Pathological axonal death through a MAPK cascade that triggers a local energy deficit.

Axonal death disrupts functional connectivity of neural circuits and is a critical feature of many neurodegenerative disorders. Pathological axon degeneration often occurs independently of known programmed death pathways, but the underlying molecular mechanisms remain largely unknown. Using traumatic injury as a model, we systematically investigate mitogen-activated protein kinase (MAPK) families and delineate a MAPK cascade that represents the early degenerative response to axonal injury. The adaptor protein Sarm1 is required for activation of this MAPK cascade, and this Sarm1-MAPK pathway disrupts axonal energy homeostasis, leading to ATP depletion before physical breakdown of damaged axons. The protective cytoNmnat1/Wld(s) protein inhibits activation of this MAPK cascade. Further, MKK4, a key component in the Sarm1-MAPK pathway, is antagonized by AKT signaling, which modulates the degenerative response by limiting activation of downstream JNK signaling. Our results reveal a regulatory mechanism that integrates distinct signals to instruct pathological axon degeneration.

An Anterior Second Heart Field Enhancer Regulates the Gene Regulatory Network of the Cardiac Outflow Tract.

BACKGROUND: Conotruncal defects due to developmental abnormalities of the outflow tract (OFT) are an important cause of cyanotic congenital heart disease. Dysregulation of transcriptional programs tuned by NKX2-5 (NK2 homeobox 5), GATA6 (GATA binding protein 6), and TBX1 (T-box transcription factor 1) have been implicated in abnormal OFT morphogenesis. However, there remains no consensus on how these transcriptional programs function in a unified gene regulatory network within the OFT. METHODS: We generated mice harboring a 226-nucleotide deletion of a highly conserved cardiac enhancer containing 2 GATA-binding sites located ≈9.4 kb upstream of the transcription start site of Nkx2-5 (Nkx2-5∆enh) using CRISPR-Cas9 gene editing and assessed phenotypes. Cardiac defects in Nkx2-5∆enh/∆enh mice were structurally characterized using histology and scanning electron microscopy, and physiologically assessed using electrocardiography, echocardiography, and optical mapping. Transcriptome analyses were performed using RNA sequencing and single-cell RNA sequencing data sets. Endogenous GATA6 interaction with and activity on the NKX2-5 enhancer was studied using chromatin immunoprecipitation sequencing and transposase-accessible chromatin sequencing in human induced pluripotent stem cell-derived cardiomyocytes. RESULTS: Nkx2-5∆enh/∆enh mice recapitulated cyanotic conotruncal defects seen in patients with NKX2-5, GATA6, and TBX1 mutations. Nkx2-5∆enh/∆enh mice also exhibited defects in right Purkinje fiber network formation, resulting in right bundle-branch block. Enhancer deletion reduced embryonic Nkx2-5 expression selectively in the right ventricle and OFT of mutant hearts, indicating that enhancer activity is localized to the anterior second heart field. Transcriptional profiling of the mutant OFT revealed downregulation of important genes involved in OFT rotation and septation, such as Tbx1, Pitx2, and Sema3c. Endogenous GATA6 interacted with the highly conserved enhancer in human induced pluripotent stem cell-derived cardiomyocytes and in wild-type mouse hearts. We found critical dose dependency of cardiac enhancer accessibility on GATA6 gene dosage in human induced pluripotent stem cell-derived cardiomyocytes. CONCLUSIONS: Our results using human and mouse models reveal an essential gene regulatory network of the OFT that requires an anterior second heart field enhancer to link GATA6 with NKX2-5-dependent rotation and septation gene programs.

PFTK1 kinase regulates axogenesis during development via RhoA activation.

BACKGROUND: PFTK1/Eip63E is a member of the cyclin-dependent kinases (CDKs) family and plays an important role in normal cell cycle progression. Eip63E expresses primarily in postnatal and adult nervous system in Drosophila melanogaster but its role in CNS development remains unknown. We sought to understand the function of Eip63E in the CNS by studying the fly ventral nerve cord during development. RESULTS: Our results demonstrate that Eip63E regulates axogenesis in neurons and its deficiency leads to neuronal defects. Functional interaction studies performed using the same system identify an interaction between Eip63E and the small GTPase Rho1. Furthermore, deficiency of Eip63E homolog in mice, PFTK1, in a newly generated PFTK1 knockout mice results in increased axonal outgrowth confirming that the developmental defects observed in the fly model are due to defects in axogenesis. Importantly, RhoA phosphorylation and activity are affected by PFTK1 in primary neuronal cultures. We report that GDP-bound inactive RhoA is a substrate of PFTK1 and PFTK1 phosphorylation is required for RhoA activity. CONCLUSIONS: In conclusion, our work establishes an unreported neuronal role of PFTK1 in axon development mediated by phosphorylation and activation of GDP-bound RhoA. The results presented add to our understanding of the role of Cdks in the maintenance of RhoA-mediated axon growth and its impact on CNS development and axonal regeneration.

Catheter ablation of atrioventricular nodal reentrant tachycardia with an irrigated contact-force sensing radiofrequency ablation catheter.

INTRODUCTION: Radiofrequency ablation (RFA) slow pathway modification for catheter ablation of atrioventricular nodal reentrant tachycardia (AVNRT) is traditionally performed using a 4-mm nonirrigated (NI) RF ablation catheter. Slow pathway modification using irrigated, contact-force sensing (ICFS) RFA catheters has been described in case reports, but the outcomes have not been systematically evaluated. METHODS: Acute procedural outcomes of 200 consecutive patients undergoing slow pathway modification for AVNRT were analyzed. A 3.5-mm ICFS RFA catheter (ThermoCool SmartTouch STSF, Biosense Webster, Inc.) was utilized in 134 patients, and a 4-mm NI RFA catheter (EZ Steer, Biosense Webster, Inc.) was utilized in 66 patients. Electroanatomic maps were retrospectively analyzed in a blinded fashion to determine the proximity of ablation lesions to the His region. RESULTS: The baseline characteristics of patients in both groups were similar. Total RF time was significantly lower in the ICFS group compared to the NI group (5.53 ± 4.6 vs. 6.24 ± 4.9 min, p = 0.03). Median procedure time was similar in both groups (ICFS, 108.0 (87.5-131.5) min vs. NI, 100.0 (85.0-125.0) min; p = 0.2). Ablation was required in closer proximity to the His region in the NI group compared to the ICFS group (14.4 ± 5.9 vs. 16.7 ± 6.4 mm, respectively, p = 0.01). AVNRT was rendered noninducible in all patients, and there was no arrhythmia recurrence during follow-up in both groups. Catheter ablation was complicated by AV block in one patient in the NI group. CONCLUSION: Slow pathway modification for catheter ablation of AVNRT using an ICFS RFA catheter is feasible, safe, and may facilitate shorter duration ablation while avoiding ablation in close proximity to the His region.

Cdk5-mediated JIP1 phosphorylation regulates axonal outgrowth through Notch1 inhibition.

BACKGROUND: Activated Cdk5 regulates a number of processes during nervous system formation, including neuronal differentiation, growth cone stabilization, and axonal growth. Cdk5 phosphorylates its downstream substrates located in axonal growth cones, where the highly expressed c-Jun N-terminal kinase (JNK)-interacting protein1 (JIP1) has been implicated as another important regulator of axonal growth. In addition, stringent control of the level of intracellular domain of Notch1 (Notch1-IC) plays a regulatory role in axonal outgrowth during neuronal differentiation. However, whether Cdk5-JIP1-Notch1 cooperate to regulate axonal outgrowth, and the mechanism of such joint contribution to this pathway, is presently unknown, and here we explore their potential interaction. RESULTS: Our interactome screen identified JIP1 as an interactor of p35, a Cdk5 activator, and we sought to explore the relationship between Cdk5 and JIP1 on the regulation of axonal outgrowth. We demonstrate that JIP1 phosphorylated by Cdk5 at Thr205 enhances axonal outgrowth and a phosphomimic JIP1 rescues the axonal outgrowth defects in JIP1-/- and p35-/- neurons. Axonal outgrowth defects caused by the specific increase of Notch1 in JIP1-/- neurons are rescued by Numb-mediated inhibition of Notch1. Finally, we demonstrate that Cdk5 phosphorylation of JIP1 further amplifies the phosphorylation status of yet another Cdk5 substrate E3-ubiquitin ligase Itch, resulting in increased Notch1 ubiquitination. CONCLUSIONS: Our findings identify a potentially critical signaling axis involving Cdk5-JIP1-Itch-Notch1, which plays an important role in the regulation of CNS development. Future investigation into the way this pathway integrates with additional pathways regulating axonal growth will further our knowledge of normal central nervous system development and pathological conditions.

Characteristics of the Ontario Neurodegenerative Disease Research Initiative cohort.

INTRODUCTION: Understanding synergies between neurodegenerative and cerebrovascular pathologies that modify dementia presentation represents an important knowledge gap. METHODS: This multi-site, longitudinal, observational cohort study recruited participants across prevalent neurodegenerative diseases and cerebrovascular disease and assessed participants comprehensively across modalities. We describe univariate and multivariate baseline features of the cohort and summarize recruitment, data collection, and curation processes. RESULTS: We enrolled 520 participants across five neurodegenerative and cerebrovascular diseases. Median age was 69 years, median Montreal Cognitive Assessment score was 25, median independence in activities of daily living was 100% for basic and 93% for instrumental activities. Spousal study partners predominated; participants were often male, White, and more educated. Milder disease stages predominated, yet cohorts reflect clinical presentation. DISCUSSION: Data will be shared with the global scientific community. Within-disease and disease-agnostic approaches are expected to identify markers of severity, progression, and therapy targets. Sampling characteristics also provide guidance for future study design.

Cardiac Pressure Overload Decreases ETV1 Expression in the Left Atrium, Contributing to Atrial Electrical and Structural Remodeling.

BACKGROUND: Elevated intracardiac pressure attributable to heart failure induces electrical and structural remodeling in the left atrium (LA) that begets atrial myopathy and arrhythmias. The underlying molecular pathways that drive atrial remodeling during cardiac pressure overload are poorly defined. The purpose of this study is to characterize the response of the ETV1 (ETS translocation variant 1) signaling axis in the LA during cardiac pressure overload in humans and mouse models and explore the role of ETV1 in atrial electrical and structural remodeling. METHODS: We performed gene expression profiling in 265 left atrial samples from patients who underwent cardiac surgery. Comparative gene expression profiling was performed between 2 murine models of cardiac pressure overload, transverse aortic constriction banding and angiotensin II infusion, and a genetic model of Etv1 cardiomyocyte-selective knockout (Etv1f/fMlc2aCre/+). RESULTS: Using the Cleveland Clinic biobank of human LA specimens, we found that ETV1 expression is decreased in patients with reduced ejection fraction. Consistent with its role as an important mediator of the NRG1 (Neuregulin 1) signaling pathway and activator of rapid conduction gene programming, we identified a direct correlation between ETV1 expression level and NRG1, ERBB4, SCN5A, and GJA5 levels in human LA samples. In a similar fashion to patients with heart failure, we showed that left atrial ETV1 expression is downregulated at the RNA and protein levels in murine pressure overload models. Comparative analysis of LA RNA sequencing datasets from transverse aortic constriction and angiotensin II-treated mice showed a high Pearson correlation, reflecting a highly ordered process by which the LA undergoes electrical and structural remodeling. Cardiac pressure overload produced a consistent downregulation of ErbB4, Etv1, Scn5a, and Gja5 and upregulation of profibrotic gene programming, which includes Tgfbr1/2, Igf1, and numerous collagen genes. Etv1f/fMlc2aCre/+ mice displayed atrial conduction disease and arrhythmias. Correspondingly, the LA from Etv1f/fMlc2aCre/+ mice showed downregulation of rapid conduction genes and upregulation of profibrotic gene programming, whereas analysis of a gain-of-function ETV1 RNA sequencing dataset from neonatal rat ventricular myocytes transduced with Etv1 showed reciprocal changes. CONCLUSIONS: ETV1 is downregulated in the LA during cardiac pressure overload, contributing to both electrical and structural remodeling.

Ionic Mechanisms of Impulse Propagation Failure in the FHF2-Deficient Heart.

RATIONALE: FHFs (fibroblast growth factor homologous factors) are key regulators of sodium channel (NaV) inactivation. Mutations in these critical proteins have been implicated in human diseases including Brugada syndrome, idiopathic ventricular arrhythmias, and epileptic encephalopathy. The underlying ionic mechanisms by which reduced Nav availability in Fhf2 knockout (Fhf2KO) mice predisposes to abnormal excitability at the tissue level are not well defined. OBJECTIVE: Using animal models and theoretical multicellular linear strands, we examined how FHF2 orchestrates the interdependency of sodium, calcium, and gap junctional conductances to safeguard cardiac conduction. METHODS AND RESULTS: Fhf2KO mice were challenged by reducing calcium conductance (gCaV) using verapamil or by reducing gap junctional conductance (Gj) using carbenoxolone or by backcrossing into a cardiomyocyte-specific Cx43 (connexin 43) heterozygous background. All conditions produced conduction block in Fhf2KO mice, with Fhf2 wild-type (Fhf2WT) mice showing normal impulse propagation. To explore the ionic mechanisms of block in Fhf2KO hearts, multicellular linear strand models incorporating FHF2-deficient Nav inactivation properties were constructed and faithfully recapitulated conduction abnormalities seen in mutant hearts. The mechanisms of conduction block in mutant strands with reduced gCaV or diminished Gj are very different. Enhanced Nav inactivation due to FHF2 deficiency shifts dependence onto calcium current (ICa) to sustain electrotonic driving force, axial current flow, and action potential (AP) generation from cell-to-cell. In the setting of diminished Gj, slower charging time from upstream cells conspires with accelerated Nav inactivation in mutant strands to prevent sufficient downstream cell charging for AP propagation. CONCLUSIONS: FHF2-dependent effects on Nav inactivation ensure adequate sodium current (INa) reserve to safeguard against numerous threats to reliable cardiac impulse propagation.

Age-associated insolubility of parkin in human midbrain is linked to redox balance and sequestration of reactive dopamine metabolites.

The mechanisms by which parkin protects the adult human brain from Parkinson disease remain incompletely understood. We hypothesized that parkin cysteines participate in redox reactions and that these are reflected in its posttranslational modifications. We found that in post mortem human brain, including in the Substantia nigra, parkin is largely insoluble after age 40 years; this transition is linked to its oxidation, such as at residues Cys95 and Cys253. In mice, oxidative stress induces posttranslational modifications of parkin cysteines that lower its solubility in vivo. Similarly, oxidation of recombinant parkin by hydrogen peroxide (H2O2) promotes its insolubility and aggregate formation, and in exchange leads to the reduction of H2O2. This thiol-based redox activity is diminished by parkin point mutants, e.g., p.C431F and p.G328E. In prkn-null mice, H2O2 levels are increased under oxidative stress conditions, such as acutely by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine toxin exposure or chronically due to a second, genetic hit; H2O2 levels are also significantly increased in parkin-deficient human brain. In dopamine toxicity studies, wild-type parkin, but not disease-linked mutants, protects human dopaminergic cells, in part through lowering H2O2. Parkin also neutralizes reactive, electrophilic dopamine metabolites via adduct formation, which occurs foremost at the primate-specific residue Cys95. Further, wild-type but not p.C95A-mutant parkin augments melanin formation in vitro. By probing sections of adult, human midbrain from control individuals with epitope-mapped, monoclonal antibodies, we found specific and robust parkin reactivity that co-localizes with neuromelanin pigment, frequently within LAMP-3/CD63+ lysosomes. We conclude that oxidative modifications of parkin cysteines are associated with protective outcomes, which include the reduction of H2O2, conjugation of reactive dopamine metabolites, sequestration of radicals within insoluble aggregates, and increased melanin formation. The loss of these complementary redox effects may augment oxidative stress during ageing in dopamine-producing cells of mutant PRKN allele carriers, thereby enhancing the risk of Parkinson's-linked neurodegeneration.

Early onset epilepsy and sudden unexpected death in epilepsy with cardiac arrhythmia in mice carrying the early infantile epileptic encephalopathy 47 gain-of-function FHF1(FGF12) missense mutation.

OBJECTIVE: Fibroblast growth factor homologous factors (FHFs) are brain and cardiac sodium channel-binding proteins that modulate channel density and inactivation gating. A recurrent de novo gain-of-function missense mutation in the FHF1(FGF12) gene (p.Arg52His) is associated with early infantile epileptic encephalopathy 47 (EIEE47; Online Mendelian Inheritance in Man database 617166). To determine whether the FHF1 missense mutation is sufficient to cause EIEE and to establish an animal model for EIEE47, we sought to engineer this mutation into mice. METHODS: The Arg52His mutation was introduced into fertilized eggs by CRISPR (clustered regularly interspaced short palindromic repeats) editing to generate Fhf1R52H/F+ mice. Spontaneous epileptiform events in Fhf1R52H/+ mice were assessed by cortical electroencephalography (EEG) and video monitoring. Basal heart rhythm and seizure-induced arrhythmia were recorded by electrocardiography. Modulation of cardiac sodium channel inactivation by FHF1BR52H protein was assayed by voltage-clamp recordings of FHF-deficient mouse cardiomyocytes infected with adenoviruses expressing wild-type FHF1B or FHF1BR52H protein. RESULTS: All Fhf1R52H/+ mice experienced seizure or seizurelike episodes with lethal ending between 12 and 26 days of age. EEG recordings in 19-20-day-old mice confirmed sudden unexpected death in epilepsy (SUDEP) as severe tonic seizures immediately preceding loss of brain activity and death. Within 2-53 s after lethal seizure onset, heart rate abruptly declined from 572 ± 16 bpm to 108 ± 15 bpm, suggesting a parasympathetic surge accompanying seizures that may have contributed to SUDEP. Although ectopic overexpression of FHF1BR52H in cardiomyocytes induced a 15-mV depolarizing shift in voltage of steady-state sodium channel inactivation and slowed the rate of channel inactivation, heart rhythm was normal in Fhf1R52H/+ mice prior to seizure. SIGNIFICANCE: The Fhf1 missense mutation p.Arg52His induces epileptic encephalopathy with full penetrance in mice. Both Fhf1 (p.Arg52His) and Scn8a (p.Asn1768Asp) missense mutations enhance sodium channel Nav 1.6 currents and induce SUDEP with bradycardia in mice, suggesting an FHF1/Nav 1.6 functional axis underlying altered brain sodium channel gating in epileptic encephalopathy.

Endoscopy-guided transesophageal echocardiography for large esophageal varices: Use of the "Double Barrel" technique.

Rhythm control strategies in patients with esophageal varices and atrial arrhythmias pose a unique challenge. The left atrium should be imaged for a thrombus prior to attempting cardioversion or ablation, but the presence of varices is a relative contraindication for transesophageal echocardiography. We present a safe, novel technique of evaluating for left atrial thrombus with simultaneous transesophageal echocardiography and esophagogastroduodenoscopy using slim probes in a patient with large, high-risk esophageal varices, and symptomatic atrial flutter with rapid ventricular rates despite medical therapy.

Regulation of myeloid cell phagocytosis by LRRK2 via WAVE2 complex stabilization is altered in Parkinson's disease.

Leucine-rich repeat kinase 2 (LRRK2) has been implicated in both familial and sporadic Parkinson's disease (PD), yet its pathogenic role remains unclear. A previous screen in Drosophila identified Scar/WAVE (Wiskott-Aldrich syndrome protein-family verproline) proteins as potential genetic interactors of LRRK2 Here, we provide evidence that LRRK2 modulates the phagocytic response of myeloid cells via specific modulation of the actin-cytoskeletal regulator, WAVE2. We demonstrate that macrophages and microglia from LRRK2-G2019S PD patients and mice display a WAVE2-mediated increase in phagocytic response, respectively. Lrrk2 loss results in the opposite effect. LRRK2 binds and phosphorylates Wave2 at Thr470, stabilizing and preventing its proteasomal degradation. Finally, we show that Wave2 also mediates Lrrk2-G2019S-induced dopaminergic neuronal death in both macrophage-midbrain cocultures and in vivo. Taken together, a LRRK2-WAVE2 pathway, which modulates the phagocytic response in mice and human leukocytes, may define an important role for altered immune function in PD.

The pro-death role of Cited2 in stroke is regulated by E2F1/4 transcription factors.

We previously reported that the cell cycle-related cyclin-dependent kinase 4-retinoblastoma (RB) transcriptional corepressor pathway is essential for stroke-induced cell death both in vitro and in vivo However, how this signaling pathway induces cell death is unclear. Previously, we found that the cyclin-dependent kinase 4 pathway activates the pro-apoptotic transcriptional co-regulator Cited2 in vitro after DNA damage. In the present study, we report that Cited2 protein expression is also dramatically increased following stroke/ischemic insult. Critically, utilizing conditional knockout mice, we show that Cited2 is required for neuronal cell death, both in culture and in mice after ischemic insult. Importantly, determining the mechanism by which Cited2 levels are regulated, we found that E2F transcription factor (E2F) family members participate in Cited2 regulation. First, E2F1 expression induced Cited2 transcription, and E2F1 deficiency reduced Cited2 expression. Moreover, determining the potential E2F-binding regions on the Cited2 gene regulatory sequence by ChIP analysis, we provide evidence that E2F1/4 proteins bind to this DNA region. A luciferase reporter assay to probe the functional outcomes of this interaction revealed that E2F1 activates and E2F4 inhibits Cited2 transcription. Moreover, we identified the functional binding motif for E2F1 in the Cited2 gene promoter by demonstrating that mutation of this site dramatically reduces E2F1-mediated Cited2 transcription. Finally, E2F1 and E2F4 regulated Cited2 expression in neurons after stroke-related insults. Taken together, these results indicate that the E2F-Cited2 regulatory pathway is critically involved in stroke injury.

Rapid pacing and high-frequency jet ventilation additively improve catheter stability during atrial fibrillation ablation.

INTRODUCTION: Catheter stability during atrial fibrillation ablation is associated with higher ablation success rates. Rapid cardiac pacing and high-frequency jet ventilation (HFJV) independently improve catheter stability. Simultaneous modulation of cardiac and respiratory motion has not been previously studied. The objective of this study was to determine the effect of simultaneous heart rate and respiratory rate modulation on catheter stability. METHODS: Forty patients undergoing paroxysmal atrial fibrillation ablation received ablation lesions at 15 prespecified locations (12 left atria, 3 right atria). Patients were randomly assigned to undergo rapid atrial pacing for either the first or the second half of each lesion. Within each group, half of the patients received HFJV and the other half standard ventilation. Contact force and ablation data for all lesions were compared among the study groups. Standard deviation of contact force was the primary endpoint defined to examine contact force variability. RESULTS: Lesions with no pacing and standard ventilation had the greatest contact force standard deviation (5.86 ± 3.08 g), compared to lesions with pacing and standard ventilation (5.45 ± 3.28 g; P < .01) or to lesions with no pacing and HFJV (4.92 ± 3.00 g; P < .01). Lesions with both pacing and HFJV had the greatest reduction in contact force standard deviation (4.35 ± 2.81 g; P < .01), confirming an additive benefit of each maneuver. Pacing and HFJV together was also associated with a reduction in the proportion of lesions with excessive maximum contact force (P < .001). DISCUSSION: Rapid pacing and HFJV additively improve catheter stability. Simultaneous pacing with HFJV further improves catheter stability over pacing or HFJV alone to optimize ablation lesions.

Comparative analysis of Parkinson's disease-associated genes in mice reveals altered survival and bioenergetics of Parkin-deficient dopamine neurons.

Many mutations in genes encoding proteins such as Parkin, PTEN-induced putative kinase 1 (PINK1), protein deglycase DJ-1 (DJ-1 or PARK7), leucine-rich repeat kinase 2 (LRRK2), and α-synuclein have been linked to familial forms of Parkinson's disease (PD). The consequences of these mutations, such as altered mitochondrial function and pathological protein aggregation, are starting to be better understood. However, little is known about the mechanisms explaining why alterations in such diverse cellular processes lead to the selective loss of dopamine (DA) neurons in the substantia nigra (SNc) in the brain of individuals with PD. Recent work has shown that one of the reasons for the high vulnerability of SNc DA neurons is their high basal rate of mitochondrial oxidative phosphorylation (OXPHOS), resulting from their highly complex axonal arborization. Here, we examined whether axonal growth and basal mitochondrial function are altered in SNc DA neurons from Parkin-, Pink1-, or DJ-1-KO mice. We provide evidence for increased basal OXPHOS in Parkin-KO DA neurons and for reduced survival of DA neurons that have a complex axonal arbor. The surviving smaller neurons exhibited reduced vulnerability to the DA neurotoxin and mitochondrial complex I inhibitor MPP+, and this reduction was associated with reduced expression of the DA transporter. Finally, we found that glial cells play a role in the reduced resilience of DA neurons in these mice and that WT Parkin overexpression rescues this phenotype. Our results provide critical insights into the complex relationship between mitochondrial function, axonal growth, and genetic risk factors for PD.

Pink1 regulates FKBP5 interaction with AKT/PHLPP and protects neurons from neurotoxin stress induced by MPP.

Loss of function mutations in the PTEN-induced putative kinase 1 (Pink1) gene have been linked with an autosomal recessive familial form of early onset Parkinson's disease (PD). However, the underlying mechanism(s) responsible for degeneration remains elusive. Presently, using co-immunoprecipitation in HEK (Human embryonic kidney) 293 cells, we show that Pink1 endogenously interacts with FK506-binding protein 51 (FKBP51 or FKBP5), FKBP5 and directly phosphorylates FKBP5 at Serine in an in vitro kinase assay. Both FKBP5 and Pink1 have been previously associated with protein kinase B (AKT) regulation. We provide evidence using primary cortical cultured neurons from Pink1-deficient mice that Pink1 increases AKT phosphorylation at Serine 473 (Ser473) challenged by 1-methyl-4-phenylpyridinium (MPP+ ) and that over-expression of FKBP5 using an adeno-associated virus delivery system negatively regulates AKT phosphorylation at Ser473 in murine-cultured cortical neurons. Interestingly, FKBP5 over-expression promotes death in response to MPP+ in the absence of Pink1. Conversely, shRNA-mediated knockdown of FKBP5 in cultured cortical neurons is protective and this effect is reversed with inhibition of AKT signaling. In addition, shRNA down-regulation of PH domain leucine-rich repeat protein phosphatase (PHLPP) in Pink1 WT neurons increases neuronal survival, while down-regulation of PHLPP in Pink1 KO rescues neuronal death in response to MPP+ . Finally, using co-immunoprecipitation, we show that FKBP5 interacts with the kinase AKT and phosphatase PHLPP. This interaction is increased in the absence of Pink1, both in Mouse Embryonic Fibroblasts (MEF) and in mouse brain tissue. Expression of kinase dead Pink1 (K219M) enhances FKBP5 interaction with both AKT and PHLPP. Overall, our results suggest a testable model by which Pink1 could regulate AKT through phosphorylation of FKBP5 and interaction of AKT with PHLPP. Our results suggest a potential mechanism by which PINK1-FKBP5 pathway contributes to neuronal death in PD. OPEN SCIENCE BADGES: This article has received a badge for *Open Materials* because it provided all relevant information to reproduce the study in the manuscript. The complete Open Science Disclosure form for this article can be found at the end of the article. More information about the Open Practices badges can be found at https://cos.io/our-services/open-science-badges/.

Mitochondrial dysfunction underlies cognitive defects as a result of neural stem cell depletion and impaired neurogenesis.

Mitochondrial dysfunction is a common feature of many genetic disorders that target the brain and cognition. However, the exact role these organelles play in the etiology of such disorders is not understood. Here, we show that mitochondrial dysfunction impairs brain development, depletes the adult neural stem cell (NSC) pool and impacts embryonic and adult neurogenesis. Using deletion of the mitochondrial oxidoreductase AIF as a genetic model of mitochondrial and neurodegenerative diseases revealed the importance of mitochondria in multiple steps of the neurogenic process. Developmentally, impaired mitochondrial function causes defects in NSC self-renewal, neural progenitor cell proliferation and cell cycle exit, as well as neuronal differentiation. Sustained mitochondrial dysfunction into adulthood leads to NSC depletion, loss of adult neurogenesis and manifests as a decline in brain function and cognitive impairment. These data demonstrate that mitochondrial dysfunction, as observed in genetic mitochondrial and neurodegenerative diseases, underlies the decline of brain function and cognition due to impaired stem cell maintenance and neurogenesis.

LRRK2(I2020T) functional genetic interactors that modify eye degeneration and dopaminergic cell loss in Drosophila.

Progressive degeneration of dopaminergic neurons in the substantia nigra pars compacta is the primary cause for motor symptoms observed in Parkinson's disease (PD). Mutations in leucine-rich repeat kinase 2 (LRRK2) are the most commonly linked contributor to familial PD. LRRK2 is suggested to be involved in a wide variety of cellular processes, but deciphering its role in the pathogenesis of PD has been difficult. Modelling PD in rodents has been a persistent challenge for the field. However, the fruit fly has been exploited to recapitulate PD gene related dopaminergic cell loss. Using the GAL4-UAS system and established models of hLRRK2 induced eye degeneration in Drosophila, we conducted an unbiased suppressor/enhancer screen to uncover genetic modifiers of LRRK2. We have identified 36 candidate interactors that modify LRRK2 induced toxicity in the Drosophila eye. Importantly, we determined that a subset of these interactors also modified hLRRK2(I2020T) induced dopaminergic neuronal loss in the fly brain and uncovered 16 candidates that modify dopaminergic cell loss. Our results suggest LRRK2 may be involved in a wide variety of cellular processes and the results from this screen provide an important genetic resource for further evaluation of LRRK2 function.

Cdc25A Is a Critical Mediator of Ischemic Neuronal Death In Vitro and In Vivo.

Dysregulation of cell cycle machinery is implicated in a number of neuronal death contexts, including stroke. Increasing evidence suggests that cyclin-dependent kinases (Cdks) are inappropriately activated in mature neurons under ischemic stress conditions. We previously demonstrated a functional role for the cyclin D1/Cdk4/pRb (retinoblastoma tumor suppressor protein) pathway in delayed neuronal death induced by ischemia. However, the molecular signals leading to cyclin D/Cdk4/pRb activation following ischemic insult are presently not clear. Here, we investigate the cell division cycle 25 (Cdc25) dual-specificity phosphatases as potential upstream regulators of ischemic neuronal death and Cdk4 activation. We show that a pharmacologic inhibitor of Cdc25 family members (A, B, and C) protects mouse primary neurons from hypoxia-induced delayed death. The major contributor to the death process appears to be Cdc25A. shRNA-mediated knockdown of Cdc25A protects neurons in a delayed model of hypoxia-induced death in vitro Similar results were observed in vivo following global ischemia in the rat. In contrast, neurons singly or doubly deficient for Cdc25B/C were not significantly protective. We show that Cdc25A activity, but not level, is upregulated in vitro following hypoxia and global ischemic insult in vivo Finally, we show that shRNA targeting Cdc25A blocks Ser795 pRb phosphorylation. Overall, our results indicate a role for Cdc25A in delayed neuronal death mediated by ischemia.SIGNIFICANCE STATEMENT A major challenge in stroke is finding an effective neuroprotective strategy to treat cerebral ischemic injury. Cdc25 family member A (Cdc25A) is a phosphatase normally activated during cell division in proliferating cells. We found that Cdc25A is activated in neurons undergoing ischemic stress mediated by hypoxia in vitro and global cerebral ischemia in rats in vivo We show that pharmacologic or genetic inhibition of Cdc25A activity protects neurons from delayed death in vitro and in vivo Downregulation of Cdc25A led to reduction in retinoblastoma tumor suppressor protein (pRb) phosphorylation. An increase in pRb phosphorylation has been previously linked to ischemic neuronal death. Our results identify Cdc25A as a potential target for neuroprotectant strategy for the treatment of delayed ischemic neuronal death.

OPA1-dependent cristae modulation is essential for cellular adaptation to metabolic demand.

Cristae, the organized invaginations of the mitochondrial inner membrane, respond structurally to the energetic demands of the cell. The mechanism by which these dynamic changes are regulated and the consequences thereof are largely unknown. Optic atrophy 1 (OPA1) is the mitochondrial GTPase responsible for inner membrane fusion and maintenance of cristae structure. Here, we report that OPA1 responds dynamically to changes in energetic conditions to regulate cristae structure. This cristae regulation is independent of OPA1's role in mitochondrial fusion, since an OPA1 mutant that can still oligomerize but has no fusion activity was able to maintain cristae structure. Importantly, OPA1 was required for resistance to starvation-induced cell death, for mitochondrial respiration, for growth in galactose media and for maintenance of ATP synthase assembly, independently of its fusion activity. We identified mitochondrial solute carriers (SLC25A) as OPA1 interactors and show that their pharmacological and genetic blockade inhibited OPA1 oligomerization and function. Thus, we propose a novel way in which OPA1 senses energy substrate availability, which modulates its function in the regulation of mitochondrial architecture in a SLC25A protein-dependent manner.