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 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

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

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