Slingshot homolog-1-mediated Nrf2 sequestration tips the balance from neuroprotection to neurodegeneration in Alzheimer's disease.

Oxidative damage in the brain is one of the earliest drivers of pathology in Alzheimer's disease (AD) and related dementias, both preceding and exacerbating clinical symptoms. In response to oxidative stress, nuclear factor erythroid 2-related factor 2 (Nrf2) is normally activated to protect the brain from oxidative damage. However, Nrf2-mediated defense against oxidative stress declines in AD, rendering the brain increasingly vulnerable to oxidative damage. Although this phenomenon has long been recognized, its mechanistic basis has been a mystery. Here, we demonstrate through in vitro and in vivo models, as well as human AD brain tissue, that Slingshot homolog-1 (SSH1) drives this effect by acting as a counterweight to neuroprotective Nrf2 in response to oxidative stress and disease. Specifically, oxidative stress-activated SSH1 suppresses nuclear Nrf2 signaling by sequestering Nrf2 complexes on actin filaments and augmenting Kelch-like ECH-associated protein 1 (Keap1)-Nrf2 interaction, independently of SSH1 phosphatase activity. We also show that Ssh1 elimination in AD models increases Nrf2 activation, which mitigates tau and amyloid-ß accumulation and protects against oxidative injury, neuroinflammation, and neurodegeneration. Furthermore, loss of Ssh1 preserves normal synaptic function and transcriptomic patterns in tauP301S mice. Importantly, we also show that human AD brains exhibit highly elevated interactions of Nrf2 with both SSH1 and Keap1. Thus, we demonstrate here a unique mode of Nrf2 blockade that occurs through SSH1, which drives oxidative damage and ensuing pathogenesis in AD. Strategies to inhibit SSH1-mediated Nrf2 suppression while preserving normal SSH1 catalytic function may provide new neuroprotective therapies for AD and related dementias.

X-linked ubiquitin-specific peptidase 11 increases tauopathy vulnerability in women.

Although women experience significantly higher tau burden and increased risk for Alzheimer's disease (AD) than men, the underlying mechanism for this vulnerability has not been explained. Here, we demonstrate through in vitro and in vivo models, as well as human AD brain tissue, that X-linked ubiquitin specific peptidase 11 (USP11) augments pathological tau aggregation via tau deubiquitination initiated at lysine-281. Removal of ubiquitin provides access for enzymatic tau acetylation at lysines 281 and 274. USP11 escapes complete X-inactivation, and female mice and people both exhibit higher USP11 levels than males. Genetic elimination of usp11 in a tauopathy mouse model preferentially protects females from acetylated tau accumulation, tau pathology, and cognitive impairment. USP11 levels also strongly associate positively with tau pathology in females but not males. Thus, inhibiting USP11-mediated tau deubiquitination may provide an effective therapeutic opportunity to protect women from increased vulnerability to AD and other tauopathies.

PERK is a critical metabolic hub for immunosuppressive function in macrophages.

Chronic inflammation triggers compensatory immunosuppression to stop inflammation and minimize tissue damage. Studies have demonstrated that endoplasmic reticulum (ER) stress augments the suppressive phenotypes of immune cells; however, the molecular mechanisms underpinning this process and how it links to the metabolic reprogramming of immunosuppressive macrophages remain elusive. In the present study, we report that the helper T cell 2 cytokine interleukin-4 and the tumor microenvironment increase the activity of a protein kinase RNA-like ER kinase (PERK)-signaling cascade in macrophages and promote immunosuppressive M2 activation and proliferation. Loss of PERK signaling impeded mitochondrial respiration and lipid oxidation critical for M2 macrophages. PERK activation mediated the upregulation of phosphoserine aminotransferase 1 (PSAT1) and serine biosynthesis via the downstream transcription factor ATF-4. Increased serine biosynthesis resulted in enhanced mitochondrial function and α-ketoglutarate production required for JMJD3-dependent epigenetic modification. Inhibition of PERK suppressed macrophage immunosuppressive activity and could enhance the efficacy of immune checkpoint programmed cell death protein 1 inhibition in melanoma. Our findings delineate a previously undescribed connection between PERK signaling and PSAT1-mediated serine metabolism critical for promoting immunosuppressive function in M2 macrophages.

Reducing acetylated tau is neuroprotective in brain injury.

Traumatic brain injury (TBI) is the largest non-genetic, non-aging related risk factor for Alzheimer's disease (AD). We report here that TBI induces tau acetylation (ac-tau) at sites acetylated also in human AD brain. This is mediated by S-nitrosylated-GAPDH, which simultaneously inactivates Sirtuin1 deacetylase and activates p300/CBP acetyltransferase, increasing neuronal ac-tau. Subsequent tau mislocalization causes neurodegeneration and neurobehavioral impairment, and ac-tau accumulates in the blood. Blocking GAPDH S-nitrosylation, inhibiting p300/CBP, or stimulating Sirtuin1 all protect mice from neurodegeneration, neurobehavioral impairment, and blood and brain accumulation of ac-tau after TBI. Ac-tau is thus a therapeutic target and potential blood biomarker of TBI that may represent pathologic convergence between TBI and AD. Increased ac-tau in human AD brain is further augmented in AD patients with history of TBI, and patients receiving the p300/CBP inhibitors salsalate or diflunisal exhibit decreased incidence of AD and clinically diagnosed TBI.

Dopamine D1R-neuron cacna1c deficiency: a new model of extinction therapy-resistant post-traumatic stress.

Post-traumatic stress disorder (PTSD) is characterized by persistent fear memory of remote traumatic events, mental re-experiencing of the trauma, long-term cognitive deficits, and PTSD-associated hippocampal dysfunction. Extinction-based therapeutic approaches acutely reduce fear. However, many patients eventually relapse to the original conditioned fear response. Thus, understanding the underlying molecular mechanisms of this condition is critical to developing new treatments for patients. Mutations in the neuropsychiatric risk gene CACNA1C, which encodes the Cav1.2 isoform of the L-type calcium channel, have been implicated in both PTSD and highly comorbid neuropsychiatric conditions, such as anxiety and depression. Here, we report that male mice with global heterozygous loss of cacna1c exhibit exacerbated contextual fear that persists at remote time points (up to 180 days after shock), despite successful acute extinction training, reminiscent of PTSD patients. Because dopamine has been implicated in contextual fear memory, and Cav1.2 is a downstream target of dopamine D1-receptor (D1R) signaling, we next generated mice with specific deletion of cacna1c from D1R-expressing neurons (D1-cacna1cKO mice). Notably, D1-cacna1cKO mice also show the same exaggerated remote contextual fear, as well as persistently elevated anxiety-like behavior and impaired spatial memory at remote time points, reminiscent of chronic anxiety in treatment-resistant PTSD. We also show that D1-cacna1cKO mice exhibit elevated death of young hippocampal neurons, and that treatment with the neuroprotective agent P7C3-A20 eradicates persistent remote fear. Augmenting survival of young hippocampal neurons may thus provide an effective therapeutic approach for promoting durable remission of PTSD, particularly in patients with CACNA1C mutations or other genetic aberrations that impair calcium signaling or disrupt the survival of young hippocampal neurons.

P7C3-A20 treatment one year after TBI in mice repairs the blood-brain barrier, arrests chronic neurodegeneration, and restores cognition.

Chronic neurodegeneration in survivors of traumatic brain injury (TBI) is a major cause of morbidity, with no effective therapies to mitigate this progressive and debilitating form of nerve cell death. Here, we report that pharmacologic restoration of the blood-brain barrier (BBB), 12 mo after murine TBI, is associated with arrested axonal neurodegeneration and cognitive recovery, benefits that persisted for months after treatment cessation. Recovery was achieved by 30 d of once-daily administration of P7C3-A20, a compound that stabilizes cellular energy levels. Four months after P7C3-A20, electron microscopy revealed full repair of TBI-induced breaks in cortical and hippocampal BBB endothelium. Immunohistochemical staining identified additional benefits of P7C3-A20, including restoration of normal BBB endothelium length, increased brain capillary pericyte density, increased expression of BBB tight junction proteins, reduced brain infiltration of immunoglobulin, and attenuated neuroinflammation. These changes were accompanied by cessation of TBI-induced chronic axonal degeneration. Specificity for P7C3-A20 action on the endothelium was confirmed by protection of cultured human brain microvascular endothelial cells from hydrogen peroxide-induced cell death, as well as preservation of BBB integrity in mice after exposure to toxic levels of lipopolysaccharide. P7C3-A20 also protected mice from BBB degradation after acute TBI. Collectively, our results provide insights into the pathophysiologic mechanisms behind chronic neurodegeneration after TBI, along with a putative treatment strategy. Because TBI increases the risks of other forms of neurodegeneration involving BBB deterioration (e.g., Alzheimer's disease, Parkinson's disease, vascular dementia, chronic traumatic encephalopathy), P7C3-A20 may have widespread clinical utility in the setting of neurodegenerative conditions.

Identification of chronic brain protein changes and protein targets of serum auto-antibodies after blast-mediated traumatic brain injury.

In addition to needing acute emergency management, blast-mediated traumatic brain injury (TBI) is also a chronic disorder with delayed-onset symptoms that manifest and progress over time. While the immediate consequences of acute blast injuries are readily apparent, chronic sequelae are harder to recognize. Indeed, the identification of individuals with mild-TBI or TBI-induced symptoms is greatly impaired in large part due to the lack of objective and robust biomarkers. The purpose of this study was to address these need by identifying candidates for serum-based biomarkers of blast TBI, and also to identify unique or differentially regulated protein expression in the thalamus in C57BL/6J mice exposed to blast using high throughput qualitative screens of protein expression. To identify thalamic proteins differentially or uniquely associated with blast exposure, we utilized an antibody-based affinity-capture strategy (referred to as "proteomics-based analysis of depletomes"; PAD) to deplete thalamic lysates from blast-treated mice of endogenous thalamic proteins also found in control mice. Analysis of this "depletome" detected 75 unique proteins, many with associations to the myelin sheath. To identify blast-associated proteins eliciting production of circulating autoantibodies, serum antibodies of blast-treated mice were immobilized, and their immunogens subsequently identified by proteomic analysis of proteins specifically captured following incubation with thalamic lysates (a variant of a strategy referred to as "proteomics-based expression library screening"; PELS). This analysis identified 46 blast-associated immunogenic proteins, including 6 shared in common with the PAD analysis (ALDOA, PHKB, HBA-A1, DPYSL2, SYN1, and CKB). These proteins and their autoantibodies are appropriate for further consideration as biomarkers of blast-mediated TBI.

Pathogenic troponin T mutants with opposing effects on myofilament Ca2+ sensitivity attenuate cardiomyopathy phenotypes in mice.

Mutations in cardiac troponin T (TnT) associated with hypertrophic cardiomyopathy generally lead to an increase in the Ca2+ sensitivity of contraction and susceptibility to arrhythmias. In contrast, TnT mutations linked to dilated cardiomyopathy decrease the Ca2+ sensitivity of contraction. Here we tested the hypothesis that two TnT disease mutations with opposite effects on myofilament Ca2+ sensitivity can attenuate each other's phenotype. We crossed transgenic mice expressing the HCM TnT-I79N mutation (I79N) with a DCM knock-in mouse model carrying the heterozygous TnT-R141W mutation (HET). The results of the Ca2+ sensitivity in skinned cardiac muscle preparations ranked from highest to lowest were as follow: I79N > I79N/HET > NTg > HET. Echocardiographic measurements revealed an improvement in hemodynamic parameters in I79N/HET compared to I79N and normalization of left ventricular dimensions and volumes compared to both I79N and HET. Ex vivo testing showed that the I79N/HET mouse hearts had reduced arrhythmia susceptibility compared to I79N mice. These results suggest that two disease mutations in TnT that have opposite effects on the myofilament Ca2+ sensitivity can paradoxically ameliorate each other's disease phenotype. Normalizing myofilament Ca2+ sensitivity may be a promising new treatment approach for a variety of diseases.

Effect of aerobic exercise training on non-alcoholic fatty liver disease induced by a high fat diet in C57BL/6 mice.

PURPOSE: The aim of this study was to investigate the effects of aerobic exercise training on a high fat diet (HFD)-induced fatty liver and its metabolic complications in C57BL/6 mice. METHODS: Mice at 5-month old (n = 30) were randomly assigned to standard chow (SC + CON, n = 10) and high-fat diet (HFD, n = 20), and they were subjected to SC and HFD, respectively, for 23-week. After 15-week of HFD, mice in the HFD group were further assigned to HFD (HFD + CON, n = 10) or exercise training (HFD + EX, n = 10) groups. The HFD + EX mice were subjected to aerobic treadmill running during the last 8-week of the 23-week HFD course. Outcomes included hepatic steatosis, insulin resistance, and expression of genes involved in mitochondrial function and/or fatty oxidation as well as de novo lipogenesis and/or triacylglycerol (TAG) synthesis. RESULTS: Treadmill running ameliorated impaired glucose tolerance and insulin resistance secondary to the HFD. The beneficial effects of treadmill running were associated with enhanced molecular markers of mitochondrial function and/or fatty acids oxidation (i.e., PPARα and CPT1a mRNAs, pAMPK/AMPK, pACC, and SIRT1 protein) as well as suppressed expression of de novo lipogenesis and/or TAG synthesis (i.e., SREBP1c, lipin1 and FAS mRNAs) in the liver. CONCLUSION: The current findings suggest that aerobic exercise training is an effective and non-pharmacological means to combat fatty liver and its metabolic complications in HFD-induced obese mice.