Maintenance of respiratory chain function in mouse hearts with severely impaired mtDNA transcription

The basal mitochondrial transcription machinery is essential for biogenesis of the respiratory chain and consists of mitochondrial RNA polymerase, mitochondrial transcription factor A (TFAM) and mitochondrial transcription factor B2. This triad of proteins is sufficient and necessary for mtDNA transcription initiation. Abolished mtDNA transcription caused by tissue-specific knockout of TFAM in the mouse heart leads to early onset of a severe mitochondrial cardiomyopathy with lethality within the first post-natal weeks. Here, we describe a mouse model expressing human TFAM instead of the endogenous mouse TFAM in heart. These rescue mice have severe reduction in mtDNA transcription initiation, but, surprisingly, are healthy at the age of 52 weeks with near-normal steady-state levels of transcripts. In addition, we demonstrate that heavy-strand mtDNA transcription normally terminates at the termination-associated sequence in the control region. This termination is abolished in rescue animals resulting in heavy (H)-strand transcription of the entire control region. In conclusion, we demonstrate here the existence of an unexpected mtDNA transcript stabilization mechanism that almost completely compensates for the severely reduced transcription initiation in rescue hearts. Future elucidation of the underlying molecular mechanism may provide a novel pathway to treat mitochondrial dysfunction in human pathology

Mitochondrial DNA polymerase gamma is essential for mammalian embryogenesis,”

Abstract Mitochondrial DNA polymerase gamma (Polg) is a heterodimeric enzyme containing a Pol I-like catalytic core (PolgA) and an accessory subunit (PolgB). Mutations in POLGA, affecting the stability of mtDNA, have been identified in several human pathologies such as progressive external ophthalmoplegia (PEO) and Alpers' syndrome. Extensive literature shows mitochondrial toxicity effects nucleoside analogue reverse transcriptase inhibitors (NRTIs) used in the treatment of HIV and chronic hepatitis B as a consequence of an inhibitory effect on Polg. We have previously shown that mice with an error-prone version of PolgA accumulate higher levels of somatic mtDNA mutations resulting in a premature aging phenotype. In the present paper we demonstrate PolgA deficiency in mouse embryos causes an early developmental arrest between embryonic day 7.5-8.5 associated with severe mtDNA depletion. Heterozygous knockout mice have half the wild-type levels of PolgA transcripts and a slight reduction in mtDNA levels, but develop normally. Surprisingly, amounts of PolgA transcripts in heterozygous knockout mice are increased in response to artificially elevated mtDNA copy number, revealing a possible regulatory link between mtDNA maintenance and PolgA expression. Our results show that Polg indeed is the only DNA polymerase capable of maintaining mtDNA in mammalian mitochondria. Furthermore, presence of Polg is absolutely essential for the organogenesis during mammalian embryonic development.

Viral labeling of neurons synaptically connected to nucleus accumbens somatostatin interneurons.

The nucleus accumbens, a key brain reward region, receives synaptic inputs from a range of forebrain and brainstem regions. Many of these projections have been established using electrophysiology or fluorescent tract tracing. However, more recently developed viral tracing techniques have allowed for fluorescent labeling of synaptic afferents in a cell type-specific manner. Since the NAc is comprised of multiple cell types, these methods have enabled the delineation of the cell type-specific connectivity of principal medium spiny neurons in the region. The synaptic connectivity of somatostatin interneurons, which account for <5% of the neurons in the region, has been inferred from electrophysiological and immunohistochemical data, but has not yet been visualized using modern viral tracing techniques. Here, we use the pseudorabies virus (PRV)-Introvert-GFP virus, an alphaherpes virus previously shown to label synaptic afferents in a cell type-specific manner, to label first order afferents to NAc somatostatin interneurons. While we find GFP(+) labeling in several well established projections to the NAc, we also observe that several known projections to NAc did not contain GFP(+) cells, suggesting they do not innervate somatostatin interneurons in the region. A subset of the GFP(+) afferents are c-FOS(+) following acute administration of cocaine, showing that NAc somatostatin interneurons are innervated by some cells that respond to rewarding stimuli. These results provide a foundation for future studies aimed toward elucidating the cell type-specific connectivity of the NAc, and identify specific circuits that warrant future functional characterization

Molecular Profiling of Neurons Based on Connectivity

The complexity and cellular heterogeneity of neural circuitry presents a major challenge to understanding the role of discrete neural populations in controlling behavior. While neuroanatomical methods enable high-resolution mapping of neural circuitry, these approaches do not allow systematic molecular profiling of neurons based on their connectivity. Here, we report the development of an approach for molecularly profiling projective neurons. We show that ribosomes can be tagged with a camelid nanobody raised against GFP and that this system can be engineered to selectively capture translating mRNAs from neurons retrogradely labeled with GFP. Using this system, we profiled neurons projecting to the nucleus accumbens. We then used an AAV to selectively profile midbrain dopamine neurons projecting to the nucleus accumbens. By comparing the captured mRNAs from each experiment, we identified a number of markers specific to VTA dopaminergic projection neurons. The current method provides a means for profiling neurons based on their projections

Molecular Profiling of Activated Neurons by Phosphorylated Ribosome Capture

The mammalian brain is composed of thousands of interacting neural cell types. Systematic approaches to establish the molecular identity of functional populations of neurons would advance our understanding of neural mechanisms controlling behavior. Here, we show that ribosomal protein S6, a structural component of the ribosome, becomes phosphorylated in neurons activated by a wide range of stimuli. We show that these phosphorylated ribosomes can be captured from mouse brain homogenates, thereby enriching directly for the mRNAs expressed in discrete subpopulations of activated cells. We use this approach to identify neurons in the hypothalamus regulated by changes in salt balance or food availability. We show that galanin neurons are activated by fasting and that prodynorphin neurons restrain food intake during scheduled feeding. These studies identify elements of the neural circuit that controls food intake and illustrate how the activity-dependent capture of cell-type-specific transcripts can elucidate the functional organization of a complex tissue

Rapid Molecular Profiling of Defined Cell Types Using Viral TRAP

Summary: Translational profiling methodologies enable the systematic characterization of cell types in complex tissues, such as the mammalian brain, where neuronal isolation is exceptionally difficult. Here, we report a versatile strategy for profiling CNS cell types in a spatiotemporally restricted fashion by engineering a Cre-dependent adeno-associated virus expressing an EGFP-tagged ribosomal protein (AAV-FLEX-EGFPL10a) to access translating mRNAs by translating ribosome affinity purification (TRAP). We demonstrate the utility of this AAV to target a variety of genetically and anatomically defined neural populations expressing Cre recombinase and illustrate the ability of this viral TRAP (vTRAP) approach to recapitulate the molecular profiles obtained by bacTRAP in corticothalamic neurons across multiple serotypes. Furthermore, spatially restricting adeno-associated virus (AAV) injections enabled the elucidation of regional differences in gene expression within this cell type. Altogether, these results establish the broad applicability of the vTRAP strategy for the molecular dissection of any CNS or peripheral cell type that can be engineered to express Cre. : Nectow et al. describe vTRAP, a technology to purify translating mRNAs from genetically defined cell types in a spatiotemporally restricted fashion. Multiplexing vTRAP with other technologies offers a comprehensive strategy to interrogate the precise role of individual, cell-type-specific genes in neural circuit function. Keywords: AAV, molecular profiling, TRAP, MCH, corticothalamic, Ntsr1, RNA-se