Bruce A. Citron

Minocycline neuroprotects, reduces microgliosis, and inhibits caspase protease expression early after spinal cord injury

Minocycline, a clinically used tetracycline for over 40 years, crosses the blood–brain barrier and prevents caspase up‐regulation. It reduces apoptosis in mouse models of Huntingtons disease and familial amyotrophic lateral sclerosis (ALS) and is in clinical trial for sporadic ALS. Because apoptosis also occurs after brain and spinal cord (SCI) injury, its prevention may be useful in improving recovery. We analyzed minocyclines neuroprotective effects over 28 days following contusion SCI and found significant functional recovery compared to tetracycline. Histology, immunocytochemistry, and image analysis indicated statistically significant tissue sparing, reduced apoptosis and microgliosis, and less activated caspase‐3 and substrate cleavage. Since our original report in abstract form, others have published both positive and negative effects of minocycline in various rodent models of SCI and with various routes of administration. We have since found decreased tumor necrosis factor‐α, as well as caspase‐3 mRNA expression, as possible mechanisms of action for minocyclines ameliorative action. These results support reports that modulating apoptosis, caspases, and microglia provide promising therapeutic targets for prevention and/or limiting the degree of functional loss after CNS trauma. Minocycline, and more potent chemically synthesized tetracyclines, may find a place in the therapeutic arsenal to promote recovery early after SCI in humans.

Participation of protease-activated receptor-1 in thrombin-induced microglial activation

Activation of microglia, the resident macrophages in the CNS, plays a significant role in neuronal death or degeneration in a broad spectrum of CNS disorders. Recent studies indicate that nanomolar concentrations of the serine protease, thrombin, can activate microglia in culture. However, in contrast to other neural cells responsive to thrombin, the participation of novel protease‐activated receptors (PARs), such as the prototypic thrombin receptor PAR1, in thrombin‐induced microglial activation was cast in doubt. In this report, by utilizing primary microglial cultures from PAR1 knockout (PAR1–/–) mice, application of the PAR1 active peptide TRAP‐6 (SFLLRN) in comparison to a scrambled peptide (LFLNR), we have unambiguously demonstrated that murine microglia constitutively express PAR1 mRNA that is translated into fully functional protein. Activation of the microglial PAR1 induces a rapid cytosolic free [Ca2+]i increase and transient activation of both p38 and p44/42 mitogen‐activated protein kinases. Moreover, although in part, this PAR1 activation directly contributes to thrombin‐induced microglial proliferation. Furthermore, although not directly inducing tumor necrosis factor‐α (TNF‐α) release, PAR1 activation up‐regulates microglial CD40 expression and potentiates CD40 ligand‐induced TNF‐α production, thus indirectly contributing to microglial activation. Taken together, these results demonstrate an essential role of PAR1 in thrombin‐induced microglial activation. In addition, strategies aimed at blocking thrombin signaling through PAR1 may be therapeutically valuable for diseases associated with cerebral vascular damage and significant inflammation with microglial activation.

Rapid Tau Aggregation and Delayed Hippocampal Neuronal Death Induced by Persistent Thrombin Signaling

Tau hyperphosphorylation, leading to self-aggregation, is widely held to underlie the neurofibrillary degeneration found in Alzheimers disease (AD) and other tauopathies. However, it is unclear exactly what environmental factors may trigger this pathogenetic tau hyperphosphorylation. From several perspectives, the coagulation serine protease, thrombin, has been implicated in AD and activates several different protein kinase pathways but has not previously been shown how it may contribute to AD pathogenesis. Here we report that nanomolar thrombin induced rapid tau hyperphosphorylation and aggregation in murine hippocampal neurons via protease-activated receptors, which was followed by delayed synaptophysin reduction and apoptotic neuronal death. Mechanistic study revealed that a persistent thrombin signaling via protease-activated receptor 4 and prolonged downstream p44/42 mitogenactivated protein kinase activation are at least in part responsible. These results pathogenetically linked thrombin to subpopulations of AD and other tauopathies associated with cerebrovascular damage. Such knowledge may be instrumental in transforming therapeutic paradigms.

Transcription factor Sp1 dysregulation in Alzheimer's disease.

Altered gene expression occurs in central nervous system disorders, including Alzheimers disease (AD). Transcription factor Sp1 may be involved insofar as it can regulate the expression of several AD‐related proteins, including amyloid precursor protein (APP) and tau. Sp1 could itself be regulated by inflammatory and other factors associated with AD, such as interleukin‐1β. We measured an almost threefold elevation in the number of mRNA molecules of this cytokine in the AD frontal cortex. Sp1 mRNA was found to be up‐regulated in these AD brains (along with Sp1‐regulated COX‐2), and the Sp1 increase was also seen at the protein level by Western immunoblotting. To determine whether this would also occur in transgenic mice developing AD pathology, we examined the expression of Sp1 in the cortex and hippocampus and observed higher levels of Sp1 mRNA and protein. These results indicate that elements of regulatory pathways involving transcription factor Sp1 may be useful targets for therapeutic intervention to prevent or reverse AD.

Abnormality of G-Protein-Coupled Receptor Kinases at Prodromal and Early Stages of Alzheimer's Disease: An Association with Early β-Amyloid Accumulation

Overwhelming evidence indicates that the effects of β-amyloid (Aβ) are dose dependent both in vitro and in vivo, which implies that Aβ is not directly detrimental to brain cells until it reaches a threshold concentration. In an effort to understand early Alzheimers disease (AD) pathogenesis, this study focused on the effects of subthreshold soluble Aβ and the underlying molecular mechanisms in murine microglial cells and an AD transgenic mouse model. We found that there were two phases of dose-dependent Aβ effects on microglial cells: at the threshold of 5 μm and above, Aβ directly induced tumor necrosis factor-α (TNF-α) release, and at subthreshold doses, Aβ indirectly potentiated TNF-α release induced by certain G-protein-coupled receptor (GPCR) activators. Mechanistic studies revealed that subthreshold Aβ pretreatment in vitro reduced membrane GPCR kinase-2/5 (GRK2/5), which led to retarded GPCR desensitization, prolonged GPCR signaling, and cellular hyperactivity to GPCR agonists. Temporal analysis in an early-onset AD transgenic model, CRND8 mice, revealed that the membrane (functional) GRK2/5 in brain cortices were significantly reduced. More importantly, such a GRK abnormality took place before cognitive decline and changed in a manner corresponding with the mild to moderate soluble Aβ accumulation in these transgenic mice. Together, this study not only discovered a novel link between subthreshold Aβ and GRK dysfunction, it also demonstrated that the GRK abnormality in vivo occurs at prodromal and early stages of AD.

Thrombin: A Potential Proinflammatory Mediator in Neurotrauma and Neurodegenerative Disorders

Thrombin is well known in its function as the ultimate serine protease in the coagulation cascade. Emerging evidence indicates that thrombin also functions as a potent signaling molecule that regulates physiologic and pathogenic responses alike in a large variety of cell populations and tissues. Accompanying CNS injury and other cerebral vascular damages, prothrombin activation and leakage of active thrombin into CNS parenchyma has been documented. Due to the irreplaceable feature of neurons, over-reactive inflammatory reactions in the CNS often cause irreversible neuronal damage. Therefore, particular attention is required to develop strategies that restrict CNS inflammatory responses to beneficial, in contrast to neurotoxic ones. In this regard, thrombin not only activates endothelial cells and induces leukocyte infiltration and edema but also activates astrocytes, and particularly microglia, as recently demonstrated, to propagate the focal inflammation and produce potential neurotoxic effects. Recently revealed molecular mechanisms underlying these thrombin effects appear to involve proteolytic activation of two different thrombin-responsive, protease-activated receptors (PARs), PAR1 and PAR4, possibly in concert. Potential therapeutic strategies based on appreciation of the current understanding of molecular mechanisms underlying thrombin-induced CNS inflammation are also discussed.

Neural Thrombin and Protease Nexin I Kinetics After Murine Peripheral Nerve Injury

Abstract: We addressed the balance between thrombin and its serpin protease nexin I (PNI) after sciatic nerve injury in the mouse. Prothrombin levels increased twofold 24 h after nerve crush, as measured by a specific chromogenic assay, and peaked at day 3. Thrombin activity also increased 2–4 days after injury in distal sciatic nerve segments. Nerve RNA analysis using reverse transcriptase‐polymerase chain reaction (RT‐PCR) assay confirmed that prothrombin was synthesized locally. We also monitored PNI levels in these injured nerve samples by complex formation with an 125I‐labeled target protease and found peak activity occurring later, 6–9 days after the thrombin induction. These data indicate that nerve injury first induces the synthesis of prothrombin, which is subsequently converted to active thrombin. Nerve crush‐induced thrombin is followed by the generation of functionally active PNI and may be directly responsible for its induction. By immunocytochemistry with anti‐PNI antibody, we found that activated Schwann cells were the source of induced PNI. These results support the concept that the balance between serine proteases and their serpins is dysregulated during nerve injury and suggests a role for its reestablishment in nerve damage repair.

The nuclear factor erythroid 2-like 2 activator, tert-butylhydroquinone, improves cognitive performance in mice after mild traumatic brain injury

Traumatic Brain injury affects at least 1.7 million people in the United States alone each year. The majority of injuries are categorized as mild but these still produce lasting symptoms that plague the patient and the medical field. Currently treatments are aimed at reducing a patients symptoms, but there is no effective method to combat the source of the problem, neuronal loss. We tested a mild, closed head traumatic brain injury model for the effects of modulation of the antioxidant transcription factor Nrf2 by the chemical activator, tert-butylhydroquinone (tBHQ). We found that post-injury visual memory was improved by a 7 day course of treatment and that the level of activated caspase-3 in the hippocampus was reduced. The injury-induced memory loss was also reversed by a single injection at 30 min after injury. Since the protective stress response molecule, HSP70, can be upregulated by Nrf2, we examined protein levels in the hippocampus, and found that HSP70 was elevated by the injury and then further increased by the treatment. To test the possible role of HSP70, model neurons in culture exposed to a mild injury and treated with the Nrf2 activator displayed improved survival that was blocked by the HSP70 inhibitor, VER155008. Following mild traumatic brain injury, there may be a partial protective response and patients could benefit from directed enhancement of regulatory pathways such as Nrf2 for neuroprotection.

Wobbler mice modeling motor neuron disease display elevated transactive response DNA binding protein.

Wobbler mice model motor neuron disease with a substantial decline in motor neurons. TDP-43 is a nucleic acid binding protein that accumulates, along with ubiquitin, in the cytoplasm of amyotrophic lateral sclerosis (ALS) motor neurons. Recently, it was reported that Cu/Zn superoxide dismutase type 1 (SOD1) familial amyotrophic lateral sclerosis (fALS) model mice do not mimic the TDP-43 changes seen in sporadic ALS, although they share a large number of other properties with the human disorder. We examined ubiquitin inclusions and TDP-43 expression in wobbler mice. TDP-43 mRNA, measured by quantitative reverse transcription-coupled PCR, was elevated in the wobbler spinal cord. Immunohistochemistry revealed intracellular ubiquitin inclusions and abnormal distribution of TDP-43 into the cytoplasm in wobblers similar to the staining reported in ALS. Finally, nuclear and cytoplasmic fractions, examined by Western immunoblotting, confirmed a delocalization of TDP-43 in the neurodegenerative wobbler. These observations indicate that wobbler mice, which suffer motor neuron loss at 21 days, undergo TDP-43 and ubiquitin changes characteristic of sporadic ALS.

Injury-induced ‘switch’ from GTP-regulated to novel GTP-independent isoform of tissue transglutaminase in the rat spinal cord

We recently found that alternative transcripts of tissue transglutaminase (tTG or TG2) were present in hippocampal brain regions of Alzheimers disease (AD), but not in control, non‐demented, age‐matched brains. Since antecedent non‐severe trauma has been implicated in AD and other neurodegenerative diseases, such as Parkinsons disease (PD) and amyotrophic lateral sclerosis (ALS), we were interested in whether alternative transcripts might be detected in a model of neurotrauma, controlled‐contusion spinal cord injury (SCI) in the rat. Implicated in diverse roles from growth and differentiation to apoptotic cell death, only bifunctional tTG, of the nine member TG family, has dual catalytic activities: guanine trinucleotide (GTP) hydrolyzing activity (GTPase), as well as protein cross‐linking. These functions imply two physiological functions: programmed cell life and death. These may have profound roles in the nervous system since studies in cultured astrocytes found tTG short (S) mRNA transcripts induced by treatment with injury‐related cytokines. In the developing rat spinal cord, tTG activity is concentrated in ventral horn alpha motoneurons, but neither studies of spinal cord tTG gene expression, nor evaluation of the GTP‐regulated isoforms in tissues, have been reported. We now report increased tTG protein and gene expression occurring rapidly after SCI. In parallel, novel appearance of a second, short form transcript, in addition to the normal long (L) isoform, occurs by 8 h of injury. Up‐regulation of tTG message and activity following neural injury. with appearance of a truncated GTP‐unregulated S form, may represent new approaches to drug targets in neurotrauma.