Craniofacial anomalies in schizophrenia-relevant GFAP.HMOX10-12m mice.

Subtle craniofacial dysmorphology has been reported in schizophrenia patients. This dysmorphology includes midline facial elongation, frontonasal anomalies and a sexually dimorphic deviation from normal directional asymmetry of the face, with male patients showing reduced and female patients showing enhanced facial asymmetry relative to healthy control subjects. GFAP.HMOX10-12m transgenic mice (Mus musculus) that overexpress heme oxygenase-1 in astrocytes recapitulate many schizophrenia-relevant neurochemical, neuropathological and behavioral features. As morphogenesis of the brain, skull and face are highly interrelated, we hypothesized that GFAP.HMOX10-12m mice may exhibit craniofacial anomalies similar to those reported in persons with schizophrenia. We examined craniofacial anatomy in male GFAP.HMOX10-12m mice and wild-type control mice at the early adulthood age of 6-8 months. We used computer vision techniques for the extraction and analysis of mouse head shape parameters from systematically acquired 2D digital images, and confirmed our results with landmark-based geometric morphometrics. We performed skull bone morphometry using digital calipers to take linear distance measurements between known landmarks. Relative to controls, adult male GFAP.HMOX10-12m mice manifested craniofacial dysmorphology including elongation of the nasal bones, alteration of head shape anisotropy and reduction of directional asymmetry in facial shape features. These findings demonstrate that GFAP.HMOX10-12m mice exhibit craniofacial anomalies resembling those described in schizophrenia patients, implicating heme oxygenase-1 in their development. As a preclinical mouse model, GFAP.HMOX10-12m mice provide a novel opportunity for the study of the etiopathogenesis of craniofacial and other anomalies in schizophrenia and related disorders.

Heme oxygenase-1 in blood and saliva during acute psychosis: A pilot study.

Despite the extensive prevalence of psychosis and schizophrenia spectrum disorders, their biological underpinnings remain largely unexplained. Recently, the overproduction of heme oxygenase-1 (HO-1), an enzyme that catalyzes the degradation of heme, was associated with oxidative stress and a neurologic phenotype similar to schizophrenia in transgenic mice. We sought to evaluate, by comparing patients experiencing an acute psychotic episode, and age/sex-matched healthy control participants, whether there was an association between HO-1 overexpression and psychosis. This cross-sectional pilot study included 16 patients and 17 control participants. Enzyme-linked immunosorbent assay and quantitative real-time polymerase chain reaction were used to quantify HO-1 expression in blood and saliva. Four psychiatric questionnaires were used to measure psychiatric symptoms in participants. Higher levels of salivary HO-1 expression were detected in patients experiencing an acute psychotic episode when compared to control participants (84.01 vs. 61.26 ng/ml, p = 0.026), but plasma and lymphocyte HO-1 expression did not significantly differ between groups. Overexpression of HO-1 in saliva specimens was also positively associated with psychiatric symptom severity and disability. The overexpression of HO-1 in the saliva of patients with psychosis suggests that it may serve as a potential biomarker for this symptom which should be explored in larger clinical trials.

Characterization and heme oxygenase-1 content of extracellular vesicles in human biofluids.

Heme oxygenase-1 (HO-1), a highly inducible stress protein that degrades heme to biliverdin, carbon monoxide, and free ferrous iron, is increased in blood and other biofluids of subjects with various systemic and neurological disorders. HO-1 does not contain an N-terminal signal peptide and the mechanism responsible for its secretion remains unknown. Extracellular vesicles (EVs) are membrane-bound inclusions that transport microRNAs, messenger RNAs, lipids, and proteins among diverse cellular and extracellular compartments. The objective of the current study was to determine whether EVs in human biofluids contain HO-1, and whether the latter may be transported in EVs from brain to periphery. Total, L1 cell adhesion molecule protein (L1CAM)-enriched (neuron-derived), and glutamate aspartate transporter 1 (GLAST)-enriched (astrocyte-derived) EVs were purified from five different human biofluids (saliva [n = 40], plasma [n = 14], serum [n = 10], urine [n = 10], and cerebrospinal fluid [n = 11]) using polymer precipitation and immuno-affinity-based capture methods. L1CAM-enriched, GLAST-enriched, and L1CAM/GLAST-depleted (LGD) EV, along with EV-depleted (EVD), fractions were validated by nanoparticle tracking analysis, enzyme-linked immunosorbent assay (ELISA), and western blot. HO-1 was assayed in all fractions using ELISA and western blot. The majority of HO-1 protein was localized to LGD, L1CAM-enriched, and GLAST-enriched EVs of all human biofluids surveyed after adjusting for age and sex, with little HO-1 protein detected in EVD fractions. HO-1 protein in human biofluids is predominantly localized to EV compartments. A substantial proportion of EV HO-1 in peripheral human biofluids is derived from the central nervous system and may contribute to the systemic manifestations of various neurological conditions.

Strategic Timing of Glial HMOX1 Expression Results in Either Schizophrenia-Like or Parkinsonian Behavior in Mice.

Aims: In this original research communication, we assess the impact of shifting the window of glial HMOX1 overexpression in mice from early-to-midlife to mid-to-late life, resulting in two disparate conditions modeling schizophrenia (SCZ) and Parkinson's disease (PD). Mesolimbic hyperdopaminergia is a widely accepted feature of SCZ, while nigrostriatal hypodopaminergia is the sine qua non of idiopathic PD. Although the advent of parkinsonian features in SCZ patients after treatment with antidopaminergic agents is intuitive, subtle features of parkinsonism commonly observed in young, drug-naïve schizophrenics are not. Similarly, emergent psychosis in PD subjects receiving levodopa replacement is not unusual, whereas spontaneous hallucinosis in nonmedicated persons with idiopathic PD is enigmatic. Investigations using GFAP.HMOX1 mice may shed light on these clinical paradoxes. Results: Astroglial heme oxygenase-1 (HO-1) overexpression in mice throughout embryogenesis until 6 or 12 months of age resulted in hyperdopaminergia, hyperkinesia/stereotypy ameliorated with clozapine, deficient prepulse inhibition of the acoustic startle response, reduced preference for social novelty, impaired nest building, and cognitive dysfunction reminiscent of SCZ. On the contrary, astroglial HO-1 overexpression between 8.5 and 19 months of age yielded a PD-like behavioral phenotype with hypodopaminergia, altered gait, locomotor incoordination, and reduced olfaction. Innovation: We conjecture that region-specific disparities in the susceptibility of dopaminergic and other circuitry to the trophic and degenerative influences of glial HMOX1 induction may permit the concomitant expression of mixed SCZ and PD traits within affected individuals. Conclusion: Elucidation of these converging mechanisms may (i) help better understand disease pathogenesis and (ii) identify HO-1 as a potential therapeutic target in neurodevelopmental and neurodegenerative disorders.

Glial HMOX1 expression promotes central and peripheral α-synuclein dysregulation and pathogenicity in parkinsonian mice.

α-Synuclein is a key player in the pathogenesis of Parkinson disease (PD). Expression of human heme oxygenase-1 (HO-1) in astrocytes of GFAP.HMOX1 transgenic (TG) mice between 8.5 and 19 months of age results in a parkinsonian phenotype characterized by neural oxidative stress, nigrostriatal hypodopaminergia associated with locomotor incoordination, and overproduction of α-synuclein. We identified two microRNAs (miR-), miR-153 and miR-223, that negatively regulate α-synuclein in the basal ganglia of male and female GFAP.HMOX1 mice. Serum concentrations of both miRNAs progressively declined in the wild-type (WT) and GFAP.HMOX1 mice between 11 and 19 months of age. Moreover, at each time point surveyed, circulating levels of miR-153 were significantly lower in the TG animals compared to WT controls, while α-synuclein protein concentrations were elevated in erythrocytes of the GFAP.HMOX1 mice at 19 months of age relative to WT values. Primary WT neurons co-cultured with GFAP.HMOX1 astrocytes exhibited enhanced protein oxidation, mitophagy and apoptosis, aberrant expression of genes regulating the dopaminergic phenotype, and an imbalance in gene expression profiles governing mitochondrial fission and fusion. Many, but not all, of these neuronal abnormalities were abrogated by small interfering RNA (siRNA) knockdown of α-synuclein, implicating α-synuclein as a potent, albeit partial, mediator of HO-1's neurodystrophic effects in these parkinsonian mice. Overexpression of HO-1 in stressed astroglia has previously been documented in the substantia nigra of idiopathic PD and may promote α-synuclein production and toxicity by downmodulating miR-153 and/or miR-223 both within the CNS and in peripheral tissues.

Evaluation of salivary heme oxygenase-1 as a potential biomarker of early Parkinson's disease.

BACKGROUND AND HYPOTHESIS: To date, there are no chemical analytes, including biochemical indices of oxidative stress, metabolites of α-synuclein protein, and differential protein expression patterns on proteomic profiling, for use in clinics as a diagnostic biomarker of idiopathic PD. OBJECTIVES: Heme oxygenase-1 has been implicated in the pathogenesis of PD. The objective of this study is to ascertain whether salivary heme oxygenase-1 may serve as a biomarker for early idiopathic PD. METHODS: Fifty-eight PD patients and 59 non-neurological disease controls were recruited. Levels of heme oxygenase-1 expression were assayed using enzyme-linked immunosorbent assay and western blot analysis of whole, unstimulated saliva. Analyses were adjusted by sex, l-dopa exposure, and relevant comorbidities. RESULTS: We documented: (1) the presence of 32-kDa heme oxygenase-1 protein in human saliva; (2) significantly higher mean heme oxygenase-1 protein concentrations in saliva of PD patients relative to control values; (3) no variability in salivary heme oxygenase-1 levels with sex, age, l-dopa equivalence, or comorbidities; and (4) significantly higher mean salivary heme oxygenase-1 concentrations in patients with H & Y stage 1 PD (early) than control subjects and stage 2 and stage 3 PD patients. The area under the receiver operating characteristic curve that separated controls from PD H & Y stage 1 was 76% (95% confidence interval: 63-90). CONCLUSIONS: Salivary heme oxygenase-1 concentrations may provide a useful, noninvasive, and relatively inexpensive biomarker of early idiopathic PD. © 2018 International Parkinson and Movement Disorder Society.

Parkinsonian features in aging GFAP.HMOX1 transgenic mice overexpressing human HO-1 in the astroglial compartment.

Epigenetic influences mediating brain iron deposition, oxidative mitochondrial injury, and macroautophagy in Parkinson disease and related conditions remain enigmatic. Here, we show that selective overexpression of the stress protein, heme oxygenase-1 (HO-1) in astrocytes of GFAP.HMOX1 transgenic mice between 8.5 and 19 months of age results in nigrostriatal hypodopaminergia associated with locomotor incoordination and stereotypy; downregulation of tyrosine hydroxylase, DAT, LMX1B, Nurr1, Pitx3 and DJ-1 mRNA and/or protein; overproduction of α-synuclein and ubiquitin; oxidative stress; basal ganglia siderosis; mitochondrial damage/mitophagy; and augmented GABAergic systems (increased GABA, GAD67 and reelin). The neurophenotype of these GFAP.HMOX18.5-19m mice is highly consistent with parkinsonism and differs dramatically from the schizophrenia-like features previously documented in younger GFAP.HMOX10-12m mice. Common stressors may elicit either early-onset developmental (schizophrenia) or later-life degenerative (PD) brain disorders depending on whether the glial HO-1 response is engaged prior to or following the maturation of dopaminergic circuitry. Curtailment of glial HO-1 transduction at strategic points of the life course may confer neuroprotection in human degenerative and developmental central nervous system disorders.

Cysteine-rich whey protein isolate (Immunocal®) ameliorates deficits in the GFAP.HMOX1 mouse model of schizophrenia.

Schizophrenia is a neuropsychiatric disorder that features neural oxidative stress and glutathione (GSH) deficits. Oxidative stress is augmented in brain tissue of GFAP.HMOX1 transgenic mice which exhibit schizophrenia-relevant characteristics. The whey protein isolate, Immunocal® serves as a GSH precursor upon oral administration. In this study, we treated GFAP.HMOX1 transgenic mice daily with either Immunocal (33mg/ml drinking water) or equivalent concentrations of casein (control) between the ages of 5 and 6.5 months. Immunocal attenuated many of the behavioral, neurochemical and redox abnormalities observed in GFAP.HMOX1 mice. In addition to restoring GSH homeostasis in the CNS of the transgenic mice, the whey protein isolate augmented GSH reserves in the brains of wild-type animals. These results demonstrate that consumption of whey protein isolate augments GSH stores and antioxidant defenses in the healthy and diseased mammalian brain. Whey protein isolate supplementation (Immunocal) may constitute a safe and effective modality for the management of schizophrenia, an unmet clinical imperative.

Astroglial heme oxygenase-1 and the origin of corpora amylacea in aging and degenerating neural tissues.

BACKGROUND: Corpora amylacea (CA) are glycoproteinaceous (predominantly glial and extracellular) inclusions that accumulate in normal aging brain and, to a greater extent, in Alzheimer disease (AD). Previous pharmacological evidence suggested that up-regulation of endogenous heme oxygenase-1 (HO-1) in astrocytes promotes transformation of normal mitochondria to CA-like inclusions. Here, we determined whether 1) HMOX1 transfection fosters the accumulation of CA-like inclusions in cultured rat astroglia; 2) the HMOX1 transgene promotes CA formation in the brains of aging GFAP.HMOX1 mice; and 3) brain mitochondrial damage and CA biogenesis are augmented in persons with mild cognitive impairment (MCI), a harbinger of AD. METHODS: CA were ascertained in (i) neonatal rat astroglia transfected with flag-tagged human HO-1 cDNA, (ii) brain sections derived from 19month-old GFAP.HMOX1 and wild-type (WT) mice, and (iii) post-mortem hippocampal sections from individuals with mild (MCI) and no cognitive impairment (NCI) after staining with PAS or antisera against HO-1, ubiquitin (Ub), manganese superoxide dismutase (MnSOD), and α-synuclein or tyrosine hydroxylase (TH). RESULTS: HMOX1 transfection induced cytoplasmic vacuolation and the accumulation of PAS+ inclusions in cultured astroglia. Numerous CA-like inclusions stained with PAS and immunoreactive for HO-1, Ub and MnSOD were observed in the brains of GFAP.HMOX1 mice, but were rarely encountered in age-matched, WT controls. Numbers of HO-1-positive CA were significantly increased in certain hippocampal strata of MCI subjects relative to NCI preparations. MnSOD and Ub proteins co-localized to CA in both the control and MCI specimens. CONCLUSIONS: HO-1 promotes mitochondrial damage and CA biogenesis in astrocyte cultures and in the intact aging brain. CA formation is enhanced in the MCI hippocampus and thus occurs relatively early in the pathogenesis of AD. Glial HO-1 suppression may attenuate bioenergetic failure and slow disease progression in AD and other neurodegenerative conditions featuring accelerated accumulation of CA.

Schizophrenia-like features in transgenic mice overexpressing human HO-1 in the astrocytic compartment.

Delineation of key molecules that act epigenetically to transduce diverse stressors into established patterns of disease would facilitate the advent of preventive and disease-modifying therapeutics for a host of neurological disorders. Herein, we demonstrate that selective overexpression of the stress protein heme oxygenase-1 (HO-1) in astrocytes of novel GFAP.HMOX1 transgenic mice results in subcortical oxidative stress and mitochondrial damage/autophagy; diminished neuronal reelin content (males); induction of Nurr1 and Pitx3 with attendant suppression of their targeting miRNAs, 145 and 133b; increased tyrosine hydroxylase and α-synuclein expression with downregulation of the targeting miR-7b of the latter; augmented dopamine and serotonin levels in basal ganglia; reduced D1 receptor binding in nucleus accumbens; axodendritic pathology and altered hippocampal cytoarchitectonics; impaired neurovascular coupling; attenuated prepulse inhibition (males); and hyperkinetic behavior. The GFAP.HMOX1 neurophenotype bears resemblances to human schizophrenia and other neurodevelopmental conditions and implicates glial HO-1 as a prime transducer of inimical (endogenous and environmental) influences on the development of monoaminergic circuitry. Containment of the glial HO-1 response to noxious stimuli at strategic points of the life cycle may afford novel opportunities for the effective management of human neurodevelopmental and neurodegenerative conditions.

Unregulated brain iron deposition in transgenic mice over-expressing HMOX1 in the astrocytic compartment.

The mechanisms responsible for pathological iron deposition in the aging and degenerating mammalian CNS remain poorly understood. The stress protein, HO-1 mediates the degradation of cellular heme to biliverdin/bilirubin, free iron, and CO and is up-regulated in the brains of persons with Alzheimer's disease and Parkinson's disease. HO-1 induction in primary astroglial cultures promotes deposition of non-transferrin iron, mitochondrial damage and macroautophagy, and predisposes cocultured neuronal elements to oxidative injury. To gain a better appreciation of the role of glial HO-1 in vivo, we probed for aberrant brain iron deposition using Perls' method and dynamic secondary ion mass spectrometry in novel, conditional GFAP.HMOX1 transgenic mice that selectively over-express human HO-1 in the astrocytic compartment. At 48 weeks, the GFAP.HMOX1 mice exhibited increased deposits of glial iron in hippocampus and other subcortical regions without overt changes in iron-regulatory and iron-binding proteins relative to age-matched wild-type animals. Dynamic secondary ion mass spectrometry revealed abundant FeO⁻ signals in the transgenic, but not wild-type, mouse brain that colocalized to degenerate mitochondria and osmiophilic cytoplasmic inclusions (macroautophagy) documented by TEM. Sustained up-regulation of HO-1 in astrocytes promotes pathological brain iron deposition and oxidative mitochondrial damage characteristic of Alzheimer's disease-affected neural tissues. Curtailment of glial HO-1 hyperactivity may limit iron-mediated cytotoxicity in aging and degenerating neural tissues.

ARAC--The Montreal Jewish General Hospital Alzheimer Risk Assessment Clinic.

INTRODUCTION: In parallel with robust efforts world-wide to develop effective neuroprotection for established disease, resources are being mobilized to delineate risk factors and implement preventive measures in a concerted effort to forestall the anticipated Alzheimer disease (AD) epidemic. A review of heritable and 'acquired' dementia risk factors, many operating at midlife, is presented in a companion paper. OBJECTIVES: In 2009, an Alzheimer Risk Assessment Clinic (ARAC) was established at the Jewish General Hospital (Montreal) to address the concerns increasingly being voiced by active middle-aged individuals at risk for AD. A positive family history of AD and/or perceived changes in personal cognitive function (predominantly short-term memory) are main reasons for referral. The primary objectives of ARAC are to (i) ascertain, inform and mitigate the risks of developing AD in cognitively-healthy persons aged 40-65 based on best available medical and epidemiological evidence, (ii) conduct scientific research on midlife dementia risk and prevention in this population and (iii) provide instruction in dementia risk assessment and management to health professionals, clinical/research fellows, medical residents and students. ARAC infrastructure, evaluation protocol, risk profile classification scheme, interventions, knowledge dissemination program, case vignettes, and seminal research projects are described. CONCLUSIONS: It is hoped that ARAC and similar initiatives will help prevent or delay dementia by innovating effective interventions based on increasingly nuanced estimation of modifiable AD risk in presymptomatic persons.

HO-1-mediated macroautophagy: a mechanism for unregulated iron deposition in aging and degenerating neural tissues.

Oxidative stress, deposition of non-transferrin iron, and mitochondrial insufficiency occur in the brains of patients with Alzheimer disease (AD) and Parkinson disease (PD). We previously demonstrated that heme oxygenase-1 (HO-1) is up-regulated in AD and PD brain and promotes the accumulation of non-transferrin iron in astroglial mitochondria. Herein, dynamic secondary ion mass spectrometry (SIMS) and other techniques were employed to ascertain (i) the impact of HO-1 over-expression on astroglial mitochondrial morphology in vitro, (ii) the topography of aberrant iron sequestration in astrocytes over-expressing HO-1, and (iii) the role of iron regulatory proteins (IRP) in HO-1-mediated iron deposition. Astroglial hHO-1 over-expression induced cytoplasmic vacuolation, mitochondrial membrane damage, and macroautophagy. HO-1 promoted trapping of redox-active iron and sulfur within many cytopathological profiles without impacting ferroportin, transferrin receptor, ferritin, and IRP2 protein levels or IRP1 activity. Thus, HO-1 activity promotes mitochondrial macroautophagy and sequestration of redox-active iron in astroglia independently of classical iron mobilization pathways. Glial HO-1 may be a rational therapeutic target in AD, PD, and other human CNS conditions characterized by the unregulated deposition of brain iron.

Brain erythropoietin receptor expression in Alzheimer disease and mild cognitive impairment.

Cellular mechanisms conferring neuroprotection in the brains of patients with Alzheimer disease (AD) remain incompletely understood. Erythropoietin (Epo) and the erythropoietin receptor (EpoR) are expressed in neural tissues and protect against oxidative and other stressors in various models of brain injury and disease. Our objective in this study was to determine whether EpoR is upregulated in the brains of persons with sporadic AD and mild cognitive impairment (MCI). Postmortem hippocampus and temporal cortex from subjects with AD, MCI, and no cognitive impairment (NCI) were procured from the Religious Orders Study. Total immunoreactive EpoR protein was determined by Western blotting. Astrocytes expressing immunoreactive EpoR were quantified in 4 temporal and 6 hippocampal regions, and correlated with clinical, neuropsychologic, and neuropathologic indices. Total immunoreactive EpoR protein was markedly increased in AD and MCI temporal cortex versus NCI tissues. Composite measures of glial EpoR expression in temporal cortex layers I to IV were significantly greater in the MCI group compared with the NCI and AD groups. Hippocampal EpoR scores were increased in persons with MCI and AD relative to those with NCI. There was substantial subregional heterogeneity in disease-related EpoR expression patterns in AD and MCI temporal cortex and hippocampus. There was no association of EpoR-positive astrocytes with summary measures of global cognition or AD pathology. We conclude that upregulation of EpoR in temporal cortical and hippocampal astrocytes is an early, potentially neuroprotective, event in the pathogenesis of sporadic AD.

Characterization of alpha1-antitrypsin as a heme oxygenase-1 suppressor in Alzheimer plasma.

Heme oxygenase-1 (HO-1) mRNA and protein levels are diminished in Alzheimer disease (AD) blood, cerebrospinal fluid (CSF) and choroid plexus. Herein, the presence of a heme oxygenase-1 suppressor (HOS) factor was ascertained by astroglial bioassay, biochemical techniques and immunofluorescence confocal microscopy. We report significantly augmented plasma HOS activity in AD patients relative to healthy elderly and neurological controls. The HOS factor was determined to be a 50-100 kDa heat-labile, heparin-binding glycoprotein that is unrelated to antioxidant ingestion, plasma total antioxidant capacity, circulating cortisol levels or apolipoprotein E epsilon4 carrier status. HOS bioactivity was recapitulated by exogenous alpha(1)-antitrypsin. alpha(1)-antitrypsin levels were significantly increased in AD plasma and correlated with HOS activity and MMSE scores. alpha(1)-antitrypsin immunodepletion attenuated HOS activity of AD plasma. In AD brain, alpha(1)-antitrypsin immunoreactivity was augmented and co-distributed with HO-1. HOS activity of alpha(1)-antitrypsin may curtail HO-1-dependent derangement of cerebral iron homeostasis and account for diminished HO-1 expression in AD peripheral tissues.

Glial heme oxygenase-1 expression in Alzheimer disease and mild cognitive impairment.

We determined whether oxidative stress is an early event in the pathogenesis of sporadic Alzheimer disease (AD), and correlated oxidative stress with neuropsychological functions and neurofibrillary pathology in AD and mild cognitive impairment (MCI). Oxidative stress was measured as the percentage of astrocytes expressing heme oxygenase-1 (HO-1) in post mortem temporal cortex and hippocampus after dual HO-1/glial fibrillary acidic protein (GFAP) immunohistochemistry. Glial HO-1 expression in the MCI temporal cortex and hippocampus was significantly greater than in the non-demented group and did not differ from AD values. Astroglial HO-1 expression in the temporal cortex was associated with decreased scores for global cognition, episodic memory, semantic memory and working memory. Hippocampal astroglial HO-1 expression was associated with lower scores for global cognition, semantic memory and perceptual speed. Glial HO-1 immunoreactivity in the temporal cortex, but not hippocampus, correlated with the burden of neurofibrillary pathology. Cortical and hippocampal oxidative stress is a very early event in the pathogenesis of sporadic AD and correlates with the development of specific cognitive deficits in this condition.