The Evolving Concept of Neuro-Thromboinflammation for Neurodegenerative Disorders and Neurotrauma: A Rationale for PAR1-Targeting Therapies.

Interest in the role of coagulation and fibrinolysis in the nervous system was active in several laboratories dating back before cloning of the functional thrombin receptor in 1991. As one of those, our attention was initially on thrombin and plasminogen activators in synapse formation and elimination in the neuromuscular system, with orientation towards diseases such as amyotrophic lateral sclerosis (ALS) and how clotting and fibrinolytic pathways fit into its pathogenesis. This perspective is on neuro-thromboinflammation, emphasizing this emerging concept from studies and reports over more than three decades. It underscores how it may lead to novel therapeutic approaches to treat the ravages of neurotrauma and neurodegenerative diseases, with a focus on PAR1, ALS, and parmodulins.

Thrombin and the Coag-Inflammatory Nexus in Neurotrauma, ALS, and Other Neurodegenerative Disorders.

This review details our current understanding of thrombin signaling in neurodegeneration, with a focus on amyotrophic lateral sclerosis (ALS, Lou Gehrig's disease) as well as future directions to be pursued. The key factors are multifunctional and involved in regulatory pathways, namely innate immune and the coagulation cascade activation, that are essential for normal nervous system function and health. These two major host defense systems have a long history in evolution and include elements and regulators of the coagulation pathway that have significant impacts on both the peripheral and central nervous system in health and disease. The clotting cascade responds to a variety of insults to the CNS including injury and infection. The blood brain barrier is affected by these responses and its compromise also contributes to these detrimental effects. Important molecules in signaling that contribute to or protect against neurodegeneration include thrombin, thrombomodulin (TM), protease activated receptor 1 (PAR1), damage associated molecular patterns (DAMPs), such as high mobility group box protein 1 (HMGB1) and those released from mitochondria (mtDAMPs). Each of these molecules are entangled in choices dependent upon specific signaling pathways in play. For example, the particular cleavage of PAR1 by thrombin vs. activated protein C (APC) will have downstream effects through coupled factors to result in toxicity or neuroprotection. Furthermore, numerous interactions influence these choices such as the interplay between HMGB1, thrombin, and TM. Our hope is that improved understanding of the ways that components of the coagulation cascade affect innate immune inflammatory responses and influence the course of neurodegeneration, especially after injury, will lead to effective therapeutic approaches for ALS, traumatic brain injury, and other neurodegenerative disorders.

Proximate Mediators of Microvascular Dysfunction at the Blood-Brain Barrier: Neuroinflammatory Pathways to Neurodegeneration.

Current projections are that by 2050 the numbers of people aged 65 and older with Alzheimer's disease (AD) in the US may increase threefold while dementia is projected to double every 20 years reaching ~115 million by 2050. AD is clinically characterized by progressive dementia and neuropathologically by neuronal and synapse loss, accumulation of amyloid plaques, and neurofibrillary tangles (NFTs) in specific brain regions. The preclinical or presymptomatic stage of AD-related brain changes may begin over 20 years before symptoms occur, making development of noninvasive biomarkers essential. Distinct from neuroimaging and cerebrospinal fluid biomarkers, plasma or serum biomarkers can be analyzed to assess (i) the presence/absence of AD, (ii) the risk of developing AD, (iii) the progression of AD, or (iv) AD response to treatment. No unifying theory fully explains the neurodegenerative brain lesions but neuroinflammation (a lethal stressor for healthy neurons) is universally present. Current consensus is that the earlier the diagnosis, the better the chance to develop treatments that influence disease progression. In this article we provide a detailed review and analysis of the role of the blood-brain barrier (BBB) and damage-associated molecular patterns (DAMPs) as well as coagulation molecules in the onset and progression of these neurodegenerative disorders.

HMGB1 and thrombin mediate the blood-brain barrier dysfunction acting as biomarkers of neuroinflammation and progression to neurodegeneration in Alzheimer's disease.

BACKGROUND: The blood-brain barrier (BBB) dysfunction represents an early feature of Alzheimer's disease (AD) that precedes the hallmarks of amyloid beta (amyloid ß) 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.

Membrane lipid peroxidation in neurodegeneration: Role of thrombin and proteinase-activated receptor-1.

Thrombin and membrane lipid peroxidation (MLP) have been implicated in various central nervous system (CNS) disorders from CNS trauma to stroke, Alzheimer's (AD) and Parkinson's (PD) diseases. Because thrombin also induces MLP in platelets and its involvement in neurodegenerative diseases we hypothesized that its deleterious effects might, in part, involve formation of MLP in neuronal cells. We previously showed that thrombin induced caspase-3 mediated apoptosis in motor neurons, via a proteinase-activated receptor (PAR1). We have now investigated thrombin's influence on the oxidative state of neurons leading to induction of MLP-protein adducts. Translational relevance of thrombin-induced MLP is supported by increased levels of 4-hydroxynonenal-protein adducts (HNEPA) in AD and PD brains. We now report for the first time that thrombin dose-dependently induces formation of HNEPA in NSC34 mouse motor neuron cells using anti-HNE and anti-acrolein monoclonal antibodies. The most prominent immunoreactive band, in SDS-PAGE, was at ∼54kDa. Membrane fractions displayed higher amounts of the protein-adduct than cytosolic fractions. Thrombin induced MLP was mediated, at least in part, through PAR1 since a PAR1 active peptide, PAR1AP, also elevated HNEPA levels. Of interest, glutamate and Fe2SO4 also increased the ∼54kDa HNEPA band in these cells but to a lesser extent. Taken together our results implicate the involvement of thrombin and MLP in neuronal cell loss observed in various CNS degenerative and traumatic pathologies.

Mitochondrial lysates induce inflammation and Alzheimer's disease-relevant changes in microglial and neuronal cells.

Neuroinflammation occurs in Alzheimer's disease (AD). While AD genetic studies implicate inflammation-relevant genes and fibrillar amyloid-ß protein promotes inflammation, our understanding of AD neuroinflammation nevertheless remains incomplete. In this study we hypothesized damage-associated molecular pattern (DAMP) molecules arising from mitochondria, intracellular organelles that resemble bacteria, could contribute to AD neuroinflammation. To preliminarily test this possibility, we exposed neuronal and microglial cell lines to enriched mitochondrial lysates. BV2 microglial cells treated with mitochondrial lysates showed decreased TREM2 mRNA, increased TNFα mRNA, increased MMP-8 mRNA, increased IL-8 mRNA, redistribution of NFκB to the nucleus, and increased p38 MAPK phosphorylation. SH-SY5Y neuronal cells treated with mitochondrial lysates showed increased TNFα mRNA, increased NFκB protein, decreased IκBα protein, increased AßPP mRNA, and increased AßPP protein. Enriched mitochondrial lysates from SH-SY5Y cells lacking detectable mitochondrial DNA (ρ0 cells) failed to induce any of these changes, while mtDNA obtained directly from mitochondria (but not PCR-amplified mtDNA) increased BV2 cell TNFα mRNA. These results indicate at least one mitochondrial-derived DAMP molecule, mtDNA, can induce inflammatory changes in microglial and neuronal cell lines. Our data are consistent with the hypothesis that a mitochondrial-derived DAMP molecule or molecules could contribute to AD neuroinflammation.

Bioenergetic dysfunction and inflammation in Alzheimer's disease: a possible connection.

Inflammation is observed in Alzheimer's disease (AD) subject brains. Inflammation-relevant genes are increasingly implicated in AD genetic studies, and inflammatory cytokines to some extent even function as peripheral biomarkers. What underlies AD inflammation is unclear, but no "foreign" agent has been implicated. This suggests that internally produced damage-associated molecular pattern (DAMPs) molecules may drive inflammation in AD. A more complete characterization and understanding of AD-relevant DAMPs could advance our understanding of AD and suggest novel therapeutic strategies. In this review, we consider the possibility that mitochondria, intracellular organelles that resemble bacteria in many ways, trigger and maintain chronic inflammation in AD subjects. Data supporting the possible nexus between AD-associated bioenergetic dysfunction are discussed.

Designing drugs that encourage spinal cord injury healing.

INTRODUCTION: Spinal cord injury (SCI) occurs within seconds, but host responses may take years. Although life expectancy has increased, half of the survivors end up as paraplegics, with many indeed as quadriplegics. Host responses may have multiple effects ranging from inhibiting inflammation to preventing repair and regeneration. Efforts seek to translate exciting results from unsatisfactory animal models to humans. AREAS COVERED: The author reviews the current basic and translational research for SCI treatment that: i) limits secondary injury; and ii) enhances endogenous repair and regeneration. The article provides an emphasis on pro-inflammatory factors (TNF-α, high-mobility group B protein 1) that are targeted to encourage healing. EXPERT OPINION: Research in the coming years needs to be directed at therapeutic agents - whether drugs, biologics or biomaterials - that encourage repair and regeneration and, at the same time, limit cell death and axonal lesions. The author believes that the process of harnessing the power of these innate responses will remain the challenge for the future in the quest for effective therapeutic options.

Novel mutation in VCP gene causes atypical amyotrophic lateral sclerosis.

OBJECTIVE: To identify the genetic variant that causes autosomal dominantly inherited motor neuron disease in a 4-generation Israeli-Arab family using genetic linkage and whole exome sequencing. METHODS: Genetic linkage analysis was performed in this family using Illumina single nucleotide polymorphism chips. Whole exome sequencing was then undertaken on DNA samples from 2 affected family members using an Illumina 2000 HiSeq platform in pursuit of potentially pathogenic genetic variants that comigrate with the disease in this pedigree. Variants meeting these criteria were then screened in all affected individuals. RESULTS: A novel mutation (p.R191G) in the valosin-containing protein (VCP) gene was identified in the index family. Direct sequencing of the VCP gene in a panel of DNA from 274 unrelated individuals with familial amyotrophic lateral sclerosis (FALS) revealed 5 additional mutations. Among them, 2 were previously identified in pedigrees with a constellation of inclusion body myopathy with Paget disease of the bone and frontotemporal dementia (IBMPFD) and in FALS, and 2 other mutations (p.R159C and p.R155C) in IBMPFD alone. We did not detect VCP gene mutations in DNA from 178 cases of sporadic amyotrophic lateral sclerosis. CONCLUSIONS: We report a novel VCP mutation identified in an amyotrophic lateral sclerosis family (p.R191G) with atypical clinical features. In our experience, VCP mutations arise in approximately 1.5% of FALS cases. Our study supports the view that motor neuron disease is part of the clinical spectrum of VCP-associated disease.

Soluble thrombomodulin levels in plasma of multiple sclerosis patients and their implication.

Thrombomodulin (TM) on the cell-surface of cerebrovascular endothelial cells (CECs) is released into blood upon CEC damage. TM promotes activation of protein C (APC), an anticoagulant, anti-inflammatory, neuroprotective molecule that protects CECs and impedes inflammatory cell migration across the blood-brain barrier (BBB). Multiple sclerosis (MS) is associated with CEC damage and BBB dysfunction. We evaluated soluble TM (sTM) levels as a biomarker of BBB integrity and whether glatiramer acetate (GA) influenced sTM levels in MS patients. sTM levels quantified by 2-site ELISA from sera of healthy controls and systemic lupus erythematosus (SLE) patients (CEC-damage positive control) were compared with levels from patients with relapsing-remitting (RRMS) or secondary-progressive MS (SPMS), stratified as: RRMS/GA/no relapse, RRMS/GA/in relapse, RRMS no GA/no relapse, RRMS/no GA/in relapse; and SPMS/no GA. Additionally, soluble endothelial protein C receptor (sEPCR) levels were assessed in the non-stratified MS group, SLE patients, and controls. sTM levels were highest in RRMS patients taking GA with or without relapse, followed in decreasing order by SLE, RRMS/no GA/in relapse, SPMS, RRMS/no GA/no relapse, healthy controls. sEPCR levels were highest in MS patients, then SLE, then controls. sTM may be a useful biomarker of BBB integrity in RRMS patients. Further evaluation of sEPCR is needed. The finding that the highest sTM levels were in RRMS patients taking GA is interesting and warrants further investigation.

Neuroprotective effects of caspase-3 inhibition on functional recovery and tissue sparing after acute spinal cord injury.

STUDY DESIGN: We used gene microarrays and found that caspase-related death genes were upregulated. We tested caspase inhibition and evaluated its effect on the spinal cord after traumatic injury. OBJECTIVE: The logical extension of previous studies was to determine whether downstream CASP genes might also be involved and whether inhibition might prevent injury-induced cell death. SUMMARY OF BACKGROUND DATA: Apoptotic cell death occurs in all endogenous cellular compartments of the spinal cord, peaking at 3 days after injury in neurons, astrocytes, and microglia. The downstream effector caspase-3 cleaves several important cellular sites after being activated by upstream initiator caspases. Along with others, we have previously identified caspase signature cleavage of PARP, alpha-fodrin, and DFF45/ICAD in the injured rat spinal cord. We also showed rapid upregulation of caspase-3 gene expression along with localization of active caspase-3 in neurons and activated microglia after SCI. Others have reported that a more general active-site mimetic peptide ketone, benzylocarbonyl-Val-Ala-Asp-fluromethylketone (zVAD-fmk) was neuroprotective after rat spinal cord injury (SCI). METHODS: In this study, we administered the caspase-3 subfamily tetrapeptide cell permeable inhibitor Z-Asp(O-Me)-Glu(O-Me)-Val-Asp(O-Me) fluoromethyl ketone (DEVD-fmk) intraperitoneally 1 hour after laminectomy and moderate (25 g cm force) SCI in rats. RESULTS.: We used the open field locomotor rating (LRS) over a 14-day course and found statistically significant improvement in DEVD-fmk-treated rats, LRS, 9.8 +/- 0.93 SEM, compared with vehicle, 6.6 +/- 0.4 (P < 0.05). Histologic analysis of percent spinal cord tissue volume spared was 50% greater for DEVD-fmk versus control (P < 0.5). CONCLUSION: These results indicate neuroprotection at both the cellular level and with substantial functional recovery, suggesting caspase-3 inhibition may be a viable therapy in the early hours after experimental SCI.

Longitudinal magnetic resonance imaging of spinal cord injury in mouse: changes in signal patterns associated with the inflammatory response.

Contusion-type spinal cord injury (SCI) in mice was followed longitudinally using in vivo magnetic resonance (MR) imaging along with neurobehavioral tests performed on postinjury Days 1, 7, 14 and 28. Magnetic resonance images were acquired from seven injured wild-type mice using a 9.4-T scanner and presented in sagittal and axial views to reflect the current state of the injured cord neuropathology on each day. The data were analyzed individually to gain more insights on the neuroinflammatory response unique to the mouse, to characterize the spatiotemporal evolution of the lesion and to quantify the changes in lesion volume and length with time. The MR intensity patterns on Day 1 showed acute injuries as focal in one group of three mice and as diffuse in the remaining group of four mice. The focal injuries appeared as a region of hypointensity with well-defined boundaries. These injuries first enlarged on Day 7, but then shrunk slightly by Days 14 and 28. In contrast, the diffuse injuries were initially obscure on Day 1, mainly because of loss of contrast between gray and white matters. On Day 7, lesions expanded asymptotically in both rostral and caudal directions with respect to the epicenter, and maintained its size on Days 14 and 28. Previous studies based on postmortem histological analysis have reported lesions behaving more like in the focal group. However, this new injury with diffuse characteristics may have important implications for SCI research carried out with mice. Unique experiments on genetically engineered mice with altered neuroinflammatory response should help clarify the origin of these differences in the lesion formation.

GRK5 deficiency leads to early Alzheimer-like pathology and working memory impairment.

G-protein coupled receptor kinase-5 (GRK5) deficiency has been linked to early Alzheimer's disease in humans and mouse models of the disease. To determine potential roles of GRK5 in the disease pathogenesis, the GRK5 knockout mouse was evaluated at pathological and behavioral levels. We found that these mice displayed an age-dependent increase in hippocampal axonal defects characterized by clusters of axonal swellings that accumulated abnormal amounts of molecular motor proteins, microtubule-associated proteins, intracellular beta-amyloid, and subcellular organelles. In severe cases, extracellular beta-amyloid fibrillar deposits were occasionally observed, along with degenerating axonal components, and were tightly surrounded by reactive astrocytes. Moreover, significant loss of synaptic proteins and early signs of cholinoceptive neurodegeneration were evident in the hippocampus as well. Consistent with the moderate level of pathologic change, aged GRK5 knockout mice displayed selective working memory impairment, with other cognitive domains unaffected. Taken together, these findings not only strongly support an important role of GRK5 deficiency in early Alzheimer's pathogenesis, but also promote the GRK5 knockout mouse as an additional model for early Alzheimer-related studies.

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 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-alpha, 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.

Magnetic resonance imaging of mouse spinal cord.

The feasibility of performing high-resolution in vivo MRI on mouse spinal cord (SC) at 9.4 T magnetic field strength is demonstrated. The MR properties of the cord tissue were measured and the characteristics of water diffusion in the SC were quantified. The data indicate that the differences in the proton density (PD) and transverse relaxation time between gray matter (GM) and white matter (WM) dominate the contrast seen on the mouse SC images at 9.4 T. However, on heavily T(2)-weighted images these differences result in a reversal of contrast. The diffusion of water in the cord is anisotropic, but the WM exhibits greater anisotropy and principal diffusivity than the GM. The quantitative data presented here should establish a standard for comparing similar measurements obtained from the SCs of genetically engineered mouse or mouse models of SC injury (SCI).

Tissue transglutaminase during mouse central nervous system development: lack of alternative RNA processing and implications for its role(s) in murine models of neurotrauma and neurodegeneration.

Tissue transglutaminase (tTG) is a member of a multigene family principally involved in catalyzing the formation of protein cross-links. Unlike other members of the transglutaminase family, tTG is multifunctional since it also serves as a guanosine triphosphate (GTP) binding protein (Galpha(h)) and participates in cell adhesion. Different isoforms of tTG can be produced by proteolysis or alternative splicing. We find that tTG mRNA is expressed at low levels in the mouse CNS relative to other tissues, and at lower levels in the CNS of mouse in comparison to that of human or rat. tTG mRNA levels are higher in the heart compared to the CNS, for example, and much higher in the liver. Within the CNS, tTG message is lowest in the adult cerebellum and thalamus and highest in the frontal cortex and striatum. In the hippocampus, tTG expression is highest during embryonic development and falls off dramatically after 1 week of life. We did not find alternative splicing of the mouse tTG. At the protein level, the predominant isoform is approximately 62 kDa. In summary, tTG, an important factor in neuronal survival, is expressed at low levels in the mouse CNS and, unlike rat and human tTG, does not appear to be regulated by alternative splicing. These findings have implications for analyses of rodent tTG expression in human neurodegenerative and neurotrauma models where alternative processing may be an attractive pathogenetic mechanism. They further impact on drug discovery paradigms, where modulation of activity may have therapeutic value.

Neuroprotective effects of recombinant thrombomodulin in controlled contusion spinal cord injury implicates thrombin signaling.

Although the central nervous system (CNS) of mammals has had poor prospects for regeneration, recent studies suggest this might improve from blocking "secondary cell loss" or apoptosis. In this regard, intravenous activated protein C (aPC) improved neurologic outcomes in a rat compression spinal cord injury (SCI) model. Protein C activation occurs when the serine protease thrombin binds to the cell surface proteoglycan thrombomodulin (TM) forming a complex that halts coagulation. In culture, rTM blocks thrombin's activation of protease-activated receptors (PARs), that mediate thrombin killing of neurons and glial reactivity. Both PAR1 and prothrombin are rapidly upregulated after contusion SCI in rats, prior to peak apoptosis. We now report neuroprotective effects of intraperitoneal soluble recombinant human rTM on open-field locomotor rating scale (BBB) and spinal cord lesion volume when given 1 h after SCI. BBB scores from four separate experiments showed a 7.6 +/- 1.4 absolute score increase (p < 0.05) at 3 days, that lasted throughout the time course. Histological sections at 14 days were even more dramatic where a twofold reduction in lesion volume was quantified in rTM-treated rats. Thionin staining revealed significant preservation of motor neuronal profiles both at, and two segments below, the lesion epicenter. Activated caspase-3 immunocytochemistry indicated apoptosis was quite prominent in motor neurons in vehicle (saline) controls, but was dramatically reduced by rTM. Microglia, increased and activated after injury, were reduced with rTM treatment. Taken together, these and previous results support a prominent role for coagulation-inflammation signaling cascades in the subacute changes following SCI. They identify a neuroprotective role for rTM by its inhibition of thrombin generation and blockade of PAR activation.

Abnormality of G-protein-coupled receptor kinases at prodromal and early stages of Alzheimer's disease: an association with early beta-amyloid accumulation.

Overwhelming evidence indicates that the effects of beta-amyloid (Abeta) are dose dependent both in vitro and in vivo, which implies that Abeta is not directly detrimental to brain cells until it reaches a threshold concentration. In an effort to understand early Alzheimer's disease (AD) pathogenesis, this study focused on the effects of subthreshold soluble Abeta 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 Abeta effects on microglial cells: at the threshold of 5 microm and above, Abeta directly induced tumor necrosis factor-alpha (TNF-alpha) release, and at subthreshold doses, Abeta indirectly potentiated TNF-alpha release induced by certain G-protein-coupled receptor (GPCR) activators. Mechanistic studies revealed that subthreshold Abeta 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 Abeta accumulation in these transgenic mice. Together, this study not only discovered a novel link between subthreshold Abeta and GRK dysfunction, it also demonstrated that the GRK abnormality in vivo occurs at prodromal and early stages of AD.

Prospects for the pharmacotherapy of amyotrophic lateral sclerosis : old strategies and new paradigms for the third millennium.

Biomedical researchers interested in amyotrophic lateral sclerosis (ALS) must invoke newly developing technologies if we are to discover pharmaceutical treatments that will help a significant population of patients with the disease. The focus of ALS research over the last 10 years has been on reactive oxygen species (ROS) and glutamate excitotoxicity, resulting in several clinical trials and the launch of the only drug currently available for the treatment of ALS, riluzole. Unfortunately, the therapeutic benefits have been minimal, at best, and the prognosis for patients with ALS has not improved beyond very modest retardation of the disease course. By emphasising ROS and glutamate excitotoxicity, current ALS research has only partially been able to attenuate the rate of motor decline and neuronal loss associated with this illness. Clues to additional therapeutic potentialities will come from an increased understanding of the mode of cell death (apoptotic or other) and the pathways leading to neuronal demise. If death is apoptotic, inhibiting caspases may be useful. The regulatory modifications for cell death at the molecular level remain to be determined and exploited to prevent neuronal loss, although novel pathways have been recently elucidated that impact on protein aggregation and processing. Oxidative stress, seen in both familial and sporadic forms of ALS, may be only one post-translational mechanism likely to affect specific proteins essential for the health and stability of motor neurons. Protein cross-linking by transglutaminase paralleling that may lead to defects in proteasome function may also be a significant mechanism. The latest capabilities to screen protein changes in specific cells represent the kinds of advances needed to combat ALS in the third millennium.

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 Alzheimer's 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.