Ancestral regulatory mechanisms specify conserved midbrain circuitry in arthropods and vertebrates.

Corresponding attributes of neural development and function suggest arthropod and vertebrate brains may have an evolutionarily conserved organization. However, the underlying mechanisms have remained elusive. Here, we identify a gene regulatory and character identity network defining the deutocerebral-tritocerebral boundary (DTB) in Drosophila This network comprises genes homologous to those directing midbrain-hindbrain boundary (MHB) formation in vertebrates and their closest chordate relatives. Genetic tracing reveals that the embryonic DTB gives rise to adult midbrain circuits that in flies control auditory and vestibular information processing and motor coordination, as do MHB-derived circuits in vertebrates. DTB-specific gene expression and function are directed by cis-regulatory elements of developmental control genes that include homologs of mammalian Zinc finger of the cerebellum and Purkinje cell protein 4Drosophila DTB-specific cis-regulatory elements correspond to regulatory sequences of human ENGRAILED-2, PAX-2, and DACHSHUND-1 that direct MHB-specific expression in the embryonic mouse brain. We show that cis-regulatory elements and the gene networks they regulate direct the formation and function of midbrain circuits for balance and motor coordination in insects and mammals. Regulatory mechanisms mediating the genetic specification of cephalic neural circuits in arthropods correspond to those in chordates, thereby implying their origin before the divergence of deuterostomes and ecdysozoans.

Lineage-specific determination of ring neuron circuitry in the central complex of Drosophila.

The ellipsoid body (EB) of the Drosophila central complex mediates sensorimotor integration and action selection for adaptive behaviours. Insights into its physiological function are steadily accumulating, however the developmental origin and genetic specification have remained largely elusive. Here we identify two stem cells in the embryonic neuroectoderm as precursor cells of neuronal progeny that establish EB circuits in the adult brain. Genetic tracing of embryonic neuroblasts ppd5 and mosaic analysis with a repressible cell marker identified lineage-related progeny as Pox neuro (Poxn)-expressing EB ring neurons, R1-R4. During embryonic brain development, engrailed function is required for the initial formation of Poxn-expressing ppd5-derived progeny. Postembryonic determination of R1-R4 identity depends on lineage-specific Poxn function that separates neuronal subtypes of ppd5-derived progeny into hemi-lineages with projections either terminating in the EB ring neuropil or the superior protocerebrum (SP). Poxn knockdown in ppd5-derived progeny results in identity transformation of engrailed-expressing hemi-lineages from SP to EB-specific circuits. In contrast, lineage-specific knockdown of engrailed leads to reduced numbers of Poxn-expressing ring neurons. These findings establish neuroblasts ppd5-derived ring neurons as lineage-related sister cells that require engrailed and Poxn function for the proper formation of EB circuitry in the adult central complex of Drosophila.

In vivo expansion of functionally integrated GABAergic interneurons by targeted increase in neural progenitors.

A central hypothesis for brain evolution is that it might occur via expansion of progenitor cells and subsequent lineage-dependent formation of neural circuits. Here, we report in vivo amplification and functional integration of lineage-specific circuitry in Drosophila Levels of the cell fate determinant Prospero were attenuated in specific brain lineages within a range that expanded not only progenitors but also neuronal progeny, without tumor formation. Resulting supernumerary neural stem cells underwent normal functional transitions, progressed through the temporal patterning cascade, and generated progeny with molecular signatures matching source lineages. Fully differentiated supernumerary gamma-amino butyric acid (GABA)-ergic interneurons formed functional connections in the central complex of the adult brain, as revealed by in vivo calcium imaging and open-field behavioral analysis. Our results show that quantitative control of a single transcription factor is sufficient to tune neuron numbers and clonal circuitry, and provide molecular insight into a likely mechanism of brain evolution.

Loss and gain of Drosophila TDP-43 impair synaptic efficacy and motor control leading to age-related neurodegeneration by loss-of-function phenotypes.

Cytoplasmic accumulation and nuclear clearance of TDP-43 characterize familial and sporadic forms of amyotrophic lateral sclerosis and frontotemporal lobar degeneration, suggesting that either loss or gain of TDP-43 function, or both, cause disease formation. Here we have systematically compared loss- and gain-of-function of Drosophila TDP-43, TAR DNA Binding Protein Homolog (TBPH), in synaptic function and morphology, motor control, and age-related neuronal survival. Both loss and gain of TBPH severely affect development and result in premature lethality. TBPH dysfunction caused impaired synaptic transmission at the larval neuromuscular junction (NMJ) and in the adult. Tissue-specific knockdown together with electrophysiological recordings at the larval NMJ also revealed that alterations of TBPH function predominantly affect pre-synaptic efficacy, suggesting that impaired pre-synaptic transmission is one of the earliest events in TDP-43-related pathogenesis. Prolonged loss and gain of TBPH in adults resulted in synaptic defects and age-related, progressive degeneration of neurons involved in motor control. Toxic gain of TBPH did not downregulate or mislocalize its own expression, indicating that a dominant-negative effect leads to progressive neurodegeneration also seen with mutational inactivation of TBPH. Together these data suggest that dysfunction of Drosophila TDP-43 triggers a cascade of events leading to loss-of-function phenotypes whereby impaired synaptic transmission results in defective motor behavior and progressive deconstruction of neuronal connections, ultimately causing age-related neurodegeneration.

Alternative oxidase rescues mitochondria-mediated dopaminergic cell loss in Drosophila.

Mitochondrial dysfunction is commonly observed in degenerative disorders, including Alzheimer's and Parkinson's disease that are characterized by the progressive and selective loss of neuronal subpopulations. It is currently unclear, however, whether mitochondrial dysfunction is primary or secondary to other pathogenic processes that eventually lead to age-related neurodegeneration. Here we establish an in vivo Drosophila model of mitochondrial dysfunction by downregulating the catalytic subunit of mitochondrial DNA (mtDNA) polymerase in cholinergic, serotonergic and dopaminergic neurons. The resulting flies are characterized by lowered respiratory chain activity, premature aging, age-related motor deficits as well as adult onset, progressive and cell-type-specific, dopaminergic neurodegeneration. Using this model, we find that associated lethality can be partially rescued by targeting PINK1/parkin signaling or Drp1, both of which have been implicated in mitochondrial dynamics and Parkinson's disease. Bypassing mitochondrial complex III/IV deficiencies with Alternative oxidase (AOX), however, fully restores ATP levels and prevents dopaminergic neurodegeneration. In contrast, ATP levels and neurodegeneration are not rescued when mitochondrial complex I deficiencies are bypassed with NADH-Q oxidoreductase. Our results demonstrate that mtDNA-mediated mitochondrial dysfunction can cause age-related and cell-type-specific neurodegeneration which AOX is able to alleviate and indicate that AOX or its surrogates may prove useful as a therapeutic tool for limiting respiratory chain deficiencies caused by mtDNA decline in healthy aging and neurodegenerative disease.