Axonal transport of late endosomes and amphisomes is selectively modulated by local Ca2+ efflux and disrupted by PSEN1 loss of function.

Dysfunction and mistrafficking of organelles in autophagy- and endosomal-lysosomal pathways are implicated in neurodegenerative diseases. Here, we reveal selective vulnerability of maturing degradative organelles (late endosomes/amphisomes) to disease-relevant local calcium dysregulation. These organelles undergo exclusive retrograde transport in axons, with occasional pauses triggered by regulated calcium efflux from agonist-evoked transient receptor potential cation channel mucolipin subfamily member 1 (TRPML1) channels-an effect greatly exaggerated by exogenous agonist mucolipin synthetic agonist 1 (ML-SA1). Deacidification of degradative organelles, as seen after Presenilin 1 (PSEN1) loss of function, induced pathological constitutive "inside-out" TRPML1 hyperactivation, slowing their transport comparably to ML-SA1 and causing accumulation in dystrophic axons. The mechanism involved calcium-mediated c-Jun N-terminal kinase (JNK) activation, which hyperphosphorylated dynein intermediate chain (DIC), reducing dynein activity. Blocking TRPML1 activation, JNK activity, or DIC1B serine-80 phosphorylation reversed transport deficits in PSEN1 knockout neurons. Our results, including features demonstrated in Alzheimer-mutant PSEN1 knockin mice, define a mechanism linking dysfunction and mistrafficking in lysosomal pathways to neuritic dystrophy under neurodegenerative conditions.

Post-Golgi carriers, not lysosomes, confer lysosomal properties to pre-degradative organelles in normal and dystrophic axons.

Lysosomal trafficking and maturation in neurons remain poorly understood and are unstudied in vivo despite high disease relevance. We generated neuron-specific transgenic mice to track vesicular CTSD acquisition, acidification, and traffic within the autophagic-lysosomal pathway in vivo, revealing that mature lysosomes are restricted from axons. Moreover, TGN-derived transport carriers (TCs), not lysosomes, supply lysosomal components to axonal organelles. Ultrastructurally distinctive TCs containing TGN and lysosomal markers enter axons, engaging autophagic vacuoles and late endosomes. This process is markedly upregulated in dystrophic axons of Alzheimer models. In cultured neurons, most axonal LAMP1 vesicles are weakly acidic TCs that shuttle lysosomal components bidirectionally, conferring limited degradative capability to retrograde organelles before they mature fully to lysosomes within perikarya. The minor LAMP1 subpopulation attaining robust acidification are retrograde Rab7+ endosomes/amphisomes, not lysosomes. Restricted lysosome entry into axons explains the unique lysosome distribution in neurons and their vulnerability toward neuritic dystrophy in disease.

Lysosome trafficking and signaling in health and neurodegenerative diseases.

Lysosomes, single-membrane organelles defined by a uniquely strong acidic lumenal pH and high content of acid hydrolases, are the shared degradative compartments of the endocytic and autophagic pathways. These pathways, and especially lysosomes, are points of particular vulnerability in many neurodegenerative diseases. Beyond the role of lysosomes in substrate degradation, new findings have ascribed to lysosomes the leading role in sensing and responding to cellular nutrients, growth factors and cellular stress. This review aims to integrate recent concepts of basic lysosome biology and pathobiology as a basis for understanding neurodegenerative disease pathogenesis. Here, we discuss the newly recognized signaling functions of lysosomes and specific aspects of lysosome biology in neurons while re-visiting the classical defining criteria for lysosomes and the importance of preserving strict definitions. Our discussion emphasizes dynein-mediated axonal transport of maturing degradative organelles, with further consideration of their roles in synaptic function. We finally examine how distinctive underlying disturbances of lysosomes in various neurodegenerative diseases result in unique patterns of auto/endolysosomal mistrafficking. The rapidly emerging understanding of lysosomal trafficking and disruptions in lysosome signaling is providing valuable clues to new targets for disease-modifying therapies.

Outcome of childhood-onset epilepsy from adolescence to adulthood: Transition issues.

This is the second of three papers that summarize the second symposium on Transition in Epilepsies held in Paris in June 2016. This paper addresses the outcome for some particularly challenging childhood-onset epileptic disorders with the goal of recommending the best approach to transition. We have grouped these disorders in five categories with a few examples for each. The first group includes disorders presenting in childhood that may have late- or adult-onset epilepsy (metabolic and mitochondrial disorders). The second group includes disorders with changing problems in adulthood (tuberous sclerosis complex, Rett syndrome, Dravet syndrome, and autism). A third group includes epilepsies that change with age (Childhood Absence Epilepsy, Juvenile Myoclonic Epilepsy, West Syndrome, and Lennox-Gastaut syndrome). A fourth group consists of epilepsies that vary in symptoms and severity depending on the age of onset (autoimmune encephalitis, Rasmussen's syndrome). A fifth group has epilepsy from structural causes that are less likely to evolve in adulthood. Finally we have included a discussion about the risk of later adulthood cerebrovascular disease and dementia following childhood-onset epilepsy. A detailed knowledge of each of these disorders should assist the process of transition to be certain that attention is paid to the most important age-related symptoms and concerns.

Gamma-Hydroxybutyrate (GHB) Content in Hair Samples Correlates Negatively with Age in Succinic Semialdehyde Dehydrogenase Deficiency.

Gamma-hydroxybutyrate (GHB) is a drug of abuse, an approved therapeutic for narcolepsy, an agent employed for facilitation of sexual assault, as well as a biomarker of succinic semialdehyde dehydrogenase deficiency (SSADHD). Our laboratory seeks to identify surrogate biomarkers in SSADHD that can shed light on the developmental course of this neurometabolic disease. Since GHB may be quantified in hair as a potential surrogate to identify victims of drug-related assault, we have opted to examine its level in SSADHD. We quantified GHB in hair derived from ten patients with SSADHD, and documented a significant negative age correlation. These findings are consistent with recent results in patient biological fluids, including plasma and red blood cells. These findings may provide additional insight into the developmental course of SSADHD (Jansen et al., J Inherit Metab Dis 39:795-800, 2016).

Aberrant mTOR signaling and disrupted autophagy: The missing link in potential vigabatrin-associated ocular toxicity?

Vigabatrin (VGB; γ-vinylGABA) is a unique antiepileptic directly elevating CNS GABA via inactivation of the GABA metabolic enzyme GABA-transaminase. VGB is effective in treating infantile spasms, a rare seizure disorder associated with significant morbidity. The potential for unexplained bilateral constriction of the visual field associated with VGB intervention can severely limit its temporal utility. Removal of this potential adverse effect with adjuvant intervention(s) would represent a significant advance in epilepsy therapeutics.

Correlation of blood biomarkers with age informs pathomechanisms in succinic semialdehyde dehydrogenase deficiency (SSADHD), a disorder of GABA metabolism.

We hypothesized that blood levels of γ-aminobutyric acid (GABA) and γ-hydroxybutyric acid (GHB), biomarkers of succinic semialdehyde dehydrogenase deficiency (SSADHD), would correlate with age. GABA and GHB were quantified in plasma and red blood cells (RBCs) from 18 patients (age range 5-41 years; median 8). Both metabolites negatively correlated with age (P < 0.05). Plasma and RBC GHB declined with age, reaching a nadir and approximate steady state by 10 years. Declining plasma GABA achieved this approximate steady state at 30-40 years of age. These biomarker relationships may reflect further GABA- and GHB-ergic neurotransmission imbalances that correlate with the onset of adolescent/adulthood neuropsychiatric morbidity and epilepsy in SSADHD.

Automated seizure detection systems and their effectiveness for each type of seizure.

Epilepsy affects almost 1% of the population and most of the approximately 20-30% of patients with refractory epilepsy have one or more seizures per month. Seizure detection devices allow an objective assessment of seizure frequency and a treatment tailored to the individual patient. A rapid recognition and treatment of seizures through closed-loop systems could potentially decrease morbidity and mortality in epilepsy. However, no single detection device can detect all seizure types. Therefore, the choice of a seizure detection device should consider the patient-specific seizure semiologies. This review of the literature evaluates seizure detection devices and their effectiveness for different seizure types. Our aim is to summarize current evidence, offer suggestions on how to select the most suitable seizure detection device for each patient and provide guidance to physicians, families and researchers when choosing or designing seizure detection devices. Further, this review will guide future prospective validation studies.

Succinic semialdehyde dehydrogenase deficiency (SSADHD): Pathophysiological complexity and multifactorial trait associations in a rare monogenic disorder of GABA metabolism.

Discovered some 35 years ago, succinic semialdehyde dehydrogenase deficiency (SSADHD) represents a rare, autosomal recessively-inherited defect in the second step of the GABA degradative pathway. Some 200 patients have been reported, with broad phenotypic and genotypic heterogeneity. SSADHD represents an unusual neurometabolic disorder in which two neuromodulatory agents, GABA (and the GABA analogue, 4-hydroxybutyrate), accumulate to supraphysiological levels. The unexpected occurrence of epilepsy in several patients is counterintuitive in view of the hyperGABAergic state, in which sedation might be expected. However, the epileptic status of some patients is most likely represented by broader imbalances of GABAergic and glutamatergic neurotransmission. Cumulative research encompassing decades of basic and clinical study of SSADHD reveal a monogenic disease with broad pathophysiological and clinical phenotypes. Numerous metabolic perturbations unmasked in SSADHD include alterations in oxidative stress parameters, dysregulation of autophagy and mitophagy, dysregulation of both inhibitory and excitatory neurotransmitters and gene expression, and unique subsets of SNP alterations of the SSADH gene (so-called ALDH5A1, or aldehyde dehydrogenase 5A1 gene) on the 6p22 chromosomal arm. While seemingly difficult to collate and interpret, these anomalies have continued to open novel pathways for pharmacotherapeutic considerations. Here, we present an update on selected aspects of SSADHD, the ALDH5A1 gene, and future avenues for research on this rare disorder of GABA metabolism.

Effect of extracorporeal CO2 removal on right ventricular and hemodynamic parameters in a patient with acute respiratory distress syndrome.

We present a female patient with severe acute respiratory distress syndrome (ARDS) necessitating intubation and mechanical ventilation on the intensive care unit (ICU). High ventilatory pressures were needed because of hypoxia and severe hypercapnia with respiratory acidosis, resulting in right ventricular dysfunction with impaired haemodynamic stability. A veno-venous extracorporeal CO2 removal (ECCO2R) circuit was initiated, effectively eliminating carbon dioxide while improving oxygenation and enabling a reduction in applied ventilatory pressures. We noted a marked improvement of right ventricular function with restoration of haemodynamic stability. Within one week, the patient was weaned from both ECCO2R and mechanical ventilation. Besides providing adequate gas exchange, extracorporeal assist devices may be helpful in ameliorating right ventricular dysfunction during ARDS.

Presenilin 1 Maintains Lysosomal Ca(2+) Homeostasis via TRPML1 by Regulating vATPase-Mediated Lysosome Acidification.

Presenilin 1 (PS1) deletion or Alzheimer's disease (AD)-linked mutations disrupt lysosomal acidification and proteolysis, which inhibits autophagy. Here, we establish that this phenotype stems from impaired glycosylation and instability of vATPase V0a1 subunit, causing deficient lysosomal vATPase assembly and function. We further demonstrate that elevated lysosomal pH in Presenilin 1 knockout (PS1KO) cells induces abnormal Ca(2+) efflux from lysosomes mediated by TRPML1 and elevates cytosolic Ca(2+). In WT cells, blocking vATPase activity or knockdown of either PS1 or the V0a1 subunit of vATPase reproduces all of these abnormalities. Normalizing lysosomal pH in PS1KO cells using acidic nanoparticles restores normal lysosomal proteolysis, autophagy, and Ca(2+) homeostasis, but correcting lysosomal Ca(2+) deficits alone neither re-acidifies lysosomes nor reverses proteolytic and autophagic deficits. Our results indicate that vATPase deficiency in PS1 loss-of-function states causes lysosomal/autophagy deficits and contributes to abnormal cellular Ca(2+) homeostasis, thus linking two AD-related pathogenic processes through a common molecular mechanism.

New insights into FAK function and regulation during spermatogenesis.

Germ cell transport across the seminiferous epithelium during the epithelial cycle is crucial to spermatogenesis, although molecular mechanism(s) that regulate these events remain unknown. Studies have shown that spatiotemporal expression of crucial regulatory proteins during the epithelial cycle represents an efficient and physiologically important mechanism to regulate spermatogenesis without involving de novo synthesis of proteins and/or expression of genes. Herein, we critically review the role of focal adhesion kinase (FAK) in coordinating the transport of spermatids and preleptotene spermatocytes across the epithelium and the BTB, respectively, along the apical ectoplasmic specialization (ES) - blood-testis barrier - basement membrane (BM) functional axis during spermatogenesis. In the testis, p-FAK-Tyr³⁸⁷ and p-FAK-Tyr⁴⁰⁷ are spatiotemporally expressed during the epithelial cycle at the actin-rich anchoring junction known as ES, regulating cell adhesion at the Sertoli-spermatid (apical ES) and Sertoli cell-cell (basal ES) interface. Phosphorylated forms of FAK exert their effects by regulating the homeostasis of F-actin at the ES, mediated via their effects on actin polymerization so that microfilaments are efficiently re-organized, such as from their "bundled" to "de-bundled/branched" configuration and vice versa during the epithelial cycle to facilitate the transport of: (i) spermatids across the epithelium, and (ii) preleptotene spermatocytes across the BTB. In summary, p-FAK-Tyr⁴⁰⁷ and p-FAK-Tyr³⁸⁷ are important regulators of spermatogenesis which serve as molecular switches that turn "on" and "off" adhesion function at the apical ES and the basal ES/BTB, mediated via their spatiotemporal expression during the epithelial cycle. A hypothetical model depicting the role of these two molecular switches is also proposed.

Cytokines, polarity proteins, and endosomal protein trafficking and signaling-the sertoli cell blood-testis barrier system in vitro as a study model.

Endosomal signaling is emerging as one of the most important cellular events that regulate signaling function in mammalian cells or an epithelium in response to changes in environment such as the presence of stimuli mediated by cytokines, toxicants, heat, ions during growth and development, and other cellular processes such as cytokinesis and spermatogenesis. Recent studies have shown that protein endocytosis-the initial step of endosomal signaling-involves the participation of polarity proteins, such as partitioning defective protein 6 (Par6), Cdc42 and 14-3-3 (also known as Par5), which in turn is regulated by cytokines (e.g., TGF-ß2, TGF-ß3) and testosterone at the Sertoli cell blood-testis barrier (BTB) in the mammalian testis. In this short method paper, we provide a detailed protocol of assessing protein endocytosis, the initial and also the most critical step of endosomal signaling at the Sertoli cell BTB. This biochemical endocytosis assay summarizes our experience for the last decade, which should likely be performed in conjunction with the dual-labeled immunofluorescence analysis to assess protein endocytosis. While we are using a Sertoli cell in vitro system that mimics the BTB in vivo, this approach should be applicable to virtually all mammalian cells.

Myc inhibition impairs autophagosome formation.

Autophagy, a major clearance route for many long-lived proteins and organelles, has long been implicated in cancer development. Myc is a proto-oncogene often found to be deregulated in many cancers, and thus is an attractive target for design of cancer therapy. Therefore, understanding the relationship between anti-Myc strategies and autophagy will be important for development of effective therapy. Here, we show that Myc depletion inhibits autophagosome formation and impairs clearance of autophagy substrates. Myc suppression has an inhibitory effect on autophagy via reduction of c-Jun N-terminal kinase 1 (JNK1) and B-cell lymphoma 2 (Bcl2) phosphorylation. Additionally, the decrease in JNK1 phosphorylation observed with Myc knockdown is associated with a reduction in ROS production. Our data suggest that targeting Myc in cancer therapy might have the additional benefit of inhibiting autophagy in the case of therapy resistance associated with chemotherapy-induced autophagy.

Palladin is a regulator of actin filament bundles at the ectoplasmic specialization in adult rat testes.

In rat testes, the ectoplasmic specialization (ES) at the Sertoli-Sertoli and Sertoli-spermatid interface known as the basal ES at the blood-testis barrier and the apical ES in the adluminal compartment, respectively, is a testis-specific adherens junction. The remarkable ultrastructural feature of the ES is the actin filament bundles that sandwiched in between the cisternae of endoplasmic reticulum and apposing plasma membranes. Although these actin filament bundles undergo extensive reorganization to switch between their bundled and debundled state to facilitate blood-testis barrier restructuring and spermatid adhesion/transport, the regulatory molecules underlying these events remain unknown. Herein we report findings of an actin filament cross-linking/bundling protein palladin, which displayed restrictive spatiotemporal expression at the apical and the basal ES during the epithelial cycle. Palladin structurally interacted and colocalized with Eps8 (epidermal growth factor receptor pathway substrate 8, an actin barbed end capping and bundling protein) and Arp3 (actin related protein 3, which together with Arp2 form the Arp2/3 complex to induce branched actin nucleation, converting bundled actin filaments to an unbundled/branched network), illustrating its role in regulating actin filament bundle dynamics at the ES. A knockdown of palladin in Sertoli cells in vitro with an established tight junction (TJ)-permeability barrier was found to disrupt the TJ function, which was associated with a disorganization of actin filaments that affected protein distribution at the TJ. Its knockdown in vivo also perturbed F-actin organization that led to a loss of spermatid polarity and adhesion, causing defects in spermatid transport and spermiation. In summary, palladin is an actin filament regulator at the ES.

Signalling pathways regulating the blood-testis barrier.

Throughout mammalian spermatogenesis, preleptotene/leptotene spermatocytes traverse the blood-testis barrier during stages VIII-XI of the seminiferous epithelial cycle while trapped within a dynamic intermediate compartment that is sealed at north and south poles by tight junctions, basal ectoplasmic specializations, desmosomes and gap junctions. In order for spermatocytes to gain entry into the adluminal compartment of the seminiferous epithelium for continued development, 'old' junctions present above migrating spermatocytes disassemble, while 'new' junctions assemble simultaneously below these germ cells. In this way, the integrity of the blood-testis barrier and the homeostasis of the seminiferous epithelium can remain intact during spermatogenesis. Previous studies have shown an array of cellular events, including protein internalization and cytoskeletal remodeling, to underline blood-testis barrier restructuring, whereas other studies have reported BTB dysfunction to associate with activation of the p38 mitogen-activated protein kinase pathway. Herein, we discuss the signaling pathways and mechanisms involved in blood-testis barrier restructuring in the mammalian testis.

Focal adhesion kinase and actin regulatory/binding proteins that modulate F-actin organization at the tissue barrier: Lesson from the testis.

Focal adhesion kinase (FAK), as its name implied, is an important mediator of integrin-based signaling function in mammalian cells at the focal adhesion complex (FAC, also known as focal contact) at the cell-extracellular matrix interface. FAK is intimately related to cell movement, such as in macrophages, fibroblasts and also tumor cells. In the testis, however, FAK and two of its phosphorylated forms, p-FAK-Tyr(407) and -Tyr(397), are not found at the FAC since there is no ultrastructure analogous or similar to FAC in the mammalian testis vs. other epithelia. Instead, FAK and its two phosphorylated forms are detected along the seminiferous epithelium in the rat testis at the cell-cell interface in a testis-specific adherens junction (AJ) known as the ectoplasmic specialization (ES). ES is an F-actin-rich ultrastructure in which bundles of actin filaments are sandwiched in-between plasma membrane and cisternae of endoplasmic reticulum not found in other mammalian epithelial/endothelial cells. The ES is restricted to the interface of Sertoli cells and spermatids (step 8-19) known as the apical ES, and to the Sertoli cell-cell interface known as the basal ES. Interestingly, the basal ES is also an integrated component of the blood-testis barrier (BTB), coexisting with tight junction (TJ) and gap junction (GJ), and it is conceivable that actin filament bundles at the ES undergo extensive organization, converting from their "bundled" to "de-bundled/branching" configuration to facilitate transport of germ cells across the epithelium and at the BTB during the epithelial cycle. A recent report (Lie et al. PNAS 109:12562-12567, 2012) has demonstrated that the stage-specific and spatiotemporal expression of p-FAK-Tyr(407) and -Tyr(397) are crucial to the regulation of these events via their stage-specific and spatiotemporal expression during the epithelial cycle mediated by their effects on the organization of the actin filament bundles at the ES, involving actin binding/regulatory proteins. In this Commentary, we will critically evaluate these findings in light of other recent reports in the field. While these ideas are based on studies in the BTB in the rat testis, this information should be applicable and helpful to investigators studying other tissue barriers.