Effects of silencing ectopically expressed hSNCA in rat substantia nigra (SN) were examined as a novel therapeutic approach to Parkinson’s disease (PD). AAV-hSNCA with or without an AAV harboring a short-hairpin (sh)RNA targeting hSNCA or luciferase was injected into one SN. At 9wks, hSNCA-expressing rats had reduced SN dopamine (DA) neurons and exhibited a forelimb deficit. AAV-shRNA-SNCA silenced hSNCA and protected against the forelimb deficit. However, AAV-shRNA-SNCA also led to DA neuron loss suggesting undesirable effects of chronic shRNA expression. Effects on nigrostriatal-projecting neurons were examined using a retrograde tract tracer. Loss of striatal-projecting DA neurons was evident in the vector injection site, whereas DA neurons outside this site were lost in hSNCA-expressing rats, but not in hSNCA-silenced rats. These observations suggest that high levels of shRNA-SNCA were toxic to DA neurons, while neighboring neurons exposed to lower levels were protected by hSNCA gene silencing. Also, data collected on DA levels suggest that neurons other than or in addition to nigrostriatal DA neurons contributed to protection of forelimb use. Our observations suggest that while hSNCA gene silencing in DA neurons holds promise as a novel PD therapy, further development of silencing technology is required.
To probe the connection between longevity and stress resistance, we compared the sensitivity of Ames long-lived dwarf mice and control littermates with paraquat, diquat, and dobutamine. In young adult animals, 95% of male and 39% of female controls died after paraquat administration, but no dwarf animals died. When the experiment was repeated at an older age or a higher dosage of paraquat, dwarf mice still showed greater resistance. Dwarf mice also were more resistant to diquat; 80% of male and 60% of female controls died compared with 40% and 20% of dwarf mice, despite greater sensitivity of dwarf liver to diquat. Dwarf mice were also less sensitive to dobutamine-induced cardiac stress and had lower levels of liver and lung F(2)-isoprostanes. This is the first direct in vivo evidence that long-lived Ames dwarf mice have enhanced resistance to chemical insult, particularly oxidative stressors.
Background Alpha-synuclein (SNCA) downregulation shows therapeutic potential for synucleinopathies, including Parkinson’s disease (PD). Previously we showed that human (h)SNCA gene silencing using a short hairpin (sh)RNA in rat substantia nigra (SN) protects against a hSNCA-induced forelimb deficit, but not dopamine (DA) neuron loss. Further, the mir-embedded hSNCA gene silencing shRNA increases cell death in vitro, but the same target sequence embedded in a microRNA30 transcript (mir30-hSNCA) does not. Objective Examine hSNCA gene silencing using mir30-hSNCA in vivo. Methods Rats were stereotaxically injected into one SN with adeno-associated virus serotype 2/8 (AAV)-hSNCA, AAV-hSNCA plus AAV-mir30-SNCA or AAV-hSNCA plus a control non-silencing mir30-embedded siRNA and DA neuron markers and associated behavior were examined. Results AAV2/8-mediated SN hSNCA expression induces a forelimb deficit and tyrosine hydroxylase-immunoreactive (TH-IR) neuron loss. hSNCA gene silencing using mir30-hSNCA protects against this forelimb deficit at 2m and ameliorates TH-IR neuron loss. Striatal (ST) TH-IR fiber density and DA markers, assessed by western blot, are unaffected by AAV-hSNCA alone. Co-expression of either silencing vector reduces ST TH-IR fibers, panTH in SN and Ser40 phosphorylated TH in SN and ST, but does not affect vesicular monoamine transporter-2. However, hSNCA gene silencing promotes partial TH-IR fiber recovery by 2m. Co-expression of either silencing vector also induces SN inflammation, although some recovery was observed by 2m in hSNCA-silenced SN. Conclusion hSNCA gene silencing with AAV-mir30-hSNCA has positive effects on forelimb behavior and SN DA neurons, which are compromised by inflammation and reduced TH expression, suggesting that AAV2/8-mir30-hSNCA-mediated gene silencing, although promising in vitro, is not a candidate for therapeutic translation for PD.
Alpha-synuclein (SNCA), an abundantly expressed presynaptic protein, is implicated in Parkinson disease (PD). Since over-expression of human SNCA (hSNCA) leads to death of dopaminergic (DA) neurons in human, rodent and fly brain, hSNCA gene silencing may reduce levels of toxic forms of SNCA and ameliorate degeneration of DA neurons in PD. To begin to develop a gene therapy for PD based on hSNCA gene silencing, two AAV gene silencing vectors were designed, and tested for efficiency and specificity of silencing, as well as toxicity in vitro. The same hSNCA silencing sequence (shRNA) was used in both vectors, but in one vector, the shRNA was embedded in a microRNA backbone and driven by a pol II promoter, and in the other the shRNA was not embedded in a microRNA and was driven by a pol III promoter. Both vectors silenced hSNCA to the same extent in 293T cells transfected with hSNCA. In DA PC12 cells, neither vector decreased expression of rat SNCA, tyrosine hydroxylase (TH), dopamine transporter (DAT) or the vesicular monoamine transporter (VMAT). However, the mir30 embedded vector was significantly less toxic to both PC12 and SH-SY5Y cells. Our in vitro data suggest that this miRNA-embedded silencing vector may be ideal for chronic in vivo SNCA gene silencing in DA neurons.
Human Immunodeficiency Virus type 1 (HIV-1) infection induces neurological and neuropsychological deficits, which are associated with dysregulation of the medial prefrontal cortex (mPFC) and other vulnerable brain regions. We evaluated the impact of HIV infection in the mPFC and the therapeutic potential of targeting over-active voltage-gated L-type Ca2+ channels (L-channel) and NMDA receptors (NMDAR), as modeled in HIV-1 transgenic (Tg) rats. Whole-cell patch-clamp recording was used to assess the membrane properties and voltage-sensitive Ca2+ potentials (Ca2+ influx) in mPFC pyramidal neurons. Neurons from HIV-1 Tg rats displayed reduced rheobase, spike amplitude and inwardly-rectifying K+ influx, increased numbers of action potentials, and a trend of aberrant firing compared to those from non-Tg control rats. Neuronal hyper-excitation was associated with abnormally-enhanced Ca2+ influx (independent of NMDAR), which was eliminated by acute L-channel blockade. Combined chronic blockade of over-active L-channels and NMDARs with open-channel blockers abolished HIV effects on spiking, aberrant firing and Ca2+ potential half-amplitude duration, though not the reduced inward rectification. In contrast, individual chronic blockade of over-active L-channels or NMDARs did not alleviate HIV-induced mPFC hyper-excitability. These studies demonstrate that HIV alters mPFC neuronal activity by dysregulating membrane excitability and Ca2+ influx through the L-channels. This renders these neurons more susceptible and vulnerable to excitatory stimuli, and could contribute to HIV-associated neuropathogenesis. Combined targeting of over-active L-channels/NMDARs alleviates HIV-induced dysfunction of mPFC pyramidal neurons, emphasizing a potential novel therapeutic strategy that may effectively decrease HIV-induced Ca2+ dysregulation in the mPFC.
These data demonstrate that ethnicity is a significant factor affecting corticotropin-releasing hormone concentrations at midgestation in the Hispanic and white populations. The use of ethnicity-specific medians to estimate the ethnicity-specific MoM for the corticotropin-releasing hormone concentrations may enhance the predictive value of midgestational maternal corticotropin-releasing hormone as a screening parameter for the prediction of preterm birth.
We assessed firing and voltage-gated Ca influx in medial prefrontal cortex (mPFC) pyramidal neurons from older (12 months old) HIV-1 transgenic (Tg) rats. We found that neurons from older Tg rats showed increased firing compared to non-Tg rats, but Ca spikes were unchanged. However, stronger excitatory stimulation was needed to evoke Ca spikes, which was associated with reduced mPFC Ca1.2 L-type Ca channel (L-channel) protein. In contrast, L-channel protein was unaltered in younger (6-7 weeks old) Tg rats, which we previously found had enhanced neuronal Ca influx. These studies demonstrate that aging alters HIV-induced Ca channel dysfunction that affects mPFC activity.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.