Tobias M. Rasse

Bruchpilot Promotes Active Zone Assembly, Ca2+ Channel Clustering, and Vesicle Release

The molecular organization of presynaptic active zones during calcium influx–triggered neurotransmitter release is the focus of intense investigation. The Drosophila coiled-coil domain protein Bruchpilot (BRP) was observed in donut-shaped structures centered at active zones of neuromuscular synapses by using subdiffraction resolution STED (stimulated emission depletion) fluorescence microscopy. At brp mutant active zones, electron-dense projections (T-bars) were entirely lost, Ca2+ channels were reduced in density, evoked vesicle release was depressed, and short-term plasticity was altered. BRP-like proteins seem to establish proximity between Ca2+ channels and vesicles to allow efficient transmitter release and patterned synaptic plasticity.

Knockdown of transactive response DNA-binding protein (TDP-43) downregulates histone deacetylase 6

TDP‐43 is an RNA/DNA‐binding protein implicated in transcriptional repression and mRNA processing. Inclusions of TDP‐43 are hallmarks of frontotemporal dementia and amyotrophic lateral sclerosis. Besides aggregation of TDP‐43, loss of nuclear localization is observed in disease. To identify relevant targets of TDP‐43, we performed expression profiling. Thereby, histone deacetylase 6 (HDAC6) downregulation was discovered on TDP‐43 silencing and confirmed at the mRNA and protein level in human embryonic kidney HEK293E and neuronal SH‐SY5Y cells. This was accompanied by accumulation of the major HDAC6 substrate, acetyl‐tubulin. HDAC6 levels were restored by re‐expression of TDP‐43, dependent on RNA binding and the C‐terminal protein interaction domains. Moreover, TDP‐43 bound specifically to HDAC6 mRNA arguing for a direct functional interaction. Importantly, in vivo validation in TDP‐43 knockout Drosophila melanogaster confirmed the specific downregulation of HDAC6. HDAC6 is necessary for protein aggregate formation and degradation. Indeed, HDAC6‐dependent reduction of cellular aggregate formation and increased cytotoxicity of polyQ‐expanded ataxin‐3 were found in TDP‐43 silenced cells. In conclusion, loss of functional TDP‐43 causes HDAC6 downregulation and might thereby contribute to pathogenesis.

Four different subunits are essential for expressing the synaptic glutamate receptor at neuromuscular junctions of Drosophila.

Three ionotropic glutamate receptor subunits, designated GluRIIA, GluRIIB, and GluRIII, have been identified at neuromuscular junctions of Drosophila. Whereas GluRIIA and GluRIIB are redundant for viability, it was shown recently that GluRIII is essential for both the synaptic localization of GluRIIA and GluRIIB and the viability of Drosophila. Here we identify a fourth and a fifth subunit expressed in the neuromuscular system, which we name GluRIID and GluRIIE. Both new subunits we show to be necessary for survival. Moreover, both GluRIID and GluRIIE are required for the synaptic expression of all other glutamate receptor subunits. All five subunits are interdependent for receptor function, synaptic receptor expression, and viability. This indicates that synaptic glutamate receptors incorporate the GluRIII, GluRIID, and GluRIIE subunit together with either GluRIIA or GluRIIB at the Drosophila neuromuscular junction. At this widely used model synapse, the assembly of four different subunits to form an individual glutamate receptor channel may thus be obligatory. This study opens the way for a further characterization of in vivo glutamate receptor assembly and trafficking using the efficient genetics of Drosophila.

Glutamate receptor dynamics organizing synapse formation in vivo

Insight into how glutamatergic synapses form in vivo is important for understanding developmental and experience-triggered changes of excitatory circuits. Here, we imaged postsynaptic densities (PSDs) expressing a functional, GFP-tagged glutamate receptor subunit (GluR-IIAGFP) at neuromuscular junctions of Drosophila melanogaster larvae for several days in vivo. New PSDs, associated with functional and structural presynaptic markers, formed independently of existing synapses and grew continuously until reaching a stable size within hours. Both in vivo photoactivation and photobleaching experiments showed that extrasynaptic receptors derived from diffuse, cell-wide pools preferentially entered growing PSDs. After entering PSDs, receptors were largely immobilized. In comparison, other postsynaptic proteins tested (PSD-95, NCAM and PAK homologs) exchanged faster and with no apparent preference for growing synapses. We show here that new glutamatergic synapses form de novo and not by partitioning processes from existing synapses, suggesting that the site-specific entry of particular glutamate receptor complexes directly controls the assembly of individual PSDs.

Activity-dependent site-specific changes of glutamate receptor composition in vivo

The subunit composition of postsynaptic non–NMDA-type glutamate receptors (GluRs) determines the function and trafficking of the receptor. Changes in GluR composition have been implicated in the homeostasis of neuronal excitability and synaptic plasticity underlying learning. Here, we imaged GluRs in vivo during the formation of new postsynaptic densities (PSDs) at Drosophila neuromuscular junctions coexpressing GluRIIA and GluRIIB subunits. GluR composition was independently regulated at directly neighboring PSDs on a submicron scale. Immature PSDs typically had large amounts of GluRIIA and small amounts of GluRIIB. During subsequent PSD maturation, however, the GluRIIA/GluRIIB composition changed and became more balanced. Reducing presynaptic glutamate release increased GluRIIA, but decreased GluRIIB incorporation. Moreover, the maturation of GluR composition correlated in a site-specific manner with the level of Bruchpilot, an active zone protein that is essential for mature glutamate release. Thus, we show that an activity-dependent, site-specific control of GluR composition can contribute to match pre- and postsynaptic assembly.

Mitochondrial proteolytic stress induced by loss of mortalin function is rescued by Parkin and PINK1

The mitochondrial chaperone mortalin was implicated in Parkinson’s disease (PD) because of its reduced levels in the brains of PD patients and disease-associated rare genetic variants that failed to rescue impaired mitochondrial integrity in cellular knockdown models. To uncover the molecular mechanisms underlying mortalin-related neurodegeneration, we dissected the cellular surveillance mechanisms related to mitochondrial quality control, defined the effects of reduced mortalin function at the molecular and cellular levels and investigated the functional interaction of mortalin with Parkin and PINK1, two PD-related proteins involved in mitochondrial homeostasis. We found that reduced mortalin function leads to: (1) activation of the mitochondrial unfolded protein response (UPR(mt)), (2) increased susceptibility towards intramitochondrial proteolytic stress, (3) increased autophagic degradation of fragmented mitochondria and (4) reduced mitochondrial mass in human cells in vitro and ex vivo. These alterations caused increased vulnerability toward apoptotic cell death. Proteotoxic perturbations induced by either partial loss of mortalin or chemical induction were rescued by complementation with native mortalin, but not disease-associated mortalin variants, and were independent of the integrity of autophagic pathways. However, Parkin and PINK1 rescued loss of mortalin phenotypes via increased lysosomal-mediated mitochondrial clearance and required intact autophagic machinery. Our results on loss of mortalin function reveal a direct link between impaired mitochondrial proteostasis, UPR(mt) and PD and show that effective removal of dysfunctional mitochondria via either genetic (PINK1 and Parkin overexpression) or pharmacological intervention (rapamycin) may compensate mitochondrial phenotypes.

The morphogene AmiC2 is pivotal for multicellular development in the cyanobacterium Nostoc punctiforme

Filamentous cyanobacteria of the order Nostocales are primordial multicellular organisms, a property widely considered unique to eukaryotes. Their filaments are composed of hundreds of mutually dependent vegetative cells and regularly spaced N2‐fixing heterocysts, exchanging metabolites and signalling molecules. Furthermore, they may differentiate specialized spore‐like cells and motile filaments. However, the structural basis for cellular communication within the filament remained elusive. Here we present that mutation of a single gene, encoding cell wall amidase AmiC2, completely changes the morphology and abrogates cell differentiation and intercellular communication. Ultrastructural analysis revealed for the first time a contiguous peptidoglycan sacculus with individual cells connected by a single‐layered septal cross‐wall. The mutant forms irregular clusters of twisted cells connected by aberrant septa. Rapid intercellular molecule exchange takes place in wild‐type filaments, but is completely abolished in the mutant, and this blockage obstructs any cell differentiation, indicating a fundamental importance of intercellular communication for cell differentiation in Nostoc. AmiC2–GFP localizes in the cell wall with a focus in the cross walls of dividing cells, implying that AmiC2 processes the newly synthesized septum into a functional cell–cell communication structure during cell division. AmiC2 thus can be considered as a novel morphogene required for cell–cell communication, cellular development and multicellularity.

The Ig cell adhesion molecule Basigin controls compartmentalization and vesicle release at Drosophila melanogaster synapses

Synapses can undergo rapid changes in size as well as in their vesicle release function during both plasticity processes and development. This fundamental property of neuronal cells requires the coordinated rearrangement of synaptic membranes and their associated cytoskeleton, yet remarkably little is known of how this coupling is achieved. In a GFP exon-trap screen, we identified Drosophila melanogaster Basigin (Bsg) as an immunoglobulin domain-containing transmembrane protein accumulating at periactive zones of neuromuscular junctions. Bsg is required pre- and postsynaptically to restrict synaptic bouton size, its juxtamembrane cytoplasmic residues being important for that function. Bsg controls different aspects of synaptic structure, including distribution of synaptic vesicles and organization of the presynaptic cortical actin cytoskeleton. Strikingly, bsg function is also required specifically within the presynaptic terminal to inhibit nonsynchronized evoked vesicle release. We thus propose that Bsg is part of a transsynaptic complex regulating synaptic compartmentalization and strength, and coordinating plasma membrane and cortical organization.

Spastic Paraplegia Mutation N256S in the Neuronal Microtubule Motor KIF5A Disrupts Axonal Transport in a Drosophila HSP Model

Hereditary spastic paraplegias (HSPs) comprise a group of genetically heterogeneous neurodegenerative disorders characterized by spastic weakness of the lower extremities. We have generated a Drosophila model for HSP type 10 (SPG10), caused by mutations in KIF5A. KIF5A encodes the heavy chain of kinesin-1, a neuronal microtubule motor. Our results imply that SPG10 is not caused by haploinsufficiency but by the loss of endogenous kinesin-1 function due to a selective dominant-negative action of mutant KIF5A on kinesin-1 complexes. We have not found any evidence for an additional, more generalized toxicity of mutant Kinesin heavy chain (Khc) or the affected kinesin-1 complexes. Ectopic expression of Drosophila Khc carrying a human SPG10-associated mutation (N256S) is sufficient to disturb axonal transport and to induce motoneuron disease in Drosophila. Neurofilaments, which have been recently implicated in SPG10 disease manifestation, are absent in arthropods. Impairments in the transport of kinesin-1 cargos different from neurofilaments are thus sufficient to cause HSP–like pathological changes such as axonal swellings, altered structure and function of synapses, behavioral deficits, and increased mortality.

PP2A and GSK-3β Act Antagonistically to Regulate Active Zone Development

The synapse is composed of an active zone apposed to a postsynaptic cluster of neurotransmitter receptors. Each Drosophila neuromuscular junction comprises hundreds of such individual release sites apposed to clusters of glutamate receptors. Here, we show that protein phosphatase 2A (PP2A) is required for the development of structurally normal active zones opposite glutamate receptors. When PP2A is inhibited presynaptically, many glutamate receptor clusters are unapposed to Bruchpilot (Brp), an active zone protein required for normal transmitter release. These unapposed receptors are not due to presynaptic retraction of synaptic boutons, since other presynaptic components are still apposed to the entire postsynaptic specialization. Instead, these data suggest that Brp localization is regulated at the level of individual release sites. Live imaging of glutamate receptors demonstrates that this disruption to active zone development is accompanied by abnormal postsynaptic development, with decreased formation of glutamate receptor clusters. Remarkably, inhibition of the serine-threonine kinase GSK-3β completely suppresses the active zone defect, as well as other synaptic morphology phenotypes associated with inhibition of PP2A. These data suggest that PP2A and GSK-3β function antagonistically to control active zone development, providing a potential mechanism for regulating synaptic efficacy at a single release site.