2005). The early AAV vectors were based on AAV serotype 2 (Kaplitt et al., 1994; Peel & Klein, 2000), but
Objective: Our aim was to investigate the relation between unilateral hippocampal and/or nigral alpha-synucleinopathy and cognitive dysfunction, anxiety and anhedonia.Materials and Methods: Female Sprague-Dawley rats were stereotactically injected adeno-associated viruses carrying alpha-synuclein (α-syn) into unilateral dentate gyrus (DG), substantia nigra (SN) or both SN + DG.The animals were tested for motor functions and memory, spatial learning, anxiety and hedony.Levels of α-syn and synaptophysin were evaluated by Western blot (WB) analysis.Results: In apomorphine-induced rotation test, a mild motor dysfunction was found in SN-α-syn group compared to control.DG-α-syn group showed memory impairment in novel object recognition test.All the α-syn injected groups spent more time to find the platform compared to controls in Morris water maze but this difference did not reach statistical significance.DG-α-syn group consumed more sucrose solution in sucrose consumption test and spent more time on the open arm in elevated plus maze, while the opposite was observed in SN-α-syn group compared to controls.We showed α-syn protein expression in the injected areas of all α-syn groups by WB and immunohistochemical staining.In WB analysis, both hippocampal and striatal synaptophysin expression levels were lower in the α-syn groups compared to controls.Conclusion: Parkinson's disease (PD) is characterized by both motor and non-motor symptoms (NMS).However, an animal model recapitulating NMS with the background of dopaminergic denervation is still lacking.This model may help to investigate hippocampal α-syn pathology correlated especially with cognitive dysfunction and other NMS of PD.
Abstract Certain neuronal populations are selectively vulnerable to alpha -synuclein pathology in Parkinson’s Disease, yet the reasons for this selectivity are unclear. Pathology affects neuronal populations that are anatomically connected although the contribution of neuronal connectivity remains to be quantitatively explored. Herein, we simulate the contribution of the connectome alone to the spread of arbitrary aggregates using a computational model of temporal spread within an abstract representation of the mouse mesoscale connectome. Our simulations are compared with the neuron-to-neuron spread of alpha -synuclein that has been observed with in vivo spreading experiments in rats. We find that neuronal connectivity appears to be compatible with the spreading pattern of alpha -synuclein pathology however, it may be per se insufficient to determine the anatomical pattern of protein spreading observed in experimental animals, suggesting a role of selective vulnerability of neuronal pathways to alpha -synuclein diffusion, accumulation and pathology. Graphical abstract
Drug-induced dyskinesias in dopamine-denervated animals are known to depend on both pre- and postsynaptic changes of the nigrostriatal circuitry. In lesion models used thus far, changes occur in both of these compartments and, therefore, it has not been possible to dissect the individual contribution of each compartment in the pathophysiology of dyskinesias. Here we silenced the nigrostriatal dopamine neurotransmission without affecting the anatomical integrity of the presynaptic terminals using a short-hairpin RNA-mediated knockdown of tyrosine hydroxylase enzyme (shTH). This treatment resulted in significant reduction (by about 70%) in extracellular dopamine concentration in the striatum as measured by on-line microdialysis. Under these conditions, the animals remained nondyskinetic after chronic L-DOPA treatment, whereas partial intrastriatal 6-hydoxydopamine lesioned rats with comparable reduction in extracellular dopamine levels developed dyskinesias. On the other hand, apomorphine caused moderate to severe dyskinesias in both groups. Importantly, single-dose L-DOPA challenge in apomorphine-primed shTH animals failed to activate the already established abnormal postsynaptic responses. Taken together, these data provide direct evidence that the status of the presynaptic, DA releasing compartment is a critical determinant of both the induction and maintenance of L-DOPA–induced dyskinesias.
Interneuronal propagation of α-synuclein has been demonstrated in a variety of experimental models and may be involved in disease progression during the course of human synucleinopathies. The aim of this study was to assess the role that neuronal injury or, vice versa, cell integrity could have in facilitating interneuronal α-synuclein transfer and consequent protein spreading in an in vivo animal model. Viral vectors carrying the DNA for human α-synuclein were injected into the rat vagus nerve to trigger protein overexpression in the medulla oblongata and consequent spreading of human α-synuclein toward pons, midbrain and forebrain. Two vector preparations sharing the same viral construct were manufactured using identical procedures with the exception of methods for their purification. They were also injected at concentrations that induced comparable levels of α-synuclein transduction/overexpression in the medulla oblongata. α-Synuclein load was associated with damage (at 6 weeks post injection) and death (at 12 weeks) of medullary neurons after treatment with only one of the two vector preparations. Of note, neuronal injury and degeneration was accompanied by a substantial reduction of caudo-rostral propagation of human α-synuclein. Interneuronal α-synuclein transfer, which underlies protein spreading from the medulla oblongata to more rostral brain regions in this rat model, is not a mere consequence of passive release from damaged or dead neurons. Neuronal injury and degeneration did not exacerbate α-synuclein propagation. In fact, data suggest that cell-to-cell passage of α-synuclein may be particularly efficient between intact, relatively healthy neurons.
Genetic studies have implicated the neuronal ubiquitin C-terminal hydrolase (UCH) protein UCH-L1 in Parkinson's disease (PD) pathogenesis. Moreover, the function of UCH-L1 may be lost in the brains of PD and Alzheimer's disease patients. We have previously reported that the UCH-L1 polymorphic variant S18Y, potentially protective against PD in population studies, demonstrates specific antioxidant functions in cell culture. Albeit genetic, biochemical and neuropathological data support an association between UCH-L1, PD, synaptic degeneration and oxidative stress, the relationship between the dopaminergic system and UCH-L1 status remains obscure. In the current study, we have examined the dopaminergic system of mice lacking endogenous UCH-L1 protein (gracile axonal dystrophy mice). Our findings show that the lack of wild-type (WT) UCH-L1 does not influence to any significant degree the dopaminergic system at baseline or following injections of the neurotoxin methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Furthermore, using a novel intrastriatal adenoviral injection protocol, we have found that mouse nigral neurons retrogradely transduced with S18Y UCH-L1, but not the WT protein, are significantly protected against MPTP toxicity. Overall, these data provide evidence for an antioxidant and neuroprotective effect of the S18Y variant of UCH-L1, but not of the WT protein, in the dopaminergic system, and may have implications for the pathogenesis of PD or related neurodegenerative conditions, in which oxidative stress might play a role.
Pregnancy-induced hypertension is one of the most important cause of maternal-fetal morbidity and mortality. Pregnancy-related hypertensive disorders are usually associated with diminished nitric oxide (NO) levels. We aimed to evaluate the role of serum NO levels and eNOS gene G894T polymorphism on hypertensive disorders of pregnancy.Eighty patients with gestational hypertension or preeclampsia, and 80 healthy pregnants were enrolled to analyze serum NO levels and G894T polymorphism of the eNOS gene. NO level was analyzed by high-performance liquid chromatography (HPLC) method. The G894T polymorphism of the eNOS gene was determined by polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP).There was no significant difference between groups in terms of G894T/eNOS genotype and allele frequencies (p > 0.05). Serum NO levels were significantly lower in the patients group. In the control group, subjects with thymine-thymine (TT) genotype had significantly lower NO levels when compared to subjects with guanine-guanine (GG) or guanine-thymine (GT) genotype (p < 0.05).We failed to demonstrate an association between eNOS gene G894T polymorphism and serum NO levels in patients with pregnancy-induced hypertensive disorders. We established a relation between pregnancy-induced hypertension and low NO levels.
Abstract The clinical progression of neurodegenerative diseases correlates with the spread of proteinopathy in the brain. Understanding of the mechanism of the proteinopathy spread is far from complete. Here, we propose that inflammation is fundamental to proteinopathy spread. A sequence variant of α-synuclein (V40G) was much less capable of fibril formation than wild-type α-synuclein (WT-syn) and, when mixed with WT-syn, interfered with its fibrillation. Yet when V40G was injected intracerebrally into mice, it induced aggregate spreading even more effectively than WT-syn. The aggregate spreading was preceded by sustained microgliosis and inflammatory responses, which were more robust with V40G than with WT-syn. Oral administration of an anti-inflammatory agent suppressed aggregate spreading, inflammation, and behavioral deficits in mice. Furthermore, exposure of cells to inflammatory cytokines increased the cell-to-cell propagation of α-synuclein. These results suggest that the inflammatory microenvironment is the major driver of the spread of synucleinopathy in the brain.
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