Microglial activating transcription factor 3 upregulation: An indirect target to attenuate inflammation in the nervous system.

Activating Transcription Factor 3 (ATF3) is upregulated in reaction to several cellular stressors found in a wide range of pathological conditions to coordinate a transcriptional response. ATF3 was first implicated in the transcriptional reaction to axotomy when its massive upregulation was measured in sensory and motor neuron cell bodies following peripheral nerve injury. It has since been shown to be critical for successful axon regeneration in the peripheral nervous system and a promising target to mitigate regenerative failure in the central nervous system. However, much of the research to date has focused on ATF3's function in neurons, leaving the expression, function, and therapeutic potential of ATF3 in glia largely unexplored. In the immunology literature ATF3 is seen as a master regulator of the innate immune system. Specifically, in macrophages following pathogen or damage associated molecular pattern receptor activation and subsequent cytokine release, ATF3 upregulation abrogates the inflammatory response. Importantly, ATF3 upregulation is not exclusively due to cellular stress exposure but has been achieved by the administration of several small molecules. In the central nervous system, microglia represent the resident macrophage population and are therefore of immediate interest with respect to ATF3 induction. It is our perspective that the potential of inducing ATF3 expression to dampen inflammatory microglial phenotype represents an unexplored therapeutic target and may have synergistic benefits when paired with concomitant neuronal ATF3 upregulation. This would be of particular benefit in pathologies that involve both detrimental inflammation and neuronal damage including spinal cord injury, multiple sclerosis, and stroke.

Systemic hypoxia mimicry enhances axonal regeneration and functional recovery following peripheral nerve injury.

Despite the ability of peripheral nerves to regenerate after injury, failure occurs due to an inability of supporting cells to maintain growth, resulting in long-term consequences such as sensorimotor dysfunction and neuropathic pain. Here, we investigate the potential of engaging the cellular adaptive response to hypoxia, via inhibiting its negative regulators, to enhance the regenerative process. Under normoxic conditions, prolyl hydroxylase domain (PHD) proteins 1, 2, and 3 hydroxylate the key metabolic regulator hypoxia inducible factor 1α (HIF1α), marking it for subsequent proteasomal degradation. We inhibited PHD protein function systemically via either individual genetic deletion or pharmacological pan-PHD inhibition using dimethyloxalylglycine (DMOG). We show enhanced axonal regeneration after sciatic nerve crush injury in PHD1-/- mice, PHD3-/- mice, and in DMOG-treated mice, and in PHD1-/- and DMOG-treated mice a reduction in hypersensitivity to cooling after permanent sciatic ligation. Electromyographically, PHD1-/- and PHD3-/- mice showed an increased CMAP amplitude one-month post-injury, probably due to protection against denervation induced muscle atrophy, while DMOG-treated and PHD2+/- mice showed reduced latencies, indicating improved motor axon function. DMOG treatment did not affect the growth of dorsal root ganglion neurites in vitro, suggesting a lack of direct effects of DMOG on axonal regrowth. Enhanced regeneration in vivo was concurrent with an increase in macrophage density, and a shift in macrophage polarization state ratios (from M1-like toward M2-like) in DMOG-treated animals. These results indicate PHD proteins as a novel therapeutic target to improve regenerative and functional outcomes after peripheral nerve injury without manipulating molecular O2.

An ATF3-CreERT2 Knock-In Mouse for Axotomy-Induced Genetic Editing: Proof of Principle.

Genome editing techniques have facilitated significant advances in our understanding of fundamental biological processes, and the Cre-Lox system has been instrumental in these achievements. Driving Cre expression specifically in injured neurons has not been previously possible: we sought to address this limitation in mice using a Cre-ERT2 construct driven by a reliable indicator of axotomy, activating transcription factor 3 (ATF3). When crossed with reporter mice, a significant amount of recombination was achieved (without tamoxifen treatment) in peripherally-projecting sensory, sympathetic, and motoneurons after peripheral nerve crush in hemizygotes (65-80% by 16 d) and was absent in uninjured neurons. Importantly, injury-induced recombination did not occur in Schwann cells distal to the injury, and with a knock-out-validated antibody we verified an absence of ATF3 expression. Functional recovery following sciatic nerve crush in ATF3-deficient mice (both hemizygotes and homozygotes) was delayed, indicating previously unreported haploinsufficiency. In a proof-of-principle experiment, we crossed the ATF3-CreERT2 line with a floxed phosphatase and tensin homolog (PTEN) line and show significantly improved axonal regeneration, as well as more complete recovery of neuromuscular function. We also demonstrate the utility of the ATF3-CreERT2 hemizygous line by characterizing recombination after lateral spinal hemisection (C8/T1), which identified specific populations of ascending spinal cord neurons (including putative spinothalamic and spinocerebellar) and descending supraspinal neurons (rubrospinal, vestibulospinal, reticulospinal and hypothalamic). We anticipate these mice will be valuable in distinguishing axotomized from uninjured neurons of several different classes (e.g., via reporter expression), and in probing the function of any number of genes as they relate to neuronal injury and regeneration.

Preserved Adrenal Function After Lumbar Spinal Cord Transection Augments Low Pressure Bladder Activity in the Rat.

Spinal cord injury (SCI) disconnects supraspinal micturition centers from the lower urinary tract resulting in immediate and long-term changes in bladder structure and function. While cervical and high thoracic SCI have a greater range of systemic effects, clinical data suggest that those with lower (suprasacral) injuries develop poorer bladder outcomes. Here we assess the impact of SCI level on acute changes in bladder activity. We used two SCI models, T3 and L2 complete transections in male Wistar rats, and compared bladder pressure fluctuations to those of naïve and bladder-denervated animals. By 2 days after L2 transection, but not T3 transection or bladder denervation, small amplitude rhythmic contractions (1 mmHg, 0.06 Hz) were present at low intravesical pressures (<6 mmHg); these were still present 1 month following injury, and at 3 months, bladders from L2 SCI animals were significantly larger than those from T3 SCI or naïve animals. Low-pressure contractions were unaffected by blocking ganglionic signaling or bladder denervation at the time of measurements. L2 (and sham surgery) but not T3 transection preserves supraspinal adrenal control, and by ELISA we show lower plasma adrenal catecholamine concentration in the latter. When an adrenalectomy preceded the L2 transection, the aberrant low-pressure contractions more closely resembled those after T3 transection, indicating that the increased bladder activity after lumbar SCI is mediated by preserved adrenal function. Since ongoing low-pressure contractions may condition the detrusor and exacerbate detrusor-sphincter dyssynergia, moderating bladder catecholamine signaling may be a clinically viable intervention strategy.