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 [Ca 2+ ] 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-a (TNF-a) release, PAR1 activation up-regulates microglial CD40 expression and potentiates CD40 ligand-induced TNF-a 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.

A chronic stress paradigm comprising exposure to predation, tail suspension and restraint induces a depressive syndrome in C57BL/6J mice that occurs in some, but not all, animals. Here, we sought to extend our behavioural studies to investigate how susceptibility (sucrose preference<65%) or resilience (sucrose preference>65%) to stress-induced anhedonia affects the 5HT system and the expression of inflammation-related genes. All chronically stressed animals, displayed increased level of anxiety, but susceptible mice exhibited an increased propensity to float in the forced swim test and demonstrate hyperactivity under stressful lighting conditions. These changes were not present in resilient or acutely stressed animals. Compared to resilient animals, susceptible mice showed elevated expression of tumour necrosis factor alpha (TNF) and the 5-HT transporter (SERT) in the pre-frontal area. Enhanced expression of 5HT(2A) and COX-1 in the pre-frontal area was observed in all stressed animals. In turn, indoleamine-2,3-dioxygenase (IDO) was significantly unregulated in the raphe of susceptible animals. At the cellular level, increased numbers of Iba-1-positive microglial cells were also present in the prefrontal area of susceptible animals compared to resilient animals. Consequently, the susceptible animals display a unique molecular profile when compared to resilient, but anxious, animals. Unexpectedly, this altered profile provides a rationale for exploring anti-inflammatory, and possibly, TNF-targeted therapy for major depression.

The objective of this study was to investigate the safety and efficacy of recombinant human insulinlike growth factor-I (rhIGF-I) in the treatment of sporadic ALS. A double-blind, placebo-controlled, randomized study of 266 patients was conducted at eight centers in North America. Placebo or rhIGF-I (0.05 mg/kg/day or 0.10 mg/kg/day) was administered for 9 months. The primary outcome measure was disease symptom progression, assessed by the rate of change (per patient slope) in the Appel ALS rating scale total score. The Sickness Impact Profile (SIP), a patient-perceived, health-related quality of life assessment, was a secondary outcome variable. Progression of functional impairment in patients receiving high-dose (0.10 mg/kg/day) rhIGF-I was 26% slower than in patients receiving placebo (p = 0.01). The high-dose treatment group was less likely to terminate the study due to protocol-defined markers of disease symptom progression, and members in this group exhibited a slower decline in quality of life, as assessed by the SIP. Patients receiving 0.05 mg/kg/day of rhIGF-I exhibited trends similar to those associated with high-dose treatment, suggesting a dose-dependent response. The incidence of clinically significant adverse experiences was comparable among the three treatment groups. Recombinant human insulin-like growth factor-I slowed the progression of functional impairment and the decline in health-related quality of life in patients with ALS with no medically important adverse effects.

Following a controlled, severe contusion lesion to the lower thoracic spinal cord in adult rats, we found that apoptosis occurred in cells located in both gray and white matter. This suggested that both nonneuronal cells, including astrocytes, oligodendroglia and microglia, as well as neurons, might participate in programmed cell death (PCD) following spinal cord injury (SCI). Determination of which cell populations participate, and the kinetics and extent of their involvement might reveal new paradigms for approaches to therapy. Consequently, we assessed the functional deficit, comparing a comprehensive locomotor rating scale (LRS) with the inclined plane test at various times after injury. Using standard histology, along with cell-specific markers, we assessed PCD in different spinal cord segments using several parameters of apoptosis. Our results indicate that hind limb motor function was lost at day 1, and then only gradually and ineffectively (about 10-15%) recovered over the next month. Evidence for increased cell number was present for astrocytes and microglia beginning at day 1 after injury. Over the postinjury time period, apoptotic cells appeared (from day 1 to 14), and peaked (in terms of apoptotic index) on day 3. About one-third were microglia, whereas neurons, both large and small, also underwent apoptosis, again peaking at day 3. However, neurons continued to die and were not replaced by proliferation, so that at day 7, three times as many neurons (as a percentage) underwent PCD compared with the glial compartment. Oligodendrocytes also underwent apoptosis, with a biphasic curve, both at days 3 and 14 following injury. Thus, in addition to immediate, passive necrosis, delayed and apoptotic PCD also occurred in all cell populations in severely injured spinal cord.

Background: The blood-brain barrier (BBB) dysfunction represents an early feature of Alzheimer's disease (AD) that precedes the hallmarks of amyloid beta (amyloid β) plaque deposition and neuronal neurofibrillary tangle (NFT) formation. A damaged BBB correlates directly with neuroinflammation involving microglial activation and reactive astrogliosis, which is associated with increased expression and/or release of high-mobility group box protein 1 (HMGB1) and thrombin. However, the link between the presence of these molecules, BBB damage, and progression to neurodegeneration in AD is still elusive. Therefore, we aimed to profile and validate non-invasive clinical biomarkers of BBB dysfunction and neuroinflammation to assess the progression to neurodegeneration in mild cognitive impairment (MCI) and AD patients. Methods: We determined the serum levels of various proinflammatory damage-associated molecules in aged control subjects and patients with MCI or AD using validated ELISA kits. We then assessed the specific and direct effects of such molecules on BBB integrity in vitro using human primary brain microvascular endothelial cells or a cell line. Results: We observed a significant increase in serum HMGB1 and soluble receptor for advanced glycation end products (sRAGE) that correlated well with amyloid beta levels in AD patients (vs. control subjects). Interestingly, serum HMGB1 levels were significantly elevated in MCI patients compared to controls or AD patients. In addition, as a marker of BBB damage, soluble thrombomodulin (sTM) antigen, and activity were significantly (and distinctly) increased in MCI and AD patients. Direct in vitro BBB integrity assessment further revealed a significant and concentration-dependent increase in paracellular permeability to dextrans by HMGB1 or α-thrombin, possibly through disruption of zona occludins-1 bands. Pre-treatment with anti-HMGB1 monoclonal antibody blocked HMGB1 effects and leaving BBB integrity intact. Conclusions: Our current studies indicate that thrombin and HMGB1 are causal proximate proinflammatory mediators of BBB dysfunction, while sTM levels may indicate BBB endothelial damage; HMGB1 and sRAGE might serve as clinical biomarkers for progression and/or therapeutic efficacy along the AD spectrum.

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 Huntington's 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 minocycline's 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-a, as well as caspase-3 mRNA expression, as possible mechanisms of action for minocycline's 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. Keywords: apoptosis, caspase inhibition, locomotor rating score, tetracycline. Spinal cord injury (SCI) in the United States is one of the leading causes of disability, with approximately 10 000 new cases reported each year (Price et al. 1994). As most new patients are young, and few reparative treatments are available, victims of this devastating condition are often incapacitated for life. The degree of dysfunction after SCI depends on several central factors, including the severity of injury and the location of the damage in the cord. Several groups, including our own, have identified a therapeutic 'window of opportunity' for strategies directed at preventing or lessening tissue loss. Other contributing factors, such as host responses and expression of stress response genes, indicate that few if any SCIs are alike, and similarly, no one treatment is expected to be universally effective. However, it is likely that early intervention will spare the maximum amount of tissue and cells, should produce the minimum degree of deficit, and result in the greatest amount of functional recovery.

Increasing evidence indicates several roles for thrombin-like serine proteases and their cognate inhibitors (serpins) in normal development and/or pathology of the nervous system. In addition to its prominent role in thrombosis and/or hemostasis, thrombin inhibits neurite outgrowth in neuroblastoma and primary neuronal cells in vitro, prevents stellation of glial cells, and induces cell death in glial and neuronal cell cultures. Thrombin is known to act via a cell surface protease-activated receptor (PAR-1), and recent evidence suggests that rodent neurons express PAR-1. Previously, we have shown that the thrombin inhibitor, protease nexin-1, significantly prevents neuronal cell death both in vitro and in vivo. Here we have examined the effects of human ␣-thrombin and the presence and/or activation of PAR-1 on the survival and differentiation of highly enriched cultures of embryonic chick spinal motoneurons. We show that thrombin significantly decreased the mean neurite length, prevented neurite branching, and induced motoneuron death by an apoptosis-like mechanism in a dose-dependent manner. These effects were prevented by cotreatment with hirudin, a specific thrombin inhibitor. Treatment of the cultures with a synthetic thrombin receptor-activating peptide (SFLLRNP) mimicked the deleterious effects of thrombin on motoneurons. Furthermore, cotreatment of the cultures with inhibitors of caspase activities completely prevented the death of motoneurons induced by either thrombin or SFLLRNP. These findings indicate that (1) embryonic avian spinal motoneurons express functional PAR-1 and (2) activation of this receptor induces neuronal cell degeneration and death via stimulation of caspases. Together with previous reports, our results suggest that thrombin, its receptor(s), and endogenous thrombin inhibitors may be important regulators of neuronal cell fate during development, after injury, and in pathology of the nervous system.

We have previously reported that thrombin, the ultimate serine protease in the coagulation cascades, is a proinflammatory agent that causes proliferation and activation of brain microglial cells. However, participation of its principal receptor, the protease-activated receptor 1 (PAR1) appears to be limited to promoting microglial proliferation and not induction of inflammatory mediators. In the present study, we now report that thrombin action in promoting inflammatory mediators from brain microglia is mediated through another thrombin receptor, PAR4. Here we show that the PAR4 agonist peptide (PAR4AP, GYPGKF), but not the PAR1AP (TRAP, SFLLRN), induced tumor necrosis factor-␣ (TNF-␣) production not only in cultured murine microglial cells in vitro but also in rat cortex in vivo. Down-regulation of PAR4 expression in microglial cultures by a specific antisense, but not a sense, oligonucleotide reduced PAR4AP-induced TNF-␣. Mechanistic studies indicated that, in comparison with PAR1 signaling, prolonged increase of [Ca 2؉ ] i and phosphorylation of p44/42 mitogen-activated protein kinases, as well as NFB activation may be responsible for PAR4AP-induced TNF-␣ production in microglia. Taken together, these results demonstrate that PAR4 activation mediates the potentially detrimental effects of thrombin on microglia, implying that perspectives of exploiting PAR1 as a potential anti-inflammatory target should be shifted toward PAR4 as a much more specific therapeutic target in brain inflammatory conditions associated with neurotrauma and neurodegenerations.