Optimization of cerebellar purkinje neuron cultures and development of a plasmid-based method for purkinje neuron-specific, miRNA-mediated protein knockdown

Abstract

We present a simple and efficient method to knock down proteins specifically in Purkinje neurons (PN) present in mixed mouse primary cerebellar cultures. This method utilizes the introduction via nucleofection of a plasmid encoding a specific miRNA downstream of the L7/Pcp2 promoter, which drives PN-specific expression. As proof-of-principle, we used this plasmid to knock down the motor protein myosin Va, which is required for the targeting of smooth endoplasmic reticulum (ER) into PN spines. Consistent with effective knockdown, transfected PNs robustly phenocopied PNs from dilute-lethal (myosin Va-null) mice with regard to the ER targeting defect. Importantly, our plasmid-based approach is less challenging technically and more specific to PNs than several alternative methods (e.g., biolistic- and lentiviral-based introduction of siRNAs). We also present a number of improvements for generating mixed cerebellar cultures that shorten the procedure and improve the total yield of PNs, and of transfected PNs, considerably. Finally, we present a method to rescue cerebellar cultures that develop large cell aggregates, a common problem that otherwise precludes the further use of the culture.

Creation of a myosin Va‐TAP‐tagged mouse and identification of potential myosin Va‐interacting proteins in the cerebellum

“…To further demonstrate that TAP-MyoVa functions normally, we asked whether SER is properly transported into the dendritic spines of PNs isolated from myosin Va TAP-tagged mice. Examination of the signals in the insets ( Figure 4, panel a2, Calbindin-D28K; panel a3, IP3R1; panel A4, overlay) shows that most of this neuron's dendritic spines are filled with SER (several examples are marked with arrowheads), consistent with previous work (Alexander & Hammer 3rd, 2016;Miyata et al, 2000;Takagishi et al, 1996;Wagner, Brenowitz, et al, 2011b). Examination of the signals in the insets ( Figure 4, panel a2, Calbindin-D28K; panel a3, IP3R1; panel A4, overlay) shows that most of this neuron's dendritic spines are filled with SER (several examples are marked with arrowheads), consistent with previous work (Alexander & Hammer 3rd, 2016;Miyata et al, 2000;Takagishi et al, 1996;Wagner, Brenowitz, et al, 2011b).…”

“…At any moment in time, the majority of dendritic spines of cerebellar PNs are filled with a tubule of SER that is required for synaptic plasticity at the granule neuron: PN synapse (Alexander & Hammer 3rd, 2016;Miyata et al, 2000;Takagishi et al, 1996;Wagner, Brenowitz, et al, 2011b). Importantly, the transport of SER into PN spines absolutely requires myosin Va (Wagner, Brenowitz, et al, 2011b).…”

“…E17-E18 embryos or P0-P2 pups were used for mixed primary cerebellar cultures as previously described (Alexander & Hammer 3rd, 2016). Timed-pregnant C57BL/6 mice were purchased from Charles River Laboratories (Wilmington, MA).…”

Myosin 18Aα targets guanine nucleotide exchange factor β-Pix to dendritic spines of cerebellar Purkinje neurons to promote spine maturation

“…Timed-pregnant C57BL/6 females of gestation day 17-18 were purchased from Charles Rivers Laboratories. E17-E18 embryos were harvested and mixed cerebellar cultures were prepared as described previously (Alexander and Hammer, 2016). Animals were anesthetized by inhalation of Isoflurane and euthanized by cervical dislocation.…”

“…With regard to which cell type to study, previous work from our lab showed that myosin Va transports tubules of smooth endoplasmic reticulum into Purkinje neuron spines to support synaptic plasticity and motor learning, thereby explaining the ataxia exhibited by dilute/myosin Va null mice (Wagner, Brenowitz and Hammer, 2011). Our subsequent efforts have resulted in improvements in culturing and transfecting Purkinje neurons, in the development of a Purkinje neuron-specific miRNA-mediated knockdown system (Alexander and Hammer, 2016), and in creating Purkinje neurons from embryonic stem cells (Alexander and Hammer, 2019). For these and other reasons we decided to investigate myosin 18A function in Purkinje neurons.…”