RESUMEN

Neural circuits, which constitute the substrate for brain processing, can be traced in the retrograde direction, from postsynaptic to presynaptic cells, using methods based on introducing modified rabies virus into genetically marked cell types. These methods have revolutionized the field of neuroscience. However, similarly reliable, transsynaptic, and non-toxic methods to trace circuits in the anterograde direction are not available. Here, we describe such a method based on an antibody-like protein selected against the extracellular N-terminus of the AMPA receptor subunit GluA1 (AMPA.FingR). ATLAS (Anterograde Transsynaptic Label based on Antibody-like Sensors) is engineered to release the AMPA.FingR and its payload, which can include Cre recombinase, from presynaptic sites into the synaptic cleft, after which it binds to GluA1, enters postsynaptic cells through endocytosis and subsequently carries its payload to the nucleus. Testing in vivo and in dissociated cultures shows that ATLAS mediates monosynaptic tracing from genetically determined cells that is strictly anterograde, synaptic, and non-toxic. Moreover, ATLAS shows activity dependence, which may make tracing active circuits that underlie specific behaviors possible.

RESUMEN

The genus Toxicodryas, historically included with the renowned Australasian cat-eyed snakes of the colubrid genus Boiga, currently includes two widespread species (T. blandingii and T. pulverulenta) in western, central, and eastern Africa. We leverage findings from a recent phylogenomic and historical demographic analysis of this genus (based on 2848-4471 Rad-seq loci from across the genome), with robust sampling from throughout the ranges of both species, to define two additional taxonomic units, with species boundaries corresponding to river barriers. Additional morphometric data from scores of examined museum specimens and literature records bolster the recognition of these two new cryptic species. We hypothesize that T. blandingii occurs west of the confluence of the Congo and Ubangi rivers, whereas a cryptic new species that is found east of this biogeographic barrier has significantly higher numbers of ventral scale counts in both sexes, additional significant differences in several scale counts, and lower venom toxicity. Toxicodryas pulverulenta occurs west of the Niger Delta in West Africa, whereas a cryptic new species that is found east of this biogeographic barrier has significantly higher numbers of subcaudal scale counts in both sexes. A review of published information regarding morphological variation, ecology, natural history, habitat, and venom is summarized for these four Toxicodryas species.

Asunto(s)

Colubridae , África Central , Animales , Colubridae/anatomía & histología , Colubridae/clasificación , Femenino , Masculino , Filogenia , Especificidad de la Especie

RESUMEN

The islet in type 2 diabetes (T2D) shares many features of the brain in protein misfolding diseases. There is a deficit of ß cells with islet amyloid derived from islet amyloid polypeptide (IAPP), a protein coexpressed with insulin. Small intracellular membrane-permeant oligomers, the most toxic form of IAPP, are more frequent in ß cells of patients with T2D and rodents expressing human IAPP. ß Cells in T2D, and affected cells in neurodegenerative diseases, share a comparable pattern of molecular pathology, including endoplasmic reticulum stress, mitochondrial dysfunction, attenuation of autophagy, and calpain hyperactivation. While this adverse functional cascade in response to toxic oligomers is well described, the sequence of events and how best to intervene is unknown. We hypothesized that calpain hyperactivation is a proximal event and tested this in vivo by ß cell-specific suppression of calpain hyperactivation with calpastatin overexpression in human IAPP transgenic mice. ß Cell-specific calpastatin overexpression was remarkably protective against ß cell dysfunction and loss and diabetes onset. The critical autophagy/lysosomal pathway for ß cell viability was protected with calpain suppression, consistent with findings in models of neurodegenerative diseases. We conclude that suppression of calpain hyperactivation is a potentially beneficial disease-modifying strategy for protein misfolding diseases, including T2D.

Asunto(s)

Proteínas de Unión al Calcio/metabolismo , Diabetes Mellitus Tipo 2/prevención & control , Células Secretoras de Insulina/metabolismo , Polipéptido Amiloide de los Islotes Pancreáticos/efectos adversos , Animales , Calpaína/metabolismo , Diabetes Mellitus Tipo 2/inducido químicamente , Femenino , Humanos , Masculino , Ratones , Ratones Transgénicos

RESUMEN

BACKGROUND: Performance-based incentives (PBIs) have garnered global attention as a promising strategy to improve healthcare delivery to vulnerable populations. However, literature gaps in the context in which an intervention is implemented and how the PBIs were developed exist. Therefore, we (1) characterized the barriers and promoters to prevention of vertical transmission of HIV (PVT) service delivery in rural Mozambique, where the vertical transmission rate is 12 %, and (2) assessed the appropriateness for a PBI's intervention and application to PVT. METHODS: We conducted 24 semi-structured interviews with nurses, volunteers, community health workers, and traditional birth attendants about the barriers and promoters they experienced delivering PVT services. We then explored emergent themes in subsequent focus group discussions (n = 7, total participants N = 92) and elicited participant perspectives on PBIs. The ecological motivation-opportunity-ability framework guided our iterative data collection and thematic analysis processes. RESULTS: The interviews revealed that while all health worker cadres were motivated intrinsically and by social recognition, they were dissatisfied with low and late remuneration. Facility-based staff were challenged by factors across the rest of the ecological levels, primarily in the opportunity domain, including the following: poor referral and record systems (work mandate), high workload, stock-outs, poor infrastructure (facility environment), and delays in obtaining patient results and donor payment discrepancies (administrative). Community-based cadres' opportunity challenges included lack of supplies, distance (work environment), lack of incorporation into the health system (administration), and ability challenges of incorrect knowledge (health worker). PBIs based on social recognition and that enable action on intrinsic motivation through training, supervision, and collaboration were thought to have the most potential for targeting improvements in record and referral systems and better integrating community-based health workers into the health system. Concerns about the implementation of incentives included neglect of non-incentivized tasks and distorted motivation among colleagues. CONCLUSIONS: We found that highly motivated health workers encountered severe opportunity challenges in their PVT mandate. PBIs have the potential to address key barriers that facility- and community-based health workers encounter when delivering PVT services, specifically by building upon existing intrinsic motivation and leveraging highly valued social recognition. We recommend a controlled intervention to monitor incentives' effects on worker motivation and non-incentivized tasks to generate insights about the feasibility of PBIs to improve the delivery of PVT services.

Asunto(s)

Actitud del Personal de Salud , Infecciones por VIH/prevención & control , Transmisión Vertical de Enfermedad Infecciosa/prevención & control , Motivación , Administración de Personal/métodos , Remuneración , Servicios de Salud Rural , Adulto , Agentes Comunitarios de Salud , Femenino , Grupos Focales , Humanos , Masculino , Partería , Mozambique , Enfermeras y Enfermeros , Investigación Cualitativa , Recompensa , Población Rural , Voluntarios

RESUMEN

Type 2 diabetes (T2D) is characterized by a deficiency in ß cell mass, increased ß cell apoptosis, and extracellular accumulation of islet amyloid derived from islet amyloid polypeptide (IAPP), which ß cells coexpress with insulin. IAPP expression is increased in the context of insulin resistance, the major risk factor for developing T2D. Human IAPP is potentially toxic, especially as membrane-permeant oligomers, which have been observed to accumulate within ß cells of patients with T2D and rodents expressing human IAPP. Here, we determined that ß cell IAPP content is regulated by autophagy through p62-dependent lysosomal degradation. Induction of high levels of human IAPP in mouse ß cells resulted in accumulation of this amyloidogenic protein as relatively inert fibrils within cytosolic p62-positive inclusions, which temporarily averts formation of toxic oligomers. Mice hemizygous for transgenic expression of human IAPP did not develop diabetes; however, loss of ß cell-specific autophagy in these animals induced diabetes, which was attributable to accumulation of toxic human IAPP oligomers and loss of ß cell mass. In human IAPP-expressing mice that lack ß cell autophagy, increased oxidative damage and loss of an antioxidant-protective pathway appeared to contribute to increased ß cell apoptosis. These findings indicate that autophagy/lysosomal degradation defends ß cells against proteotoxicity induced by oligomerization-prone human IAPP.

Asunto(s)

Autofagia/fisiología , Células Secretoras de Insulina/patología , Células Secretoras de Insulina/fisiología , Polipéptido Amiloide de los Islotes Pancreáticos/fisiología , Proteínas Adaptadoras Transductoras de Señales/deficiencia , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Diabetes Mellitus Tipo 2/patología , Diabetes Mellitus Tipo 2/fisiopatología , Proteínas de Choque Térmico/deficiencia , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Humanos , Insulina/metabolismo , Polipéptido Amiloide de los Islotes Pancreáticos/química , Polipéptido Amiloide de los Islotes Pancreáticos/genética , Lisosomas/metabolismo , Masculino , Ratones , Ratones Noqueados , Ratones Transgénicos , Factor 2 Relacionado con NF-E2/metabolismo , Estrés Oxidativo , Estructura Cuaternaria de Proteína , Proteolisis , Ratas , Ratas Transgénicas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteína Sequestosoma-1

RESUMEN

The islet in type 2 diabetes mellitus (T2DM) is characterized by a deficit in ß-cells and increased ß-cell apoptosis attributable at least in part to intracellular toxic oligomers of IAPP (islet amyloid polypeptide). ß-cells of individuals with T2DM are also characterized by accumulation of polyubiquitinated proteins and deficiency in the deubiquitinating enzyme UCHL1 (ubiquitin carboxyl-terminal esterase L1 [ubiquitin thiolesterase]), accounting for a dysfunctional ubiquitin/proteasome system. In the present study, we used mouse genetics to elucidate in vivo whether a partial deficit in UCHL1 enhances the vulnerability of ß-cells to human-IAPP (hIAPP) toxicity, and thus accelerates diabetes onset. We further investigated whether a genetically induced deficit in UCHL1 function in ß-cells exacerbates hIAPP-induced alteration of the autophagy pathway in vivo. We report that a deficit in UCHL1 accelerated the onset of diabetes in hIAPP transgenic mice, due to a decrease in ß-cell mass caused by increased ß-cell apoptosis. We report that UCHL1 dysfunction aggravated the hIAPP-induced defect in the autophagy/lysosomal pathway, illustrated by the marked accumulation of autophagosomes and cytoplasmic inclusions positive for SQSTM1/p62 and polyubiquitinated proteins with lysine 63-specific ubiquitin chains. Collectively, this study shows that defective UCHL1 function may be an early contributor to vulnerability of pancreatic ß-cells for protein misfolding and proteotoxicity, hallmark defects in islets of T2DM. Also, given that deficiency in UCHL1 exacerbated the defective autophagy/lysosomal degradation characteristic of hIAPP proteotoxicity, we demonstrate a previously unrecognized role of UCHL1 in the function of the autophagy/lysosomal pathway in ß-cells.

Asunto(s)

Autofagia/fisiología , Células Secretoras de Insulina/metabolismo , Células Secretoras de Insulina/patología , Polipéptido Amiloide de los Islotes Pancreáticos/metabolismo , Ubiquitina Tiolesterasa/deficiencia , Animales , Apoptosis , Autofagia/genética , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/patología , Humanos , Resistencia a la Insulina , Polipéptido Amiloide de los Islotes Pancreáticos/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Mutantes , Ratones Transgénicos , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Ubiquitina/metabolismo , Ubiquitina Tiolesterasa/genética , Ubiquitina Tiolesterasa/metabolismo

RESUMEN

Here we present evidence that the kinesin, Kif5B, is involved in the transportation and axonal targeting of Kv1 channels. We show that a dominant negative variant of Kif5B specifically blocks localization to the axon of expressed, tagged versions of Kv1.3 in cultured cortical slices. In addition, the dominant negative variant of Kif5B blocks axonal localization of endogenous Kv1.1, Kv1.2, and Kv1.4 in cortical neurons in dissociated cultures. We also found evidence that Kif5B interacts with Kv1 channels. Endogenous Kv1.2 colocalized with Kif5B in cortical neurons and coimmunoprecipitated with Kif5B from brain lysate. The T1 domain of Shaker K(+) channels has been shown to play a critical role in targeting the channel to the axon. We have three pieces of evidence to suggest that the T1 domain also mediates interaction between Kv1 channels and Kif5B: Addition of the T1 domain to a heterologous protein, TfR, is sufficient to cause the resulting fusion protein, TfRT1, to colocalize with Kif5B. Also, the T1 domain is necessary for interaction of Kv1.3 with Kif5B in a coimmunoprecipitation assay. Finally, dominant negative variants of Kif5B block axonal targeting of TfRT1, but have no effect on dendritic localization of TfR. Together these data suggest a model where Kif5B interacts with Kv1 channels either directly or indirectly via the T1 domain, causing the channels to be transported to axons.

Asunto(s)

Axones/metabolismo , Cinesinas/fisiología , Canal de Potasio Kv.1.1/metabolismo , Animales , Western Blotting/métodos , Células Cultivadas , Corteza Cerebral/citología , Chlorocebus aethiops , Cricetinae , Embrión de Mamíferos , Femenino , Regulación de la Expresión Génica , Proteínas Fluorescentes Verdes/metabolismo , Inmunohistoquímica/métodos , Inmunoprecipitación/métodos , Técnicas In Vitro , Proteínas Asociadas a Microtúbulos/metabolismo , Neuronas/citología , Embarazo , Transporte de Proteínas/fisiología , Ratas , Transfección

RESUMEN

Although kinesins are known to transport neuronal proteins, it is not known what role they play in the targeting of their cargos to specific subcellular compartments in neurons. Here we present evidence that the K+ channel Kv4.2, which is a major regulator of dendritic excitability, is transported to dendrites by the kinesin isoform Kif17. We show that a dominant negative construct against Kif17 dramatically inhibits localization to dendrites of both introduced and endogenous Kv4.2, but those against other kinesins found in dendrites do not. Kv4.2 colocalizes with Kif17 but not with other kinesin isoforms in dendrites of cortical neurons. Native Kv4.2 and Kif17 coimmunoprecipitate from brain lysate, and introduced, tagged versions of the two proteins coimmunoprecipitate from COS cell lysate, indicating that the two proteins interact, either directly or indirectly. The interaction between Kif17 and Kv4.2 appears to occur through the extreme C terminus of Kv4.2 and not through the dileucine motif. Thus, the dileucine motif does not determine the localization of Kv4.2 by causing the channel to interact with a specific motor protein. In support of this conclusion, we found that the dileucine motif mediates dendritic targeting of CD8 independent of Kif17. Together our data show that Kif17 is probably the motor that transports Kv4.2 to dendrites but suggest that this motor does not, by itself, specify dendritic localization of the channel.

Asunto(s)

Dendritas/metabolismo , Cinesinas/metabolismo , Proteínas Motoras Moleculares/metabolismo , Canales de Potasio Shal/metabolismo , Animales , Transporte Biológico/fisiología , Células COS , Células Cultivadas , Corteza Cerebral/citología , Chlorocebus aethiops , Femenino , Cinesinas/genética , Leucina/metabolismo , Ratones , Ratones Endogámicos , Proteínas Motoras Moleculares/genética , Neuronas/metabolismo , Neuronas/ultraestructura , Técnicas de Cultivo de Órganos , Embarazo , Ratas , Ratas Sprague-Dawley , Canales de Potasio Shal/genética , Transfección

RESUMEN

Shaker K+ channels play an important role in modulating electrical excitability of axons. Recent work has demonstrated that the T1 tetramerization domain of Kv1.2 is both necessary and sufficient for targeting of the channel to the axonal surface [Gu, C., Jan, Y.N. & Jan, L.Y. (2003) Science,301, 646-649]. Here we use a related channel, Kv1.3, as a model to investigate cellular mechanisms that mediate axonal targeting. We show that the T1 domain of Kv1.3 is necessary and sufficient to mediate targeting of the channel to the axonal surface in pyramidal neurons in slices of cortex from neonatal rat. The T1 domain is also sufficient to cause preferential axonal localization of intracellular protein, which indicates that the domain probably does not work through compartment-specific endocytosis or compartment-specific vesicle docking. To determine whether the T1 domain mediates axonal trafficking of transport vesicles, we compared the trafficking of vesicles containing green fluorescent protein-labelled transferrin receptor with those containing the same protein fused with the T1 domain in living cortical neurons. Vesicles containing the wild-type transferrin receptor did not traffic to the axon, in accord with previously published results; however, those containing the transferrin receptor fused to T1 did traffic to the axon. These results are consistent with the T1 domain of Kv1.3 mediating axonal targeting by causing transport vesicles to traffic to axons and they represent the first evidence that such a mechanism might underlie axonal targeting.

Asunto(s)

Axones/fisiología , Espacio Extracelular/fisiología , Neuronas/citología , Canales de Potasio Shaw/fisiología , Animales , Animales Recién Nacidos , Axones/efectos de los fármacos , Células Cultivadas , Corteza Cerebral/citología , Diagnóstico por Imagen/métodos , Embrión de Mamíferos , Regulación de la Expresión Génica/genética , Proteínas Fluorescentes Verdes/metabolismo , Inmunohistoquímica/métodos , Técnicas In Vitro , Modelos Moleculares , Mutagénesis/fisiología , Neuronas/fisiología , Estructura Terciaria de Proteína/fisiología , Ratas , Ratas Sprague-Dawley , Canales de Potasio Shaw/química , Transfección/métodos

RESUMEN

The molecular mechanisms underlying polarized sorting of proteins in neurons are poorly understood. Here we report the identification of a 16 amino-acid, dileucine-containing motif that mediates dendritic targeting in a variety of neuronal cell types in slices of rat brain. This motif is present in the carboxy (C) termini of Shal-family K+ channels and is highly conserved from C. elegans to humans. It is necessary for dendritic targeting of potassium channel Kv4.2 and is sufficient to target the axonally localized channels Kv1.3 and Kv1.4 to the dendrites. It can also mediate dendritic targeting of a non-channel protein, CD8.

Asunto(s)

Dendritas/metabolismo , Canales de Potasio con Entrada de Voltaje , Canales de Potasio/metabolismo , Secuencias de Aminoácidos/genética , Secuencias de Aminoácidos/fisiología , Secuencia de Aminoácidos , Animales , Antígenos CD8/genética , Antígenos CD8/metabolismo , Células Cultivadas , Secuencia Conservada , Endocitosis , Técnicas In Vitro , Canal de Potasio Kv1.3 , Canal de Potasio Kv1.4 , Leucina , Datos de Secuencia Molecular , Canales de Potasio/genética , Transporte de Proteínas/fisiología , Células Piramidales/metabolismo , Ratas , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Canales de Potasio Shal