Psora-4, a Kv1.3 Blocker, Enhances Differentiation and Maturation in Neural Progenitor Cells

Abstract

Aim: The self-repair ability of neural progenitor cells (NPCs) has been found to be activated and protected in several therapies helpful in multiple sclerosis (MS), an inflammatory demyelinating disease of the CNS. As a potential therapeutic target in MS, the role of the ion channel Kv1.3 in NPC self-repair has received limited attention. The aim of this study was to explore the effects of a selective Kv1.3 blocker on NPC neuronal differentiation and maturation.

Methods: A small-molecule selective blocker for Kv1.3, Psora-4, was added to the differentiation medium of cultured mouse NPCs to assess its effect on NPC differentiation efficiency. Both a polypeptide Kv1.3 blocker and Kv1.3-specific RNA interference were used in parallel experiments. Further, the maturity of newborn neurons in the presence of Psora-4 was measured both by morphological analysis and by whole-cell patch clamping.

Results: Psora-4 induced a significant increase in the percentage of neurons. Knockdown of Kv1.3 in NPCs also promoted neuronal differentiation. Both morphological and electrophysiological analyses suggested that NPC-derived neurons in the presence of Psora-4 were more mature.

Conclusion: Our studies reveal a crucial role for the ion channel Kv1.3 in the regulation of NPC differentiation and maturation, making Psora-4 a promising candidate molecule for MS treatment.

Keywords: Differentiation; Kv1.3; Maturation; Neural progenitor cell; Small molecule.

Figure 1

Psora‐4 or MgTX promoted the differentiation of neural progenitor cells (NPCs) toward a neuronal lineage. (A) Identification of NPCs and differentiated cells derived from NPCs by immunocytochemistry. The NPCs were labeled with antibody for nestin (green) at Day 0, and the differentiated cells were labeled with antibodies for β‐III‐tubulin (green), MAP2 (red) and GFAP (red) at Day 5. Nuclei were stained with DAPI (blue). (B) Example images of cells immunostained at Day 5 with or without Psora‐4 (10 nM). (C) and (D) Various concentrations of Psora‐4 or MgTX affect NPC neuronal differentiation. The percentage of positive cells was calculated in relation to the total number of cells, visualized by DAPI staining of the nuclei. **P < 0.01, *P < 0.05.

Figure 2

Silencing of Kv1.3 in neural progenitor cells (NPCs) by shRNA interference induced increased differentiation efficiency, consistent with Psora‐4 treatment. (A) Example images of NPCs infected with shRNA lentiviruses with red fluorescence after flow cytometric sorting. The top panel shows the light field, and the bottom panel shows the red fluorescence in the same field. The cells were plated on Matrigel‐coated slides. (B) Kv1.3 mRNA expression levels in NPCs with shRNA lentivirus. Relative Kv1.3 mRNA expression was quantified and normalized to the Nonsense control group (n = 4). Nonsense control: NPCs infected with lentivirus containing the nonsense shRNA‐sequence. Blank vector: NPCs infected with empty lentivirus vector. (C) and (D) Differentiation efficiency of NPC cell lines with Kv1.3‐shRNA toward neuron lineage or astrocyte lineage. The data were calculated at Day 5 in differentiation condition (n = 4). **P < 0.01, *P < 0.05.

Figure 3

Newborn neurons derived from neural progenitor cells (NPCs) in the presence of Psora‐4 (10 nM) developed more mature morphology. (A) Immunocytochemistry images of NPC offspring with or without Psora‐4 at days 5, 10, and 14. (B) Psora‐4 influenced the morphology of newborn neurons. Left, percentage of neurons with multiple neurites (n = 3). Right, average neurite length (n = 4). At least 150 neurons were measured using ImageJ in each independent experiment. (C) Mature‐like and immature‐like neurons at Day 5 of NPC neuronal differentiation. (a) Low‐power image of mature‐like neurons with long and extensive dendrites (orange arrow) and immature‐like neurons with thin and sparse dendrites (white arrow). (b) High‐power image of boxed region in panel a. (c) Low‐power image of neurons (green) and astrocytes (red). This shows the same field as the image in panel a. (d) High‐power image of the boxed region in panel c. (D) Psora‐4 increased the percentage of mature neuron candidates at days 5, 10, and 14 in differentiation condition. (n = 5). (E) MgTX (5 nM) increased the percentage of mature‐like neurons at Day 5 (n = 3). ***P < 0.001, **P < 0.01, *P < 0.05.

Figure 4

Neuron‐like cells differentiated with or without Psora‐4 (10 nM) displayed mature electrophysiological characteristics. (A) Representative electrophysiological recordings of neurons from control and Psora‐4 group, respectively. Spontaneous and induced continuous action potentials (APs) were recorded under current clamp (shown in a, b, e, and f). Mature positive K⁺ and negative Na⁺ currents were recorded under voltage‐clamp (shown in c, d, g, and h). (B) A microscopic image showing whole‐cell patch‐clamp recording of a neuron at Day 14.

Figure 5

Newborn neurons derived from neural progenitor cells (NPCs) in the Psora‐4 (10 nM) group developed more mature electrophysiological properties. (A) Typical electrophysiological recordings from the three main differentiation periods (days 5–10, 13–16 and 20–24). The left panel shows K⁺ and Na⁺ currents, the middle panel shows induced action potentials (APs), and the right panel shows spontaneous APs. (B) Current‐voltage relationships for Na⁺ current (I_{N a}) at days 13–16 (left) and days 20–24 (right). The I_{N a} value at days 20–24 was significantly increased in the presence of Psora‐4 (n = 19). I_{N a} was measured as the peak inward current. (C) Psora‐4 increased the percentage of neurons that fired induced continuous APs (ICAPs) and spontaneous APs (SAPs) at days 20–24. (D) Neurons differentiated with Psora‐4 developed more hyperpolarized rest membrane potentials (V_rest) both at days 13–16 (control n = 18, Psora‐4 n = 17) and at days 20–24 (control n = 19, Psora‐4 n = 18). *P < 0.05.