Synthesis and regulation of catecholamine neurotransmitters in the central nervous system are implicated in the pathogenesis of a number of neuropsychiatric disorders. To identify factors that regulate the presynaptic synthesis of catecholamines, we tested the hypothesis that the rate-limiting enzyme of the catecholamine biosynthetic pathway, tyrosine hydroxylase (TH), is locally synthesized in axons and presynaptic nerve terminals of noradrenergic neurons. To isolate pure axonal mRNA and protein, rat superior cervical ganglion sympathetic neurons were cultured in compartmentalized Campenot chambers. qRT-PCR and RNA in situ hybridization analyses showed that TH mRNA is present in distal axons. Colocalization experiments with nerve terminal marker proteins suggested that both TH mRNA and protein localize in regions of the axon that resemble nerve terminals (i.e., synaptic boutons). Analysis of polysome-bound RNA showed that TH mRNA is present in polysomes isolated from distal axons. Metabolic labeling of axonally synthesized proteins labeled with the methionine analog, L-azidohomoalanine, showed that TH is locally synthesized in axons. Moreover, the local transfection and translation of exogenous TH mRNA into distal axons facilitated axonal dopamine synthesis. Finally, using chimeric td-Tomato-tagged constructs, we identified a sequence element within the TH 3 ′ UTR that is required for the axonal localization of the reporter mRNA. Taken together, our results provide the first direct evidence that TH mRNA is trafficked to the axon and that the mRNA is locally translated. These findings raise the interesting possibility that the biosynthesis of the catecholamine neurotransmitters is locally regulated in the axon and/or presynaptic nerve terminal.

No abstract

No abstract

No abstract

Background: The cardiac dyad and associated calcium (Ca 2+ ) release units (CRUs) are integral for cardiac excitation-contraction (E-C) coupling. The cardiac dyad is a multicomponent complex composed of the transverse-tubules, junctional sarcoplasmic reticulum (jSR), and CRU-associated proteins. Proper E-C coupling requires Ca 2+ influx via long-lasting-L-type Ca 2+ channels (Cav1.2), which triggers Ca 2+ release from adjacent ryanodine receptor 2 (RyR2) located in jSR. βII-spectrin loss alters RyR2 localization, but its contribution to cardiac dyad organization and E-C coupling remains unclear. Hypothesis: We hypothesized that βII-spectrin is required for cardiac dyad organization and proper cardiomyocyte calcium signaling. Methods: Cardiomyocyte-specific βII-spectrin knockout (βII-cKO) and control mice (βII-flox) were generated and harvested for immunoblotting, qRT-PCR, and imaging by transmission electron microscopy (TEM). Mice hearts were also interrogated by echocardiography following transaortic constriction. Results: Cardiac dyad structure and CRU components are significantly altered in βII-cKO hearts. Ultrastructural analysis of βII-cKOhearts by TEM showed fragmented cardiac dyads, dilated jSR and reduced jSR contact with T-tubules. Quantitative RT-PCR showed increased expression of calsequestrin 1 and 2, decreased phospholambam, Ncx and Cacna1c (major Cav1.2 subunit), while Ryr2 , Jph2 , Serca2a are unchanged. Protein expression of NCX was decreased, while expression of CAV1.2, RYR2, CASQ2, and SERCA2A remained unchanged. Furthermore, pressure-induced overload of βII-cKO hearts promoted accelerated heart failure (HF). Conclusions: This is the first demonstration that the βII-spectrin cytoskeletal protein is critically important for maintaining the structural integrity of the cardiac dyad. βII-spectrin loss and the resulting alterations in CRU structure, genes, and protein composition promoting accelerated HF.

No abstract