A murine model of hnRNPH2-related neurodevelopmental disorder reveals a mechanism for genetic compensation by Hnrnph1.

Mutations in HNRNPH2 cause an X-linked neurodevelopmental disorder with features that include developmental delay, motor function deficits, and seizures. More than 90% of patients with hnRNPH2 have a missense mutation within or adjacent to the nuclear localization signal (NLS) of hnRNPH2. Here, we report that hnRNPH2 NLS mutations caused reduced interaction with the nuclear transport receptor Kapß2 and resulted in modest cytoplasmic accumulation of hnRNPH2. We generated 2 knockin mouse models with human-equivalent mutations in Hnrnph2 as well as Hnrnph2-KO mice. Knockin mice recapitulated clinical features of the human disorder, including reduced survival in male mice, impaired motor and cognitive functions, and increased susceptibility to audiogenic seizures. In contrast, 2 independent lines of Hnrnph2-KO mice showed no detectable phenotypes. Notably, KO mice had upregulated expression of Hnrnph1, a paralog of Hnrnph2, whereas knockin mice failed to upregulate Hnrnph1. Thus, genetic compensation by Hnrnph1 may counteract the loss of hnRNPH2. These findings suggest that HNRNPH2-related disorder may be driven by a toxic gain of function or a complex loss of HNRNPH2 function with impaired compensation by HNRNPH1. The knockin mice described here are an important resource for preclinical studies to assess the therapeutic benefit of gene replacement or knockdown of mutant hnRNPH2.

Translational Repression of G3BP in Cancer and Germ Cells Suppresses Stress Granules and Enhances Stress Tolerance.

Stress granules (SGs) are membrane-less ribonucleoprotein condensates that form in response to various stress stimuli via phase separation. SGs act as a protective mechanism to cope with acute stress, but persistent SGs have cytotoxic effects that are associated with several age-related diseases. Here, we demonstrate that the testis-specific protein, MAGE-B2, increases cellular stress tolerance by suppressing SG formation through translational inhibition of the key SG nucleator G3BP. MAGE-B2 reduces G3BP protein levels below the critical concentration for phase separation and suppresses SG initiation. Knockout of the MAGE-B2 mouse ortholog or overexpression of G3BP1 confers hypersensitivity of the male germline to heat stress in vivo. Thus, MAGE-B2 provides cytoprotection to maintain mammalian spermatogenesis, a highly thermosensitive process that must be preserved throughout reproductive life. These results demonstrate a mechanism that allows for tissue-specific resistance against stress and could aid in the development of male fertility therapies.

Selective modulation of the androgen receptor AF2 domain rescues degeneration in spinal bulbar muscular atrophy.

Spinal bulbar muscular atrophy (SBMA) is a motor neuron disease caused by toxic gain of function of the androgen receptor (AR). Previously, we found that co-regulator binding through the activation function-2 (AF2) domain of AR is essential for pathogenesis, suggesting that AF2 may be a potential drug target for selective modulation of toxic AR activity. We screened previously identified AF2 modulators for their ability to rescue toxicity in a Drosophila model of SBMA. We identified two compounds, tolfenamic acid (TA) and 1-[2-(4-methylphenoxy)ethyl]-2-[(2-phenoxyethyl)sulfanyl]-1H-benzimidazole (MEPB), as top candidates for rescuing lethality, locomotor function and neuromuscular junction defects in SBMA flies. Pharmacokinetic analyses in mice revealed a more favorable bioavailability and tissue retention of MEPB compared with TA in muscle, brain and spinal cord. In a preclinical trial in a new mouse model of SBMA, MEPB treatment yielded a dose-dependent rescue from loss of body weight, rotarod activity and grip strength. In addition, MEPB ameliorated neuronal loss, neurogenic atrophy and testicular atrophy, validating AF2 modulation as a potent androgen-sparing strategy for SBMA therapy.

CNS tau efflux via exosomes is likely increased in Parkinson's disease but not in Alzheimer's disease.

INTRODUCTION: Alzheimer's disease (AD) and Parkinson's disease (PD) involve tau pathology. Tau is detectable in blood, but its clearance from neuronal cells and the brain is poorly understood. METHODS: Tau efflux from the brain to the blood was evaluated by administering radioactively labeled and unlabeled tau intracerebroventricularly in wild-type and tau knock-out mice, respectively. Central nervous system (CNS)-derived tau in L1CAM-containing exosomes was further characterized extensively in human plasma, including by single molecule array technology with 303 subjects. RESULTS: The efflux of Tau, including a fraction via CNS-derived L1CAM exosomes, was observed in mice. In human plasma, tau was explicitly identified within L1CAM exosomes. In contrast to AD patients, L1CAM exosomal tau was significantly higher in PD patients than controls and correlated with cerebrospinal fluid tau. CONCLUSIONS: Tau is readily transported from the brain to the blood. The mechanisms of CNS tau efflux are likely different between AD and PD.

Increased CSF E-Selectin in Clinical Alzheimer's Disease without Altered CSF Aß42 and Tau.

Clinically diagnosed Alzheimer's disease (AD) is pathologically heterogeneous. In this multicenter cohort of 215 clinically diagnosed AD patients and 249 controls, E-selectin and vascular cell adhesion molecule 1 (VACM-1) were measured along with amyloid-ß peptide 1-42 (Aß42) and tau. We discovered that E-selectin, a biomarker of endothelial function/vascular injury, was inversely correlated with cerebrospinal fluid (CSF) tau/Aß42 ratio and significantly elevated in clinical AD patients without the typical AD CSF biomarker signature (i.e., low tau/Aß42 ratio) compared to those with the signature. These findings suggest that E-selectin may be an objective biomarker related to vascular mechanisms contributing to dementia.

Diagnostic Values of Cerebrospinal Fluid T-Tau and Aß42 using Meso Scale Discovery Assays for Alzheimer's Disease.

BACKGROUND: Meso Scale Discovery (MSD) recently established electrochemiluminescence-based assays to measure cerebrospinal fluid (CSF) levels of total tau (t-tau) and amyloid-ß 1-42 peptide (Aß42) that can aid in the diagnosis of Alzheimer's disease (AD). The goal of this investigation is to independently evaluate this platform and establish cut-off values of these biomarkers for AD diagnosis. OBJECTIVE: To validate the analytical and clinical performance of the MSD t-tau and Aß42 kits and propose diagnostic cut-off values for the field. METHODS: The analytical performance of the CSF t-tau and Aß42 assays was determined, followed by assessment of diagnostic performance of CSF t-tau, Aß42, and t-tau/Aß42 in three clinically characterized cohorts. RESULTS: Both MSD assays demonstrated consistent and stable analytical performance, as well as resistance to several important pre-analytic variables. Diagnostically, t-tau/Aß42 performed the best. CONCLUSIONS: Our results independently confirm the analytical and clinical performance of the MSD CSF t-tau and Aß42 assays. Based on a large, multi-center, clinically-diagnosed cohort, we propose for the first time candidate diagnostic cut-offs for MSD measured CSF t-tau, Aß42, and t-tau/Aß42. However, these values needs to be refined as more subjects are included and the assays are tested by other laboratories.

Low levels of cerebrospinal fluid complement 3 and factor H predict faster cognitive decline in mild cognitive impairment.

INTRODUCTION: Alzheimer's disease (AD) is characterized by the deposition of tau and amyloid in the brain. Although the core cerebrospinal fluid (CSF) AD biomarkers amyloid ß peptide 1-42 (Aß1-42), total tau (t-tau) and phosphorylated tau 181 (p-tau181) show good diagnostic sensitivity and specificity, additional biomarkers that can aid in preclinical diagnosis or better track disease progression are needed. Activation of the complement system, a pivotal part of inflammation, occurs at very early stages in the AD brain. Therefore, CSF levels of complement proteins that could be linked to cognitive and structural changes in AD may have diagnostic and prognostic value. METHODS: Using xMAP® technology based assays we measured complement 3 (C3) and factor H (FH) in the CSF of 110 controls (CN), 187 mild cognitive impairment (MCI) and 92 AD subjects of the AD Neuroimaging Initiative (ADNI) at baseline. All ADNI participants underwent clinical follow-up at 12 month intervals and MCI subjects had additional visits at 6 and 18 months. The association between CSF biomarkers and different outcome measures were analyzed using Cox proportional hazard models (conversion from MCI to AD), logistic regression models (classification of clinical groups) and mixed-effects models adjusted for age, gender, education, t-tau/Aß1-42 and APOE ϵ4 presence (baseline and longitudinal association between biomarkers and cognitive scores). RESULTS: Although no association was found between the complement proteins and clinical diagnosis or cognitive measures, lower levels of C3 (ß = -0.12, p = 0.041) and FH (ß = -0.075, p = 0.041) were associated with faster cognitive decline in MCI subjects as measured by the AD Assessment Scale-cognitive subscale (ADAS-Cog) test. Furthermore, lower FH levels were associated with larger lateral ventricular volume (p = 0.024), which is indicative of brain atrophy. CONCLUSIONS: Our study confirms a lack of suitability of CSF C3 and FH as diagnostic biomarkers of AD, but points to their modest potential as prognostic biomarkers and therapeutic targets in cognitively impaired patients.

Cheek cell-derived α-synuclein and DJ-1 do not differentiate Parkinson's disease from control.

Recently, α-synuclein (α-syn) and DJ-1, 2 proteins critically involved in Parkinson's disease (PD), have been shown to be present in saliva, suggesting their potential utility as biomarkers of PD. However, the origin and influence of demographic characteristics (e.g., age or sex) on these proteins are unknown. We identified cheek epithelium, which forms the majority of the cellular component of saliva and is readily accessible clinically, as 1 of several potential sources of salivary α-syn and DJ-1. However, no PD-related trend in the cellular component was present. In the supernatant collected from 198 healthy subjects, no correlation was seen between salivary DJ-1 or α-syn with age. When male and female subjects were analyzed separately, a weak age-dependent increase in DJ-1 level was present in male subjects, along with slightly increased α-syn in female subjects. These results, albeit largely negative, provide critical information for understanding the salivary gland pathology and saliva as a PD biomarker source, and must be considered in future investigations of salivary changes in PD.

CSF α-synuclein improves diagnostic and prognostic performance of CSF tau and Aß in Alzheimer's disease.

Alzheimer's disease (AD) and Lewy body diseases (LBD), e.g., Parkinson's disease (PD) dementia and dementia with Lewy bodies (DLB), are common causes of geriatric cognitive impairments. In addition, AD and LBD are often found in the same patients at autopsy; therefore, biomarkers that can detect the presence of both pathologies in living subjects are needed. In this investigation, we report the assessment of α-synuclein (α-syn) in cerebrospinal fluid (CSF) and its association with CSF total tau (t-tau), phosphorylated tau181 (p-tau181), and amyloid beta1-42 (Aß1-42) in subjects of the Alzheimer's Disease Neuroimaging Initiative (ADNI; n = 389), with longitudinal clinical assessments. A strong correlation was noted between α-syn and t-tau in controls, as well as in patients with AD and mild cognitive impairment (MCI). However, the correlation is not specific to subjects in the ADNI cohort, as it was also seen in PD patients and controls enrolled in the Parkinson's Progression Markers Initiative (PPMI; n = 102). A bimodal distribution of CSF α-syn levels was observed in the ADNI cohort, with high levels of α-syn in the subjects with abnormally increased t-tau values. Although a correlation was also noted between α-syn and p-tau181, there was a mismatch (α-syn-p-tau181-Mis), i.e., higher p-tau181 levels accompanied by lower α-syn levels in a subset of ADNI patients. We hypothesize that this α-syn-p-tau181-Mis is a CSF signature of concomitant LBD pathology in AD patients. Hence, we suggest that inclusion of measures of CSF α-syn and calculation of α-syn-p-tau181-Mis improves the diagnostic sensitivity/specificity of classic CSF AD biomarkers and better predicts longitudinal cognitive changes.

α-Synuclein in cerebrospinal fluid of Alzheimer's disease and mild cognitive impairment.

In addition to amyloid-ß (Aß) and tau, α-synuclein, best known for its role in Parkinson's disease (PD), has been suggested to be involved in cognition and pathogenesis of Alzheimer's disease (AD). We investigate the potential of α-synuclein in cerebrospinal fluid (CSF) as a biomarker of cognitive decline in AD, and its prodromal phase, mild cognitive impairment (MCI). Using an established, sensitive Luminex assay, we measured α-synuclein levels in the CSF of a cohort of close to 400 healthy control, MCI, and AD subjects obtained from the Alzheimer's Disease Neuroimaging Initiative (ADNI) and factored in APOE genotype in data analysis. CSF α-synuclein levels were significantly higher in the MCI (p = 0.005) and AD (p < 0.001) groups, compared to controls. However, receiver operating characteristic (ROC) curve analysis suggests that CSF α-synuclein level on its own only offered modest sensitivity (65%) and specificity (74%) as a diagnostic marker of AD, with an area under the curve (AUC) value of 0.719 for AD versus controls. The effect of APOE genotype, if any, was quite subtle. However, there was a significant correlation between α-synuclein and cognition (p = 0.001), with increased α-synuclein levels associated with decreased Mini-Mental State Exam scores. Our results support a role for α-synuclein even in MCI, the early phase of AD, in addition to being a potential contributor in MCI and AD diagnosis or monitoring of disease progression.

Inflammatory effects of highly pathogenic H5N1 influenza virus infection in the CNS of mice.

The A/VN/1203/04 strain of the H5N1 influenza virus is capable of infecting the CNS of mice and inducing a number of neurodegenerative pathologies. Here, we examined the effects of H5N1 on several pathological aspects affected in parkinsonism, including loss of the phenotype of dopaminergic neurons located in the substantia nigra pars compacta (SNpc), expression of monoamines and indolamines in brain, alterations in SNpc microglia number and morphology, and expression of cytokines, chemokines, and growth factors. We find that H5N1 induces a transient loss of the dopaminergic phenotype in SNpc and now report that this loss recovers by 90 d after infection. A similar pattern of loss and recovery was seen in monoamine levels of the basal ganglia. The inflammatory response in lung and different regions of the brain known to be targets of the H5N1 virus (brainstem, substantia nigra, striatum, and cortex) were examined at 3, 10, 21, 60, and 90 d after infection. In each of these brain regions, we found a significant increase in the number of activated microglia that lasted at least 90 d. We also quantified expression of IL-1α, IL-1ß, IL-2, IL-6, IL-9, IL-10, IL-12(p70), IL-13, TNF-α, IFN-γ, granulocyte-macrophage colony-stimulating factor, granulocyte colony-stimulating factor, macrophage colony-stimulating factor, eotaxin, interferon-inducible protein 10, cytokine-induced neutrophil chemoattractant, monocyte chemotactic protein-1, macrophage inflammatory protein (MIP) 1α, MIP-1ß, and VEGF, and found that the pattern and levels of expression are dependent on both brain region and time after infection. We conclude that H5N1 infection in mice induces a long-lasting inflammatory response in brain and may play a contributing factor in the development of pathologies in neurodegenerative disorders.

Alterations in glutathione S-transferase pi expression following exposure to MPP+ -induced oxidative stress in the blood of Parkinson's disease patients.

The major motor symptoms of Parkinson's disease do not occur until a majority of the dopaminergic neurons in the midbrain SNpc have already died. For this reason, it is critical to identify biomarkers that will allow for the identification of presymptomatic individuals. In this study, we examine the baseline expression of the anti-oxidant protein glutathione S-transferase pi (GSTpi) in the blood of PD patients and environmentally- and age-matched controls and compare it to GSTpi levels following exposure to 1-methyl-4-phenylpyridinium (MPP(+)), an agent that has been shown to induce oxidative stress. We find that after 4 h of exposure to MPP(+), significant increases in GSTpi levels can be observed in the leukocytes of PD patients. No changes were seen in other blood components. This suggests that GSTpi and potentially other members of this and other anti-oxidant families may be viable biomarkers for PD.

Evaluating the behavioural consequences of early maternal separation in adult C57BL/6 mice; the importance of time.

Research in rats has shown that early maternal separation can have a significant effect on stress-associated neuro- and endocrine mechanisms in adulthood. However, despite a growing body of evidence on the neurobiology of early MS, showing significant overlap in data from rat, non-human primate and human studies, there is still some uncertainty about the validity of this model in mice. Here we present evidence in support of long lasting effects of early MS on adult mouse behaviour, which were only apparent when time was included as an analytical component. In the elevated plus maze (EPM), conventional statistical strategies, which typically evaluate behaviour as a summed test-session total, were not sufficient to reveal more complex time-dependent behavioural profiles. Specifically, the spatially more complex nature of the EPM test underscored treatment-related differences in the time-dependent adjustments of open arm exploration and risk-assessment behaviours. In contrast, the open field elicited an immediate and consistent divergence in risk-assessment behaviours, between MS animals and controls. Finally, plasma corticosterone further underscored MS-associated alterations in adult mouse stress profiles, with significantly higher concentrations in the MS group, post-restraint stress. The extension of conventional analysis strategies, to include time as a significant dimension of behaviour on the EPM, identified behavioural nuances, which could reflect adaptive aspects of stress-driven behaviours in MS mice.

Increased hippocampal nitric oxide synthase activity and stress responsiveness after imipramine discontinuation: role of 5HT 2A/C-receptors.

Chronic depressive illness may cause shrinkage of the hippocampus with stress-induced release of glutamate and nitric oxide possibly causally linked to this pathology. Poor antidepressant compliance may contribute to this pathology as well as to long term morbidity. However, antidepressant withdrawal-associated symptoms in depressed patients often reflect hyperserotonergia. The effect of chronic imipramine (IMI; 15 mg/kg/d ip x 3wks) treatment and withdrawal on swim stress responsiveness was studied in Sprague-Dawley rats together with assay of hippocampal NO synthase (NOS) activity. The dependence of any biobehavioral changes following IMI withdrawal on 5HT(2A/C) receptor-mediated events was studied using the 5HT(2A/C) receptor antagonist, ritanserin (RIT; 4 mg/kg/day ip x 7 days), administered alone or during IMI withdrawal. IMI significantly inhibited the situational stress response to forced swimming while also significantly decreasing NOS activity. IMI withdrawal was associated with a significant increase in swim immobility together with a significant increase in NOS activity compared to both control and IMI-treated groups. RIT re-established the anti-immobility effects and reversed NOS hyper-function during IMI withdrawal, although alone it increased NOS activity. Antidepressant discontinuation therefore increases stress responsiveness together with disinhibition of hippocampal NOS through a mechanism involving 5HT(2A/C) receptor activation. The resulting increased nitrergic activity may have significant implications for depressive illness and its treatment.