Onset of Alzheimer disease in apolipoprotein ɛ4 carriers is earlier in butyrylcholinesterase K variant carriers.

BACKGROUND: The authors sought to examine the impact of the K-variant of butyrylcholinesterase (BCHE-K) carrier status on age-at-diagnosis of Alzheimer disease (AD) in APOE4 carriers. METHODS: Patients aged 50-74 years with cerebrospinal fluid (CSF) biomarker-confirmed AD, were recruited to clinical trial (NCT03186989 since June 14, 2017). Baseline demographics, disease characteristics, and biomarkers were evaluated in 45 patients according to BCHE-K and APOE4 allelic status in this post-hoc study. RESULTS: In APOE4 carriers (N = 33), the mean age-at-diagnosis of AD in BCHE-K carriers (n = 11) was 6.4 years earlier than in BCHE-K noncarriers (n = 22, P < .001, ANOVA). In APOE4 noncarriers (N = 12) there was no observed influence of BCHE-K. APOE4 carriers with BCHE-K also exhibited slightly higher amyloid and tau accumulations compared to BCHE-K noncarriers. A predominantly amyloid, limited tau, and limbic-amnestic phenotype was exemplified by APOE4 homozygotes with BCHE-K. In the overall population, multiple regression analyses demonstrated an association of amyloid accumulation with APOE4 carrier status (P < .029), larger total brain ventricle volume (P < .021), less synaptic injury (Ng, P < .001), and less tau pathophysiology (p-tau181, P < .005). In contrast, tau pathophysiology was associated with more neuroaxonal damage (NfL, P = .002), more synaptic injury (Ng, P < .001), and higher levels of glial activation (YKL-40, P = .01). CONCLUSION: These findings have implications for the genetic architecture of prognosis in early AD, not the genetics of susceptibility to AD. In patients with early AD aged less than 75 years, the mean age-at-diagnosis of AD in APOE4 carriers was reduced by over 6 years in BCHE-K carriers versus noncarriers. The functional status of glia may explain many of the effects of APOE4 and BCHE-K on the early AD phenotype. TRIAL REGISTRATION: NCT03186989 since June 14, 2017.

ATXN7-Related Cone-Rod Dystrophy: The Integrated Functional Evaluation of the Cerebellum (CERMOI) Study.

Importance: Reliable biomarkers with diagnostic and prognostic values are needed for upcoming gene therapy trials for spinocerebellar ataxias. Objective: To identify ophthalmological biomarkers in a sample of spinocerebellar ataxia type 7 (SCA7) carriers. Design, Setting, and Participants: This article presents baseline data from a cross-sectional natural history study conducted in Paris, France, reference centers for rare diseases from May 2020 to April 2021. Data were analyzed from September to December 2022. Fifteen adult ATXN7 pathogenic expansion carriers (9 with preataxia and 6 with ataxia) were included, all with a Scale for the Assessment and Rating of Ataxia (SARA) score of 15 of 40 or lower. Patients were recruited at the Paris Brain Institute, and all contacted patients accepted to participate in the study. Main Outcomes and Measures: Three visits (baseline, 6 months, and 12 months) were planned, including neurological examination (SARA and Composite Cerebellar Functional Severity Score), ophthalmological examination (best-corrected visual acuity, microperimetry, full-field electroretinogram, optical coherence tomography, and fundus autofluorescence imaging), and neurofilament light chain (NfL) measurements. Here we report the baseline ophthalmic data from the cohort and determine whether there is a correlation between disease scores and ophthalmic results. Results: Among the 15 included SCA7 carriers (median [range] age, 38 [18-60] years; 8 women and 7 men), 12 displayed cone or cone-rod dystrophy, with the number of CAG repeats correlating with disease severity (ρ, 0.73, 95% CI, 0.34 to 0.90; P < .001). Two patients with cone-rod dystrophy exhibited higher repeat numbers and greater ataxia scores (median [range] SARA score, 9 [7-15]) compared to those with only cone dystrophy (median [range] SARA score, 2 [0-5]). A correlation emerged for outer nuclear layer thickness with SARA score (ρ, -0.88; 95% CI, -0.96 to -0.59; P < .001) and NfL levels (ρ, -0.87; 95% CI, -0.86 to 0.96; P < .001). Moreover, ataxia severity was correlated with visual acuity (ρ: 0.89; 95% CI, 0.68 to 0.96; P < .001) and retinal sensitivity (ρ, -0.88; 95% CI, -0.96 to 0.59; P < .001). Conclusions and Relevance: In this cross-sectional study, retinal abnormalities were found at preataxic stages of the disease. Most of the carriers presented with cone dystrophy and preserved rod function. The outer nuclear layer thickness correlated with SARA score and plasma NfL levels suggesting nuclear layer thickness to be a biomarker of disease severity. These findings contribute to understanding the dynamics of SCA7-related retinal dystrophy and may help lay the groundwork for future therapeutic intervention monitoring and clinical trials. Trial Registration: ClinicalTrials.gov Identifier: NCT04288128.

Exploratory Tau Biomarker Results From a Multiple Ascending-Dose Study of BIIB080 in Alzheimer Disease: A Randomized Clinical Trial.

Importance: Accumulation of hyperphosphorylated, tangled microtubule-associated protein tau (MAPT) is a pathological hallmark of Alzheimer disease (AD) associated with disease progression and cognitive decline. Objective: To evaluate the effect of tau synthesis reduction on tau biomarkers in patients with mild AD. Design, Setting, and Participants: This randomized clinical trial was a double-blind, placebo-controlled 36-week multiple-ascending dose (MAD) phase 1b trial (October 2017 to September 2020), followed by a 64- or 71-week open-label long-term extension (LTE) (October 2019 to May 2022). After being assessed for eligibility at 12 sites in Canada and Europe, participants with mild AD and confirmed amyloid pathology were randomized 3:1 (BIIB080:placebo) in 4 dose cohorts. Intervention: Intrathecal administration of BIIB080, a MAPT-targeting antisense oligonucleotide, or placebo. Active dose arms included 10 mg every 4 weeks, 30 mg every 4 weeks, 60 mg every 4 weeks, and 115 mg every 12 weeks during the MAD period and 60 mg every 12 weeks or 115 mg every 12 weeks during the LTE. Main Outcome and Measures: The original primary end point was safety. Additionally, BIIB080, total tau (t-tau), and phosphorylated tau 181 (p-tau181) cerebrospinal fluid (CSF) concentrations were evaluated. Tau positron emission tomography (PET) was collected in a substudy, and standard uptake value ratios (SUVRs) were calculated in a priori-defined composite regions of interest. Results: Of 102 participants assessed for eligibility, 46 participants with mild AD were enrolled; 23 (50%) were female, and mean (SD) age was 65.8 (5.70) years. BIIB080 was generally well tolerated and was associated with a dose-dependent reduction in CSF t-tau and p-tau181 in the MAD period (56% reduction; 95% CI, 50% to 62%; and 51% reduction; 95% CI, 38% to 63%, of CSF t-tau in the 2 higher-dose cohorts) that continued and/or was maintained through quarterly dosing in the LTE. Tau PET demonstrated reduced accumulation vs placebo at week 25 (n = 13). At week 100, tau PET showed a reduction from baseline across all regions assessed (n = 12), with the largest reductions from baseline observed in the temporal composite (-0.71 SUVR; 95% CI, -1.40 to -0.02). A moderate correlation was observed between model-predicted cumulative CSF drug exposure and tau PET change. Conclusions and Relevance: In this randomized clinical trial, BIIB080 reduced tau biomarkers, including CSF t-tau, CSF p-tau181, and tau PET, which is associated with cognitive decline, in participants with mild AD. Effects of BIIB080 on biomarkers and clinical outcomes are being further evaluated in a phase 2 trial. Trial Registration: ClinicalTrials.gov Identifier: NCT03186989.

Tau-targeting antisense oligonucleotide MAPTRx in mild Alzheimer's disease: a phase 1b, randomized, placebo-controlled trial.

Tau plays a key role in Alzheimer's disease (AD) pathophysiology, and accumulating evidence suggests that lowering tau may reduce this pathology. We sought to inhibit MAPT expression with a tau-targeting antisense oligonucleotide (MAPTRx) and reduce tau levels in patients with mild AD. A randomized, double-blind, placebo-controlled, multiple-ascending dose phase 1b trial evaluated the safety, pharmacokinetics and target engagement of MAPTRx. Four ascending dose cohorts were enrolled sequentially and randomized 3:1 to intrathecal bolus administrations of MAPTRx or placebo every 4 or 12 weeks during the 13-week treatment period, followed by a 23 week post-treatment period. The primary endpoint was safety. The secondary endpoint was MAPTRx pharmacokinetics in cerebrospinal fluid (CSF). The prespecified key exploratory outcome was CSF total-tau protein concentration. Forty-six patients enrolled in the trial, of whom 34 were randomized to MAPTRx and 12 to placebo. Adverse events were reported in 94% of MAPTRx-treated patients and 75% of placebo-treated patients; all were mild or moderate. No serious adverse events were reported in MAPTRx-treated patients. Dose-dependent reduction in the CSF total-tau concentration was observed with greater than 50% mean reduction from baseline at 24 weeks post-last dose in the 60 mg (four doses) and 115 mg (two doses) MAPTRx groups. Clinicaltrials.gov registration number: NCT03186989 .

Pelizaeus-Merzbacher Disease: A Caregiver Assessment of Disease Impact.

Pelizaeus-Merzbacher disease is a rare X-linked leukodystrophy accompanied by central nervous system hypomyelination with a spectrum of clinical phenotypes. This is the first survey of caregivers of individuals with Pelizaeus-Merzbacher disease to investigate the presenting symptoms, path to diagnosis, identity and impact of most bothersome symptoms, and needs that future treatment should address. One hundred participants completed the survey. Results from this survey demonstrate that the majority of Pelizaeus-Merzbacher disease symptoms manifest before 2 years of age and commonly include deficits in gross and fine motor skills, speech, and communication. Caregivers rated difficulty crawling, standing, or walking as the most bothersome symptoms due to Pelizaeus-Merzbacher disease, with constipation and difficulty with sleep, manual dexterity, and speech and communication rated nearly as high. The most important treatment goals for caregivers were improved mobility and communication. The survey findings present a caregiver perspective of the impact of symptoms in Pelizaeus-Merzbacher disease and provide helpful guidance to affected families, physicians, and drug developers on the often-long path to diagnosis and the unmet medical needs of this patient population.

PreSERVE-AMI: A Randomized, Double-Blind, Placebo-Controlled Clinical Trial of Intracoronary Administration of Autologous CD34+ Cells in Patients With Left Ventricular Dysfunction Post STEMI.

RATIONALE: Despite direct immediate intervention and therapy, ST-segment-elevation myocardial infarction (STEMI) victims remain at risk for infarct expansion, heart failure, reinfarction, repeat revascularization, and death. OBJECTIVE: To evaluate the safety and bioactivity of autologous CD34+ cell (CLBS10) intracoronary infusion in patients with left ventricular dysfunction post STEMI. METHODS AND RESULTS: Patients who underwent successful stenting for STEMI and had left ventricular dysfunction (ejection fraction≤48%) ≥4 days poststent were eligible for enrollment. Subjects (N=161) underwent mini bone marrow harvest and were randomized 1:1 to receive (1) autologous CD34+ cells (minimum 10 mol/L±20% cells; N=78) or (2) diluent alone (N=83), via intracoronary infusion. The primary safety end point was adverse events, serious adverse events, and major adverse cardiac event. The primary efficacy end point was change in resting myocardial perfusion over 6 months. No differences in myocardial perfusion or adverse events were observed between the control and treatment groups, although increased perfusion was observed within each group from baseline to 6 months (P<0.001). In secondary analyses, when adjusted for time of ischemia, a consistently favorable cell dose-dependent effect was observed in the change in left ventricular ejection fraction and infarct size, and the duration of time subjects was alive and out of hospital (P=0.05). At 1 year, 3.6% (N=3) and 0% deaths were observed in the control and treatment group, respectively. CONCLUSIONS: This PreSERVE-AMI (Phase 2, randomized, double-blind, placebo-controlled trial) represents the largest study of cell-based therapy for STEMI completed in the United States and provides evidence supporting safety and potential efficacy in patients with left ventricular dysfunction post STEMI who are at risk for death and major morbidity. CLINICAL TRIAL REGISTRATION: URL: http://www.clinicaltrials.gov. Unique identifier: NCT01495364.

The RENEW Trial: Efficacy and Safety of Intramyocardial Autologous CD34(+) Cell Administration in Patients With Refractory Angina.

OBJECTIVES: This study tested whether intramyocardial (IM) administration of mobilized, purified autologous CD34(+) cells would improve total exercise time (TET) and angina frequency in patients with refractory angina. BACKGROUND: IM administration of autologous CD34(+) cells has been associated consistently with improvements in functional capacity and angina symptoms in early phase clinical trials. METHODS: RENEW (Efficacy and Safety of Targeted Intramyocardial Delivery of Auto CD34+ Stem Cells for Improving Exercise Capacity in Subjects With Refractory Angina) was a randomized, double-blind, multicenter trial comparing IM CD34(+) administration with no intervention (open-label standard of care) or IM placebo injections (active control). The primary efficacy endpoint was change in TET at 12 months. Key secondary endpoints include changes in angina frequency at 3, 6, and 12 months, and TET at 3 and 6 months. The key safety analysis was the incidence of major adverse cardiovascular events through 24 months. RESULTS: The sponsor terminated the study for strategic considerations after enrollment of 112 of planned 444 patients. The difference in TET between patients treated with cell therapy versus placebo was 61.0 s at 3 months (95% confidence interval (CI): -2.9 to 124.8; p = 0.06), 46.2 s at 6 months (95% CI: -28.0 to 120.4; p = 0.22), and 36.6 s at 12 months (95% CI: -56.1 to 129.2; p = 0.43); angina frequency was improved at 6 months (relative risk: 0.63; p = 0.05). Autologous CD34(+) cell therapy seemed to be safe compared with both open-label standard of care and active control (major adverse cardiovascular events 67.9% [standard of care], 42.9% (active control), 46.0% [CD34(+)]). CONCLUSIONS: Due to early termination, RENEW was an incomplete experiment; however, the results were consistent with observations from earlier phase studies. These findings underscore the need for a definitive trial. (Efficacy and Safety of Targeted Intramyocardial Delivery of Auto CD34(+) Stem Cells for Improving Exercise Capacity in Subjects With Refractory Angina [RENEW]: NCT01508910).

Autologous CD34+ Cell Therapy for Refractory Angina: 2-Year Outcomes From the ACT34-CMI Study.

An increasing number of patients have refractory angina despite optimal medical therapy and are without further revascularization options. Preclinical studies indicate that human CD34+ stem cells can stimulate new blood vessel formation in ischemic myocardium, improving perfusion and function. In ACT34-CMI (N = 167), patients treated with autologous CD34+ stem cells had improvements in angina and exercise time at 6 and 12 months compared to placebo; however, the longer-term effects of this treatment are unknown. ACT34 was a phase II randomized, double-blind, placebo-controlled clinical trial comparing placebo, low dose (1 × 105 CD34/kg body weight), and high dose (5 × 105 CD34/kg) using intramyocardial delivery into the ischemic zone following NOGA® mapping. To obtain longer-term safety and efficacy in these patients, we compiled data of major adverse cardiac events (MACE; death, myocardial infarction, acute coronary syndrome, or heart failure hospitalization) up to 24 months as well as angina and quality of life assessments in patients who consented for 24-month follow-up. A total of 167 patients with class III-IV refractory angina were randomized and completed the injection procedure. The low-dose-treated patients had a significant reduction in angina frequency (p = 0.02, 0.035) and improvements in exercise tolerance testing (ETT) time (p = 0.014, 0.017) compared to the placebo group at 6 and 12 months. At 24 months, patients treated with both low-and high-dose CD34+ cells had significant reduction in angina frequency (p = 0.03). At 24 months, there were a total of seven deaths (12.5%) in the control group versus one (1.8%) in the low-dose and two (3.6%) in the high-dose (p = 0.08) groups. At 2 years, MACE occurred at a rate of 33.9%, 21.8%, and 16.2% in control, low-, and high-dose patients, respectively (p = 0.08). Autologous CD34+ cell therapy was associated with persistent improvement in angina at 2 years and a trend for reduction in mortality in no-option patients with refractory angina.

A phase 3, randomized, double-blinded, active-controlled, unblinded standard of care study assessing the efficacy and safety of intramyocardial autologous CD34+ cell administration in patients with refractory angina: design of the RENEW study.

Preclinical trials indicate that CD34+ cells represent an effective angiogenic stem cell component. Early-phase clinical trials suggest that intramyocardial administration of autologous CD34+ cells may improve functional capacity and symptoms of angina. RENEW is a pivotal phase 3 trial designed to determine the efficacy of granulocyte colony-stimulating factor (G-CSF)-mobilized CD34+ stem cells for the treatment for patients with refractory angina and chronic myocardial ischemia. Patients (n = 444) receiving maximally tolerated antianginal therapies and lacking conventional revascularization options with Canadian Cardiovascular Society class III or IV angina and ischemia on stress testing will be randomized 2:1:1 to cell therapy (G-CSF-mediated stem cell mobilization, apheresis, and intramyocardial injection of 1 × 10(5) autologous CD34(+) cells/kg), active control (G-CSF-mediated stem cell mobilization, apheresis, and intramyocardial placebo injection), or open-label standard of care. The primary efficacy end point is change in exercise treadmill time in the treated vs active control patients, with 90% power to detect a 60-second difference in exercise time between cell-treated (n = 200) and active control (n = 100) patients. Key secondary end points include total number of anginal episodes per week and the incidence of independently adjudicated major adverse cardiac events and serious adverse events. RENEW will be the first adequately powered study aimed at definitively determining the efficacy of a cell therapy (intramyocardially delivered autologous CD34+ cells) for improvement of functional capacity in patients with refractory angina.

A randomized, controlled pilot study of autologous CD34+ cell therapy for critical limb ischemia.

BACKGROUND: Critical limb ischemia portends a risk of major amputation of 25% to 35% within 1 year of diagnosis. Preclinical studies provide evidence that intramuscular injection of autologous CD34+ cells improves limb perfusion and reduces amputation risk. In this randomized, double-blind, placebo-controlled pilot study, we evaluated the safety and efficacy of intramuscular injections of autologous CD34+ cells in subjects with moderate or high-risk critical limb ischemia, who were poor or noncandidates for surgical or percutaneous revascularization (ACT34-CLI). METHODS AND RESULTS: Twenty-eight critical limb ischemia subjects were randomized and treated: 7 to 1 × 10(5) (low-dose) and 9 to 1 × 10(6) (high-dose) autologous CD34+ cells/kg; and 12 to placebo (control). Intramuscular injections were distributed into 8 sites within the ischemic lower extremity. At 6 months postinjection, 67% of control subjects experienced a major or minor amputation versus 43% of low-dose and 22% of high-dose cell-treated subjects (P=0.137). This trend continued at 12 months, with 75% of control subjects experiencing any amputation versus 43% of low-dose and 22% of high-dose cell-treated subjects (P=0.058). Amputation incidence was lower in the combined cell-treated groups compared with control group (6 months: P=0.125; 12 months: P=0.054), with the low-dose and high-dose groups individually showing trends toward improved amputation-free survival at 6 months and 12 months. No adverse safety signal was associated with cell administration. CONCLUSIONS: This study provides evidence that intramuscular administration of autologous CD34+ cells was safe in this patient population. Favorable trends toward reduced amputation rates in cell-treated versus control subjects were observed. These findings warrant further exploration in later-phase clinical trials. CLINICAL TRIAL REGISTRATION: URL: http://www.clinicaltrials.gov. Unique identifier: NCT00616980.

Incidence and clinical significance of cardiac biomarker elevation during stem cell mobilization, apheresis, and intramyocardial delivery: an analysis from ACT34-CMI.

BACKGROUND: Cell therapy is a promising therapeutic for a variety of cardiovascular conditions including refractory angina. Elevation of cardiac biomarkers during cell delivery has been frequently described, but the clinical implications have never been studied. METHODS: ACT34-CMI was a randomized double-blind study assessing the use of intramyocardial delivery of autologous CD34(+) cells for the treatment of refractory angina. Patients (n = 167) underwent G-CSF-mediated (5 µg/[kg day] × 5 days) stem cell mobilization, apheresis, and intramyocardial injection of 1 × 10(5)/kg or 5 × 10(5)/kg CD34(+) cells or placebo. Troponin and creatinine kinase MB were assessed at baseline (n = 161), after cell mobilization and apheresis (n = 153 and 143, respectively), and post-intramyocardial injection (n = 155 and 141, respectively). Major adverse cardiac events (MACE) included death, myocardial infarction, acute congestive heart failure, urgent revascularization, or sustained ventricular arrhythmia. RESULTS: Seven (4.3%) subjects had troponin above the upper limits of normal (ULN) at baseline. Thirty-four (22.2%) and 11 (7.2%) subjects had troponin levels > ULN or >3× ULN after cell mobilization and apheresis, whereas 72 (46.1%) and 39 (25.2%) subjects had troponin elevations > ULN or >3× ULN, respectively, after intramyocardial injections. Age, but no other preprocedural factors, was predictive of troponin elevation. Periprocedural troponin elevation was not associated with an increased risk of MACE during 1 year, especially in cell therapy-treated patients. CONCLUSIONS: Troponin elevation is common during stem cell harvesting and intramyocardial administration, is usually asymptomatic, and does not appear to be associated with long-term MACE in subjects undergoing stem cell mobilization and intramyocardial injection.

Activation of protease activated receptor 1 increases the excitability of the dentate granule neurons of hippocampus.

Protease activated receptor-1 (PAR1) is expressed in multiple cell types in the CNS, with the most prominent expression in glial cells. PAR1 activation enhances excitatory synaptic transmission secondary to the release of glutamate from astrocytes following activation of astrocytically-expressed PAR1. In addition, PAR1 activation exacerbates neuronal damage in multiple in vivo models of brain injury in a manner that is dependent on NMDA receptors. In the hippocampal formation, PAR1 mRNA appears to be expressed by a subset of neurons, including granule cells in the dentate gyrus. In this study we investigate the role of PAR activation in controlling neuronal excitability of dentate granule cells. We confirm that PAR1 protein is expressed in neurons of the dentate cell body layer as well as in astrocytes throughout the dentate. Activation of PAR1 receptors by the selective peptide agonist TFLLR increased the intracellular Ca2+ concentration in a subset of acutely dissociated dentate neurons as well as non-neuronal cells. Bath application of TFLLR in acute hippocampal slices depolarized the dentate gyrus, including the hilar region in wild type but not in the PAR1-/- mice. PAR1 activation increased the frequency of action potential generation in a subset of dentate granule neurons; cells in which PAR1 activation triggered action potentials showed a significant depolarization. The activation of PAR1 by thrombin increased the amplitude of NMDA receptor-mediated component of EPSPs. These data suggest that activation of PAR1 during normal function or pathological conditions, such as during ischemia or hemorrhage, can increase the excitability of dentate granule cells.

The serine protease plasmin cleaves the amino-terminal domain of the NR2A subunit to relieve zinc inhibition of the N-methyl-D-aspartate receptors.

Zinc is hypothesized to be co-released with glutamate at synapses of the central nervous system. Zinc binds to NR1/NR2A N-methyl-d-aspartate (NMDA) receptors with high affinity and inhibits NMDAR function in a voltage-independent manner. The serine protease plasmin can cleave a number of substrates, including protease-activated receptors, and may play an important role in several disorders of the central nervous system, including ischemia and spinal cord injury. Here, we demonstrate that plasmin can cleave the native NR2A amino-terminal domain (NR2A(ATD)), removing the functional high affinity Zn(2+) binding site. Plasmin also cleaves recombinant NR2A(ATD) at lysine 317 (Lys(317)), thereby producing a approximately 40-kDa fragment, consistent with plasmin-induced NR2A cleavage fragments observed in rat brain membrane preparations. A homology model of the NR2A(ATD) predicts that Lys(317) is near the surface of the protein and is accessible to plasmin. Recombinant expression of NR2A with an amino-terminal deletion at Lys(317) is functional and Zn(2+) insensitive. Whole cell voltage-clamp recordings show that Zn(2+) inhibition of agonist-evoked NMDA receptor currents of NR1/NR2A-transfected HEK 293 cells and cultured cortical neurons is significantly reduced by plasmin treatment. Mutating the plasmin cleavage site Lys(317) on NR2A to alanine blocks the effect of plasmin on Zn(2+) inhibition. The relief of Zn(2+) inhibition by plasmin occurs in PAR1(-/-) cortical neurons and thus is independent of interaction with protease-activated receptors. These results suggest that plasmin can directly interact with NMDA receptors, and plasmin may increase NMDA receptor responses through disruption or removal of the amino-terminal domain and relief of Zn(2+) inhibition.

Plasmin potentiates synaptic N-methyl-D-aspartate receptor function in hippocampal neurons through activation of protease-activated receptor-1.

Protease-activated receptor-1 (PAR1) is activated by a number of serine proteases, including plasmin. Both PAR1 and plasminogen, the precursor of plasmin, are expressed in the central nervous system. In this study we examined the effects of plasmin in astrocyte and neuronal cultures as well as in hippocampal slices. We find that plasmin evokes an increase in both phosphoinositide hydrolysis (EC(50) 64 nm) and Fura-2/AM fluorescence (195 +/- 6.7% above base line, EC(50) 65 nm) in cortical cultured murine astrocytes. Plasmin also activates extracellular signal-regulated kinase (ERK1/2) within cultured astrocytes. The plasmin-induced rise in intracellular Ca(2+) concentration ([Ca(2+)](i)) and the increase in phospho-ERK1/2 levels were diminished in PAR1(-/-) astrocytes and were blocked by 1 microm BMS-200261, a selective PAR1 antagonist. However, plasmin had no detectable effect on ERK1/2 or [Ca(2+)](i) signaling in primary cultured hippocampal neurons or in CA1 pyramidal cells in hippocampal slices. Plasmin (100-200 nm) application potentiated the N-methyl-D-aspartate (NMDA) receptor-dependent component of miniature excitatory postsynaptic currents recorded from CA1 pyramidal neurons but had no effect on alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate- or gamma-aminobutyric acid receptor-mediated synaptic currents. Plasmin also increased NMDA-induced whole cell receptor currents recorded from CA1 pyramidal cells (2.5 +/- 0.3-fold potentiation over control). This effect was blocked by BMS-200261 (1 microm; 1.02 +/- 0.09-fold potentiation over control). These data suggest that plasmin may serve as an endogenous PAR1 activator that can increase [Ca(2+)](i) in astrocytes and potentiate NMDA receptor synaptic currents in CA1 pyramidal neurons.

Protease-activated receptor-1 in human brain: localization and functional expression in astrocytes.

Protease-activated receptor-1 (PAR1) is a G-protein coupled receptor that is proteolytically activated by blood-derived serine proteases. Although PAR1 is best known for its role in coagulation and hemostasis, recent findings demonstrate that PAR1 activation has actions in the central nervous system (CNS) apart from its role in the vasculature. Rodent studies have demonstrated that PAR1 is expressed throughout the brain on neurons and astrocytes. PAR1 activation in vitro and in vivo appears to influence neurodegeneration and neuroprotection in animal models of stroke and brain injury. Because of increasing evidence that PAR1 has important and diverse roles in the CNS, we explored the protein localization and function of PAR1 in human brain. PAR1 is most intensely expressed in astrocytes of white and gray matter and moderately expressed in neurons. PAR1 and GFAP co-localization demonstrates that PAR1 is expressed on the cell body and on astrocytic endfeet that invest capillaries. PAR1 activation in the U178MG human glioblastoma cell line increased PI hydrolysis and intracellular Ca(2+), indicating that PAR1 is functional in human glial-derived tumor cells. Primary cultures of human astrocytes and human glioblastoma cells respond to PAR1 activation by increasing intracellular Ca(2+). Together, these results demonstrate that PAR1 is expressed in human brain and functional in glial tumors and cultures derived from it. Because serine proteases may enter brain tissue and activate PAR1 when the blood brain barrier (BBB) breaks down, pharmacological manipulation of PAR1 signaling may provide a potential therapeutic target for neuroprotection in human neurological disorders.

The contribution of protease-activated receptor 1 to neuronal damage caused by transient focal cerebral ischemia.

The serine proteases tissue plasminogen activator, plasmin, and thrombin and their receptors have previously been suggested to contribute to neuronal damage in certain pathological situations. Here we demonstrate that mice lacking protease-activated receptor 1 (PAR1) have a 3.1-fold reduction in infarct volume after transient focal cerebral ischemia. Intracerebroventricular injection of PAR1 antagonist BMS-200261 reduced infarct volume 2.7-fold. There are no detectable differences between PAR1-/- and WT mice in cerebrovascular anatomy, capillary density, or capillary diameter, demonstrating that the neuroprotective phenotype is not likely related to congenital abnormalities in vascular development. We also show that the exogenously applied serine proteases thrombin, plasmin, and tissue plasminogen activator can activate PAR1 signaling in brain tissue. These data together suggest that if blood-derived serine proteases that enter brain tissue in ischemic situations can activate PAR1, this sequence of events may contribute to the harmful effects observed. Furthermore, PAR1 immunoreactivity is present in human brain, suggesting that inhibition of PAR1 may provide a novel potential therapeutic strategy for decreasing neuronal damage associated with ischemia and blood-brain barrier breakdown.