SIRT1 is increased in affected brain regions and hypothalamic metabolic pathways are altered in Huntington disease.

AIMS: Metabolic dysfunction is involved in modulating the disease process in Huntington disease (HD) but the underlying mechanisms are not known. The aim of this study was to investigate if the metabolic regulators sirtuins are affected in HD. METHODS: Quantitative real-time polymerase chain reactions were used to assess levels of SIRT1-3 and downstream targets in post mortem brain tissue from HD patients and control cases as well as after selective hypothalamic expression of mutant huntingtin (HTT) using recombinant adeno-associated viral vectors in mice. RESULTS: We show that mRNA levels of the metabolic regulator SIRT1 are increased in the striatum and the cerebral cortex but not in the less affected cerebellum in post mortem HD brains. Levels of SIRT2 are only increased in the striatum and SIRT3 is not affected in HD. Interestingly, mRNA levels of SIRT1 are selectively increased in the lateral hypothalamic area (LHA) and ventromedial hypothalamus (VMH) in HD. Further analyses of the LHA and VMH confirmed pathological changes in these regions including effects on SIRT1 downstream targets and reduced mRNA levels of orexin (hypocretin), prodynorphin and melanin-concentrating hormone (MCH) in the LHA and of brain-derived neurotrophic factor (BDNF) in the VMH. Analyses after selective hypothalamic expression of mutant HTT suggest that effects on BDNF, orexin, dynorphin and MCH are early and direct, whereas changes in SIRT1 require more widespread expression of mutant HTT. CONCLUSIONS: We show that SIRT1 expression is increased in HD-affected brain regions and that metabolic pathways are altered in the HD hypothalamus.

Transduced wild-type but not P301S mutated human tau shows hyperphosphorylation in transgenic mice overexpressing A30P mutated human alpha-synuclein.

Neuropathological and cell culture studies suggest that tau and α-synuclein pathologies may promote each other. To study the relevance and functional implications of these findings in vivo, we transduced hippocampal neurons of wild-type or human A30P α-synuclein transgenic mice with wild-type or P301S mutated human tau using an adeno-associated virus vector. Green fluorescent protein transduction was used as a control. We assessed spontaneous exploratory activity, anxiety and spatial learning and memory 11 weeks after the transduction and perfused the mice for histology. The transduced tau was mainly found in axon terminals and largely restricted within the hippocampi. In addition, neurons around the injection site showed cytoplasmic staining for human tau in both wild-type and A30P mice. Of these tau-positive neurons, 44% in A30P mice but only 3% in wild-type mice receiving human wild-type tau transduction formed paired helical filament-1 (PHF-1)-positive cytoplasmic densities. In contrast, only 1% of tau-positive neurons were also PHF-1 positive after transduction with P301S tau in mice of either genotype. Transduction of P301S tau reduced swimming speed but otherwise tau transduction had no significant behavioral consequences. Cytoplasmic PHF-1 densities were associated with poor spatial memory in wild-type mice but slightly improved memory in A30P mice, indicating that also tau hyperphosphorylation does not necessarily compromise neural functions. These data demonstrate that α-synuclein promotes tau hyperphosphorylation depending on the amino acids on the 301 site.

Long-term polarization of microglia upon α-synuclein overexpression in nonhuman primates.

We have previously shown that persistent α-synuclein overexpression in ventral midbrain of marmoset leads to a distinctive neurodegenerative process and motor defects. The neurodegeneration was confined to caudate putamen dopaminergic fibers in animals overexpressing wild-type (wt) α-synuclein. However, A53T α-synuclein overexpression induced neurodegeneration that resulted in nigral dopaminergic cell death. Here, we analyze the microglia population in the midbrain of these animals by stereological quantification of Iba1+ cells. Our data here show that monkeys overexpressing A53T α-synuclein showed a long-term increase in microglia presenting macrophagic morphology. However, wt α-synuclein overexpression, despite the absence of dopaminergic cell death, resulted in a permanent robust increase of the microglia population characterized by a range of distinct morphological types that persisted after 1 year. These results confirm that the microglial response differs depending on the type of α-synuclein (wt/A53T) and/or whether α-synuclein expression results in cell death or not, suggesting that microglia may play different roles during disease progression. Furthermore, the microglial response is modulated by events related to α-synuclein expression in substantia nigra and persists in the long term. The data presented here is in agreement with that previously observed in a recombinant adeno-associated virus (rAAV) α-synuclein rat model, thereby validating both the findings and the model, and highlighting the translational potential of the rodent model to higher species closer to humans.

Development and characterisation of a novel rat model of Parkinson's disease induced by sequential intranigral administration of AAV-α-synuclein and the pesticide, rotenone.

Modeling Parkinson's disease remains a major challenge for preclinical researchers, as existing models fail to reliably recapitulate all of the classic features of the disease, namely, the progressive emergence of a bradykinetic motor syndrome with underlying nigrostriatal α-synuclein protein accumulation and nigrostriatal neurodegeneration. One limitation of the existing models is that they are normally induced by a single neuropathological insult, whereas the human disease is thought to be multifactorial with genetic and environmental factors contributing to the disease pathogenesis. Thus, in order to develop a more relevant model, we sought to determine if administration of the Parkinson's disease-associated pesticide, rotenone, into the substantia nigra of rats overexpressing the Parkinson's disease-associated protein, α-synuclein, could reliably model the triad of classic features of the human disease. To do so, rats underwent stereotaxic surgery for unilateral delivery of the adeno-associated virus (AAV)-α-synuclein into the substantia nigra. This was followed 13 weeks later by delivery of rotenone into the same site. The effect of the genetic and environmental insults alone or in combination on lateralised motor performance (Corridor, Stepping, and Whisker Tests), nigrostriatal integrity (tyrosine hydroxylase immunohistochemistry), and α-synucleinopathy (α-synuclein immunohistochemistry) was assessed. We found that rats treated with either AAV-α-synuclein or rotenone developed significant motor dysfunction with underlying nigrostriatal neurodegeneration. However, when the genetic and environmental insults were sequentially administered, the detrimental impact of the combined insults on motor performance and nigrostriatal integrity was significantly greater than the impact of either insult alone. This indicates that sequential exposure to relevant genetic and environmental insults is a valid approach to modeling human Parkinson's disease in the rat.

Towards a neuroprotective gene therapy for Parkinson's disease: use of adenovirus, AAV and lentivirus vectors for gene transfer of GDNF to the nigrostriatal system in the rat Parkinson model.

During the last few years, recombinant viral vectors derived from adenovirus (Ad), adeno-associated virus (AAV) or lentivirus (LV) have been developed into highly effective vehicles for gene transfer to the adult central nervous system. In recent experiments, in the rat model of Parkinson's disease, all three vector systems have been shown to be effective for long-term delivery of glial cell line-derived neurotrophic factor (GDNF) at biologically relevant levels in the nigrostriatal system. Injection of the GDNF encoding vectors into either striatum or substantia nigra thus makes it possible to obtain a regionally restricted over-expression of GDNF within the nigrostriatal system that is sufficient to block the toxin-induced degeneration of the nigral dopamine neurons. Injection of GDNF vectors in the striatum, in particular, is effective not only in rescuing the cell bodies in the substantia nigra, but also in preserving the nigrostriatal projection and a functional striatal dopamine innervation in the rat Parkinson model. Long-term experiments using AAV-GDNF and LV-GDNF vectors show, moreover, that sustained GDNF delivery over 3-6 months can promote regeneration and significant functional recovery in both 6-OHDA-lesioned rats and MPTP-lesioned monkeys. The impressive efficacy of the novel AAV and LV vectors in rodent and primate Parkinson models suggests that the time may now be ripe to explore these vector systems as tools for neuroprotective treatments in patients with Parkinson's disease.

EGF infusion stimulates the proliferation and migration of embryonic progenitor cells transplanted in the adult rat striatum.

Immature progenitor cells (generated by in vitro propagation) may provide a useful alternative to primary cells (from dissected embryonic tissue) for transplantation if their migratory and proliferative and differentiation properties can be controlled and directed in vivo. In this study E15 murine EGF-responsive progenitor cells were transplanted to the striatum of adult rats. Simultaneously, these animals received continuous infusion of either epidermal growth factor (EGF) or vehicle, to the lateral ventricle, for 8 days. In animals that received EGF, the transplanted progenitors migrated toward the lateral ventricle and proliferated, as evidenced by bromodeoxyuridine incorporation. Progenitor cells transplanted to rats that received vehicle infusions showed neither of these responses. In all animals, transplanted progenitors expressed an immature astrocyte or oligodendrocyte phenotype, the majority of cells being astrocytes. We conclude that EGF stimulates the migration and proliferation of murine progenitor cells in vivo, either directly or indirectly, but does not influence their phenotypic differentiation.

Long-term rAAV-mediated gene transfer of GDNF in the rat Parkinson's model: intrastriatal but not intranigral transduction promotes functional regeneration in the lesioned nigrostriatal system.

Previous studies have used recombinant adeno-associated viral (rAAV) vectors to deliver glial cell line-derived neurotrophic factor (GDNF) in the substantia nigra to protect the nigral dopamine (DA) neurons from 6-hydroxydopamine-induced damage. However, no regeneration or functional recovery was observed in these experiments. Here, we have used an rAAV-GDNF vector to express GDNF long-term (6 months) in either the nigral DA neurons themselves, in the striatal target cells, or in both of these structures. The results demonstrate that both nigral and striatal transduction provide significant protection of nigral DA neurons against the toxin-induced degeneration. However, only the rats receiving rAAV-GDNF in the striatum displayed behavioral recovery, accompanied by significant reinnervation of the lesioned striatum, which developed gradually over the first 4-5 months after the lesion. GDNF transgene expression was maintained at high levels throughout this period. These results provide evidence that rAAV is a highly efficient vector system for long-term expression of therapeutic proteins in the nigrostriatal system.

Protection and regeneration of nigral dopaminergic neurons by neurturin or GDNF in a partial lesion model of Parkinson's disease after administration into the striatum or the lateral ventricle.

Both glial cell line-derived neurotrophic factor (GDNF) and its recently discovered congener, neurturin (NTN), have been shown to exert neuroprotective effects on lesioned nigral dopamine (DA) neurons when administered at the level of the substantia nigra. In the present study, we have explored the relative in vivo potency of these two neurotrophic factors using two alternative routes of administration, into the striatum or the lateral ventricle, which may be more relevant in a clinical setting. In rats subjected to an intrastriatal (IS) 6-hydroxydopamine (6-OHDA) lesion, GDNF and NTN were injected every third day for 3 weeks starting on the day after the 6-OHDA injection. GDNF provided almost complete (90-92%) protection of the lesioned nigral DA neurons after both IS and intracerebroventricular (ICV) administration. NTN, by contrast, was only partially effective after IS injection (72% sparing) and totally ineffective after ICV injection. Although the trophic factor injections protected the nigral neurons from lesion-induced cell death, the level of expression of the phenotypic marker, tyrosine hydroxylase (TH), was markedly reduced in the rescued cell bodies. The extent of 6-OHDA-induced DA denervation in the striatum was unaffected by both types of treatment; consistent with this observation, the high rate of amphetamine-induced turning seen in the lesioned control animals was unaltered by either GDNF or NTN treatment. In the GDNF-treated animals, and to a lesser extent also after IS NTN treatment, prominent axonal sprouting was observed within the globus pallidus, at the level where the lesioned nigrostriatal axons are known to end at the time of onset of the neurotrophic factor treatment. The results show that GDNF is highly effective as a neuroprotective and axon growth-stimulating agent in the IS 6-OHDA lesion model after both IS and ICV administration. The lower efficacy of NTN after IS, and particularly ICV, administration may be explained by the poor solubility and diffusion properties at neutral pH.

Acute contractile effects of epidermal growth factor on bladder smooth muscles. An in vivo and in vitro study in rats.

Chronic treatment with epidermal growth factor (EGF) stimulates growth of all wall layers of the urinary tract in pigs and rats. Herein, we investigated the acute effects of EGF on detrusor smooth muscle activity. For in vivo examination, awake rats received EGF (75 micrograms/kg) intravenously and detrusor smooth muscle activity was monitored cystometrically. The EGF bolus caused no alteration in diuresis but a doubling of the micturition frequency, a 25% increase in micturition pressures, and increased irregular baseline contractile activity. For in vitro examination detrusor smooth muscle strips were exposed to EGF (1 microgram/ml). EGF caused contraction and increase in the spontaneous activity. In conclusion, EGF increases rat detrusor smooth muscle contractile activity in vivo and in vitro. The finding suggests that a direct effect of EGF on bladder smooth muscles is part of the genesis to the growth of the detrusor smooth muscle observed after chronic EGF treatment.