Kinetics of alpha-synuclein depletion in three brain regions following conditional pan-neuronal inactivation of the encoding gene (Snca) by tamoxifen-induced Cre-recombination in adult mice

Abstract

Conditional pan-neuronal inactivation of the Snca gene in 2-month old male and female mice causes dramatic decrease in the level of the encoded protein, alpha-synuclein, in three studied brain regions, namely cerebral cortex, midbrain and striatum, 12 weeks after the last injection of tamoxifen. Kinetics of alpha-synuclein depletion is different in these brain regions with a longer lag period in the cerebral cortex where this protein is normally most abundant. Our results suggest that efficient post-developmental pan-neuronal knockout of alpha-synuclein in adult, i.e. 5- to 6-month old, animals, could be achieved by tamoxifen treatment of 2-month old mice carrying loxP-flanked Snca gene and expressing inducible Cre-ERT2 recombinase under control of the promoter of neuron-specific enolase (NSE) gene.

Early Effects of Alpha-Synuclein Depletion by Pan-Neuronal Inactivation of Encoding Gene on Electroencephalogram Coherence between Different Brain Regions in Mice

“…Indeed, the latter is in line with the results of our previous study where three months after identical TAM administration, significant depletion of alpha-synuclein levels in different brain regions (the cortex, striatum, and midbrain) was revealed [10]. Furthermore, the similarities between corresponding EEG coherent interrelations between "TAM/vehicle" and "alpha-synuclein knockout/control" groups (c.f., Figure 4c,d) confirm indirectly an associa-2023, 11, 3282 tion of these interrelations with elimination/deactivation of the brain's alpha-synuclein.…”

“…The generation of mouse lines and the breeding of experimental animals used in this study have been described earlier in [10]. Briefly, all lines were previously established and maintained on a pure C57Bl6J (Charles River) genetic background in our Bioresource Collection of the IPAC RAS Centre for collective use (covered by framework FFSN-2021-0005) and provided for this experiment.…”

“…All mice were checked for the presence of the modification in the genome by genotyping using conventional PCR, as described in [10]. Total DNA was isolated from ear biopsies (~30 mg) and used as a template in a PCR reaction with primers:…”

“…However, in eighteen-month-old mice, TAM administration was accompanied by significant changes in dopamine (DA) metabolism, in particular, by a decrease of DOPAC (3,4-dihydroxyphenylacetic acid), a product of oxidative deamination of DA, and HVA (homovanillic acid), its terminal metabolite. In young (two-month-old) conditional knockout Snca flox/∆flox-NSE/Cre-ERT2 mice, significant depletion of the alpha-synuclein protein was revealed in various brain areas (the cortex, the striatum, and the midbrain area) in the three months after TAM administration [10]. In parallel with alpha-synuclein depletion, TAM is expected to be able to inhibit the dopamine transporter (DAT) function independent of TAM activity as a selective estrogen receptor modulator [11].…”

“…Recently, the efficacy of the EEG approaches has been demonstrated in our studies on knockout mice lacking alpha-, beta-, and gamma-synucleins in all possible combinations [17] and on mice models of both Alzheimer's disease and amyotrophic lateral sclerosis [18]. In the current study, we analyze the early effects of a TAM-initiated conditional pan-neuronal inactivated Snca gene and its progression by measuring the levels of baseline EEG coherence between the cortex (MC), putamen (Pt), ventral tegmental area (VTA), and substantia nigra (SN) one, two, and three months after chronic (for five days) injections of TAM or the vehicle (corn oil) in ready-to-use two-month-old mice of a conditional knockout Snca Snca flox/∆flox-NSE/Cre-ERT2 line [10]. A combined time-dependent suppressive effect of alpha-synuclein depletion on the intracerebral EEG coherence was shown.…”