Enhancing neurogenesis after traumatic brain injury: The role of adenosine kinase inhibition in promoting neuronal survival and differentiation

Abstract

Traumatic brain injury (TBI) presents a significant public health challenge, necessitating innovative interventions for effective treatment. Recent studies have challenged conventional perspectives on neurogenesis, unveiling endogenous repair mechanisms within the adult brain following injury. However, the intricate mechanisms governing post-TBI neurogenesis remain unclear. The microenvironment of an injured brain, characterized by astrogliosis, neuroinflammation, and excessive cell death, significantly influences the fate of newly generated neurons. Adenosine kinase (ADK), the key metabolic regulator of adenosine, emerges as a crucial factor in brain development and cell proliferation after TBI. This study investigates the hypothesis that targeting ADK could enhance brain repair, promote neuronal survival, and facilitate differentiation. In a TBI model induced by controlled cortical impact, C57BL/6 male mice received intraperitoneal injections of the small molecule ADK inhibitor 5-iodotubercidin (ITU) for three days following TBI. To trace the fate of TBI-associated proliferative cells, animals received intraperitoneal injections of BrdU for seven days, beginning immediately after TBI. Our results show that ADK inhibition by ITU improved brain repair 14 days after injury as evidenced by a diminished injury size. Additionally, the number of mature neurons generated after TBI was increased in ITU-treated mice. Remarkably, the TBI-associated pathological events including astrogliosis, neuroinflammation, and cell death were arrested in ITU-treated mice. Finally, ADK inhibition modulated cell death by regulating the PERK signaling pathway. Together, these findings demonstrate a novel therapeutic approach to target multiple pathological mechanisms involved in TBI. This research contributes valuable insights into the intricate molecular mechanisms underlying neurogenesis and gliosis after TBT.

Keywords: Adenosine kinase; Astroglia; Microglia; Neurogenesis; Regeneration; Traumatic brain injury; hippocampus.

Fig. 1.. Adenosine kinase inhibition promotes recovery after TBI.

(A) Schematic representation of the experimental design for investigating the impact of adenosine kinase (ADK) inhibition on traumatic brain injury (TBI)-associated neurogenesis and cell proliferation. C57BL/6 mice were subjected to moderate-to-severe TBI using controlled cortical impact (CCI). Animals were randomly assigned to two groups, each group received 5 total i.p. injections of either 1.6 mg/kg 5-iodotubericine (ITU) or vehicle every 12 h for three consecutive days after TBI. All animal groups were injected 50 mg/kg BrdU via i.p. once a day for 7 consecutive days started immediately after TBI. Animals were sacrificed at 14 days post-TBI (DPI) for analysis. (B–C) ADK inhibition significantly reduced the injury cavity volume in ITU-treated animals compared to controls. (B) Whole brain images from ITU-and vehicle-treated mice. (C) Light microscopic images of Nissl-stained brain sections illustrating the injury cavity in ITU-and vehicle-treated mice. (D) Left panel, graphic illustrating the method used for volume measurements. Asterisk representing the cavity space. (D) Right panel, Quantitative measurement of the injury cavity volume. Scale bar is 5 mm in B and 1 mm in C. All values are presented as mean ± SEM (n = 5 mice per group). Two-tailed unpaired student t-tests were used in D. ^**** P < 0.001.

Fig. 2.. Adenosine kinase inhibition promotes neuronal survival and differentiation after TBI.

(A) Representative fluorescence images of hippocampal brain sections co-labeled with the mature neuronal marker (NeuN)(green) and bromodeoxyuridine (BrdU) (red) from ITU-treated and control mice. (B) Schematic diagram of the hippocampus in the ipsilateral hemisphere where the region of interest is highlighted by a dotted blue line. (C) Quantitative analysis of the number of BrdU-positive cells revealed no significant change in the total number of BrdU-positive cells between ITU-treated and control groups. (D) A significantly higher number of NeuN and BrdU positive neurons in the hippocampus of ITU-treated mice compared to controls, indicating enhanced neuronal survival and differentiation. Scale bar is 100 μm and 50 μm in A. All values are presented as mean ± SEM (n = 4). Two-tailed unpaired student t-tests were used in B and C. ^*** = p ≤ 0.001, ns = no significance.

Fig. 3.. Modulation of TBI-induced astrogliosis by ITU treatment.

(A) Representative fluorescence images of hippocampal brain sections co-labeled with glial fibrillary acidic protein (GFAP) (green) and BrdU (red) showed reduced GFAP-positive cells in ITU-treated mice compared to controls. (B) Quantitative analysis of GFAP-positive cells revealed a significant reduction in GFAP-positive cells in ITU-treated mice compared to control mice, indicative of decreased astrogliosis. (C) Quantitative analysis of cells double-labeled with GFAP and BrdU showed that the cell count was significantly lower in ITU-treated mice compared to controls, suggesting the modulatory effect of ITU on astrogliosis. (D & E) Quantitative analysis of BrdU-positive cells in subgranular layer (SGL), granular layer (GL), and hilus of the dentate gyrus in ITU and vehicle-treated miceThese findings highlight ITU’s potential to target astrogliosis after TBI. Scale bar is 100 μm and 50 μm in A. All values are presented as mean ± SEM (n = 4). Two-tailed unpaired student t-tests were used in B and C. ^**p = 0.007 and ^*** p < 0.001.

Fig. 4.. Suppression of microglial activity by ITU treatment.

(A) Representative fluorescence images of hippocampal brain sections labeled with the ionized calcium-binding adapter molecule 1 (IBA1) (red) and DAPI (blue), indicating reduced microglial activity in ITU-treated mice compared to controls after TBI. (B) Quantitative analysis of IBA1-positive cells revealed a significant decrease in their number in ITU-treated mice vs control. These results collectively suggest that modulating adenosine metabolism through ADK inhibition suppresses microglial activity, contributing to tissue repair after TBI. Scale bar is 200 μm and 50 μm in A. All values are presented as mean ± SEM (n = 4). Two-tailed unpaired student t-tests were used in B and C. ^** P = 0.004.

Fig. 5.. Attenuation of TBI-Induced Cell Death by ITU through PERK Pathway Inhibition.

(A) Terminal deoxynucleotidyl transferase dUTP (TUNEL) staining indicating reduced cell death in ITU-treated mice compared to controls after TBI. (B) Quantification of TUNEL stain fluorescence confirmed a significant reduction in cell death in ITU-treated mice. (C) Western blot analysis of protein bands for PAKT and PERK proteins and GAPDH as a housekeeping protein. (D) Quantitative analysis of Western blot protein bands using ImageJ V. 1.52 software and expressed as the ratio of optical densities of PAKT showed no significant change, while (E) PERK was significantly lower in ITU-treated mice compared to controls, suggesting that ADK regulates TBI-associated cell death through the inhibition of the PERK signaling pathway. All values are presented as mean ± SEM (n = 4–6 mice per group). Two-tailed unpaired student t-tests were used in B and C. ^* P ≤ 0.05, ^** P ≤ 0.01, ^*** = P ≤ 0.001, NS = no significance.