Inhaled nitric oxide suppresses neuroinflammation in experimental ischemic stroke.

Ischemic stroke is a major global health issue and characterized by acute vascular dysfunction and subsequent neuroinflammation. However, the relationship between these processes remains elusive. In the current study, we investigated whether alleviating vascular dysfunction by restoring vascular nitric oxide (NO) reduces post-stroke inflammation. Mice were subjected to experimental stroke and received inhaled NO (iNO; 50 ppm) after reperfusion. iNO normalized vascular cyclic guanosine monophosphate (cGMP) levels, reduced the elevated expression of intercellular adhesion molecule-1 (ICAM-1), and returned leukocyte adhesion to baseline levels. Reduction of vascular pathology significantly reduced the inflammatory cytokines interleukin-1ß (Il-1ß), interleukin-6 (Il-6), and tumor necrosis factor-α (TNF-α), within the brain parenchyma. These findings suggest that vascular dysfunction is responsible for leukocyte adhesion and that these processes drive parenchymal inflammation. Reversing vascular dysfunction may therefore emerge as a novel approach to diminish neuroinflammation after ischemic stroke and possibly other ischemic disorders.

Loss of function of FIP200 in human pluripotent stem cell-derived neurons leads to axonal pathology and hyperactivity.

FIP200 plays important roles in homeostatic processes such as autophagy and signaling pathways such as focal adhesion kinase (FAK) signaling. Furthermore, genetic studies suggest an association of FIP200 mutations with psychiatric disorders. However, its potential connections to psychiatric disorders and specific roles in human neurons are not clear. We set out to establish a human-specific model to study the functional consequences of neuronal FIP200 deficiency. To this end, we generated two independent sets of isogenic human pluripotent stem cell lines with homozygous FIP200KO alleles, which were then used for the derivation of glutamatergic neurons via forced expression of NGN2. FIP200KO neurons exhibited pathological axonal swellings, showed autophagy deficiency, and subsequently elevated p62 protein levels. Moreover, monitoring the electrophysiological activity of neuronal cultures on multi-electrode arrays revealed that FIP200KO resulted in a hyperactive network. This hyperactivity could be abolished by glutamatergic receptor antagonist CNQX, suggesting a strengthened glutamatergic synaptic activation in FIP200KO neurons. Furthermore, cell surface proteomic analysis revealed metabolic dysregulation and abnormal cell adhesion-related processes in FIP200KO neurons. Interestingly, an ULK1/2-specific autophagy inhibitor could recapitulate axonal swellings and hyperactivity in wild-type neurons, whereas inhibition of FAK signaling was able to normalize the hyperactivity of FIP200KO neurons. These results suggest that impaired autophagy and presumably also disinhibition of FAK can contribute to the hyperactivity of FIP200KO neuronal networks, whereas pathological axonal swellings are primarily due to autophagy deficiency. Taken together, our study reveals the consequences of FIP200 deficiency in induced human glutamatergic neurons, which might, in the end, help to understand cellular pathomechanisms contributing to neuropsychiatric conditions.

Hippo-released WWC1 facilitates AMPA receptor regulatory complexes for hippocampal learning.

Learning and memory rely on changes in postsynaptic glutamergic α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type receptor (AMPAR) number, spatial organization, and function. The Hippo pathway component WW and C2 domain-containing protein 1 (WWC1) regulates AMPAR surface expression and impacts on memory performance. However, synaptic binding partners of WWC1 and its hierarchical position in AMPAR complexes are largely unclear. Using cell-surface proteomics in hippocampal tissue of Wwc1-deficient mice and by generating a hippocampus-specific interactome, we show that WWC1 is a major regulatory platform in AMPAR signaling networks. Under basal conditions, the Hippo pathway members WWC1 and large tumor-suppressor kinase (LATS) are associated, which might prevent WWC1 effects on synaptic proteins. Reduction of WWC1/LATS binding through a point mutation at WWC1 elevates the abundance of WWC1 in AMPAR complexes and improves hippocampal-dependent learning and memory. Thus, uncoupling of WWC1 from the Hippo pathway to AMPAR-regulatory complexes provides an innovative strategy to enhance synaptic transmission.

Proteomic profiling in cerebral amyloid angiopathy reveals an overlap with CADASIL highlighting accumulation of HTRA1 and its substrates.

Cerebral amyloid angiopathy (CAA) is an age-related condition and a major cause of intracerebral hemorrhage and cognitive decline that shows close links with Alzheimer's disease (AD). CAA is characterized by the aggregation of amyloid-ß (Aß) peptides and formation of Aß deposits in the brain vasculature resulting in a disruption of the angioarchitecture. Capillaries are a critical site of Aß pathology in CAA type 1 and become dysfunctional during disease progression. Here, applying an advanced protocol for the isolation of parenchymal microvessels from post-mortem brain tissue combined with liquid chromatography tandem mass spectrometry (LC-MS/MS), we determined the proteomes of CAA type 1 cases (n = 12) including a patient with hereditary cerebral hemorrhage with amyloidosis-Dutch type (HCHWA-D), and of AD cases without microvascular amyloid pathology (n = 13) in comparison to neurologically healthy controls (n = 12). ELISA measurements revealed microvascular Aß1-40 levels to be exclusively enriched in CAA samples (mean: > 3000-fold compared to controls). The proteomic profile of CAA type 1 was characterized by massive enrichment of multiple predominantly secreted proteins and showed significant overlap with the recently reported brain microvascular proteome of patients with cerebral autosomal-dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL), a hereditary cerebral small vessel disease (SVD) characterized by the aggregation of the Notch3 extracellular domain. We found this overlap to be largely attributable to the accumulation of high-temperature requirement protein A1 (HTRA1), a serine protease with an established role in the brain vasculature, and several of its substrates. Notably, this signature was not present in AD cases. We further show that HTRA1 co-localizes with Aß deposits in brain capillaries from CAA type 1 patients indicating a pathologic recruitment process. Together, these findings suggest a central role of HTRA1-dependent protein homeostasis in the CAA microvasculature and a molecular connection between multiple types of brain microvascular disease.

SARS-CoV-2-mediated dysregulation of metabolism and autophagy uncovers host-targeting antivirals.

Viruses manipulate cellular metabolism and macromolecule recycling processes like autophagy. Dysregulated metabolism might lead to excessive inflammatory and autoimmune responses as observed in severe and long COVID-19 patients. Here we show that SARS-CoV-2 modulates cellular metabolism and reduces autophagy. Accordingly, compound-driven induction of autophagy limits SARS-CoV-2 propagation. In detail, SARS-CoV-2-infected cells show accumulation of key metabolites, activation of autophagy inhibitors (AKT1, SKP2) and reduction of proteins responsible for autophagy initiation (AMPK, TSC2, ULK1), membrane nucleation, and phagophore formation (BECN1, VPS34, ATG14), as well as autophagosome-lysosome fusion (BECN1, ATG14 oligomers). Consequently, phagophore-incorporated autophagy markers LC3B-II and P62 accumulate, which we confirm in a hamster model and lung samples of COVID-19 patients. Single-nucleus and single-cell sequencing of patient-derived lung and mucosal samples show differential transcriptional regulation of autophagy and immune genes depending on cell type, disease duration, and SARS-CoV-2 replication levels. Targeting of autophagic pathways by exogenous administration of the polyamines spermidine and spermine, the selective AKT1 inhibitor MK-2206, and the BECN1-stabilizing anthelmintic drug niclosamide inhibit SARS-CoV-2 propagation in vitro with IC50 values of 136.7, 7.67, 0.11, and 0.13 µM, respectively. Autophagy-inducing compounds reduce SARS-CoV-2 propagation in primary human lung cells and intestinal organoids emphasizing their potential as treatment options against COVID-19.

SKP2 attenuates autophagy through Beclin1-ubiquitination and its inhibition reduces MERS-Coronavirus infection.

Autophagy is an essential cellular process affecting virus infections and other diseases and Beclin1 (BECN1) is one of its key regulators. Here, we identified S-phase kinase-associated protein 2 (SKP2) as E3 ligase that executes lysine-48-linked poly-ubiquitination of BECN1, thus promoting its proteasomal degradation. SKP2 activity is regulated by phosphorylation in a hetero-complex involving FKBP51, PHLPP, AKT1, and BECN1. Genetic or pharmacological inhibition of SKP2 decreases BECN1 ubiquitination, decreases BECN1 degradation and enhances autophagic flux. Middle East respiratory syndrome coronavirus (MERS-CoV) multiplication results in reduced BECN1 levels and blocks the fusion of autophagosomes and lysosomes. Inhibitors of SKP2 not only enhance autophagy but also reduce the replication of MERS-CoV up to 28,000-fold. The SKP2-BECN1 link constitutes a promising target for host-directed antiviral drugs and possibly other autophagy-sensitive conditions.

Novel mutation in HTRA1 in a family with diffuse white matter lesions and inflammatory features.

OBJECTIVE: To investigate the possible involvement of germline mutations in a neurologic condition involving diffuse white matter lesions. METHODS: The patients were 3 siblings born to healthy parents. We performed homozygosity mapping, whole-exome sequencing, site-directed mutagenesis, and immunoblotting. RESULTS: All 3 patients showed clinical manifestations of ataxia, behavioral and mood changes, premature hair loss, memory loss, and lower back pain. In addition, they presented with inflammatory-like features and recurrent rhinitis. MRI showed abnormal diffuse demyelination lesions in the brain and myelitis in the spinal cord. We identified an insertion in high-temperature requirement A (HTRA1), which showed complete segregation in the pedigree. Functional analysis showed the mutation to affect stability and secretion of truncated protein. CONCLUSIONS: The patients' clinical manifestations are consistent with cerebral autosomal recessive arteriopathy with subcortical infarcts and leukoencephalopathy (CARASIL; OMIM #600142), which is known to be caused by HTRA1 mutations. Because some aspects of the clinical presentation deviate from those reported for CARASIL, our study expands the spectrum of clinical consequences of loss-of-function mutations in HTRA1.

CADASIL brain vessels show a HTRA1 loss-of-function profile.

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) and a phenotypically similar recessive condition (CARASIL) have emerged as important genetic model diseases for studying the molecular pathomechanisms of cerebral small vessel disease (SVD). CADASIL, the most frequent and intensely explored monogenic SVD, is characterized by a severe pathology in the cerebral vasculature including the mutation-induced aggregation of the Notch3 extracellular domain (Notch3ECD) and the formation of protein deposits of insufficiently determined composition in vessel walls. To identify key molecules and pathways involved in this process, we quantitatively determined the brain vessel proteome from CADASIL patient and control autopsy samples (n = 6 for each group), obtaining 95 proteins with significantly increased abundance. Intriguingly, high-temperature requirement protein A1 (HTRA1), the extracellular protease mutated in CARASIL, was found to be strongly enriched (4.9-fold, p = 1.6 × 10-3) and to colocalize with Notch3ECD deposits in patient vessels suggesting a sequestration process. Furthermore, the presence of increased levels of several HTRA1 substrates in the CADASIL proteome was compatible with their reduced degradation as consequence of a loss of HTRA1 activity. Indeed, a comparison with the brain vessel proteome of HTRA1 knockout mice (n = 5) revealed a highly significant overlap of 18 enriched proteins (p = 2.2 × 10-16), primarily representing secreted and extracellular matrix factors. Several of them were shown to be processed by HTRA1 in an in vitro proteolysis assay identifying them as novel substrates. Our study provides evidence for a loss of HTRA1 function as a critical step in the development of CADASIL pathology linking the molecular mechanisms of two distinct SVD forms.

Glycogen synthase kinase-3ß inhibition in the medial prefrontal cortex mediates paradoxical amphetamine action in a mouse model of ADHD.

Psychostimulants show therapeutic efficacy in the treatment of attention-deficit hyperactivity disorder (ADHD). It is generally assumed that they ameliorate ADHD symptoms via interfering with monoaminergic signaling. We combined behavioral pharmacology, neurochemistry and molecular analyses to identify mechanisms underlying the paradoxical calming effect of amphetamine in low trait anxiety behavior (LAB) mice, a novel multigenetic animal model of ADHD. Amphetamine (1 mg/kg) and methylphenidate (10 mg/kg) elicited similar dopamine and norepinephrine release in the medial prefrontal cortex (mPFC) and in the striatum of LAB mice. In contrast, amphetamine decreased, while methylphenidate increased locomotor activity. This argues against changes in dopamine and/or norepinephrine release as mediators of amphetamine paradoxical effects. Instead, the calming activity of amphetamine corresponded to the inhibition of glycogen synthase kinase 3ß (GSK3ß) activity, specifically in the mPFC. Accordingly, not only systemic administration of the GSK3ß inhibitor TDZD-8 (20 mg/kg), but also local microinjections of TDZD-8 and amphetamine into the mPFC, but not into the striatum, decreased locomotor activity in LAB mice. Amphetamine effects seem to depend on NMDA receptor signaling, since pre- or co-treatment with MK-801 (0.3 mg/kg) abolished the effects of amphetamine (1 mg/kg) on the locomotion and on the phosphorylation of GSK3ß at the level of the mPFC. Taken together, the paradoxical calming effect of amphetamine in hyperactive LAB mice concurs with a decreased GSK3ß activity in the mPFC. This effect appears to be independent of dopamine or norepinephrine release, but contingent on NMDA receptor signaling.

Association of FKBP51 with priming of autophagy pathways and mediation of antidepressant treatment response: evidence in cells, mice, and humans.

BACKGROUND: FK506 binding protein 51 (FKBP51) is an Hsp90 co-chaperone and regulator of the glucocorticoid receptor, and consequently of stress physiology. Clinical studies suggest a genetic link between FKBP51 and antidepressant response in mood disorders; however, the underlying mechanisms remain elusive. The objective of this study was to elucidate the role of FKBP51 in the actions of antidepressants, with a particular focus on pathways of autophagy. METHODS AND FINDINGS: Established cell lines, primary neural cells, human blood cells of healthy individuals and patients with depression, and mice were treated with antidepressants. Mice were tested for several neuroendocrine and behavioral parameters. Protein interactions and autophagic pathway activity were mainly evaluated by co-immunoprecipitation and Western blots. We first show that the effects of acute antidepressant treatment on behavior are abolished in FKBP51 knockout (51KO) mice. Autophagic markers, such as the autophagy initiator Beclin1, were increased following acute antidepressant treatment in brains from wild-type, but not 51KO, animals. FKBP51 binds to Beclin1, changes decisive protein interactions and phosphorylation of Beclin1, and triggers autophagic pathways. Antidepressants and FKBP51 exhibited synergistic effects on these pathways. Using chronic social defeat as a depression-relevant stress model in combination with chronic paroxetine (PAR) treatment revealed that the stress response, as well as the effects of antidepressants on behavior and autophagic markers, depends on FKBP51. In human blood cells of healthy individuals, FKBP51 levels correlated with the potential of antidepressants to induce autophagic pathways. Importantly, the clinical antidepressant response of patients with depression (n = 51) could be predicted by the antidepressant response of autophagic markers in patient-derived peripheral blood lymphocytes cultivated and treated ex vivo (Beclin1/amitriptyline: r = 0.572, p = 0.003; Beclin1/PAR: r = 0.569, p = 0.004; Beclin1/fluoxetine: r = 0.454, p = 0.026; pAkt/amitriptyline: r =  -0.416, p = 0.006; pAkt/PAR: r =  -0.355, p = 0.021; LC3B-II/PAR: r = 0.453, p = 0.02), as well as by the lymphocytic expression levels of FKBP51 (r = 0.631, p<0.0001), pAkt (r =  -0.515, p = 0.003), and Beclin1 (r = 0.521, p = 0.002) at admission. Limitations of the study include the use of male mice only and the relatively low number of patients for protein analyses. CONCLUSIONS: To our knowledge, these findings provide the first evidence for the molecular mechanism of FKBP51 in priming autophagic pathways; this process is linked to the potency of at least some antidepressants. These newly discovered functions of FKBP51 also provide novel predictive markers for treatment outcome, consistent with physiological and potential clinical relevance. Please see later in the article for the Editors' Summary.