Pascal S. Kaeser

RIM proteins tether Ca2+-channels to presynaptic active zones via a direct PDZ-domain interaction

At a synapse, fast synchronous neurotransmitter release requires localization of Ca(2+) channels to presynaptic active zones. How Ca(2+) channels are recruited to active zones, however, remains unknown. Using unbiased yeast two-hybrid screens, we here identify a direct interaction of the central PDZ domain of the active-zone protein RIM with the C termini of presynaptic N- and P/Q-type Ca(2+) channels but not L-type Ca(2+) channels. To test the physiological significance of this interaction, we generated conditional knockout mice lacking all multidomain RIM isoforms. Deletion of RIM proteins ablated most neurotransmitter release by simultaneously impairing the priming of synaptic vesicles and by decreasing the presynaptic localization of Ca(2+) channels. Strikingly, rescue of the decreased Ca(2+)-channel localization required the RIM PDZ domain, whereas rescue of vesicle priming required the RIM N terminus. We propose that RIMs tether N- and P/Q-type Ca(2+) channels to presynaptic active zones via a direct PDZ-domain-mediated interaction, thereby enabling fast, synchronous triggering of neurotransmitter release at a synapse.

Complement facilitates early prion pathogenesis

New-variant Creutzfeldt–Jakob disease and scrapie are typically initiated by extracerebral exposure to the causative agent, and exhibit early prion replication in lymphoid organs. In mouse scrapie, depletion of B-lymphocytes prevents neuropathogenesis after intraperitoneal inoculation, probably due to impaired lymphotoxin-dependent maturation of follicular dendritic cells (FDCs), which are a major extracerebral prion reservoir. FDCs trap immune complexes with Fc-γ receptors and C3d/C4b-opsonized antigens with CD21/CD35 complement receptors. We examined whether these mechanisms participate in peripheral prion pathogenesis. Depletion of circulating immunoglobulins or of individual Fc-γ receptors had no effect on scrapie pathogenesis if B-cell maturation was unaffected. However, mice deficient in C3, C1q, Bf/C2, combinations thereof or complement receptors were partially or fully protected against spongiform encephalopathy upon intraperitoneal exposure to limiting amounts of prions. Splenic accumulation of prion infectivity and PrPSc was delayed, indicating that activation of specific complement components is involved in the initial trapping of prions in lymphoreticular organs early after infection.

RIM Determines Ca2+ Channel Density and Vesicle Docking at the Presynaptic Active Zone

At presynaptic active zones, neurotransmitter release is initiated by the opening of voltage-gated Ca²+ channels close to docked vesicles. The mechanisms that enrich Ca²+ channels at active zones are, however, largely unknown, possibly because of the limited presynaptic accessibility of most synapses. Here, we have established a Cre-lox based conditional knockout approach at a presynaptically accessible central nervous system synapse, the calyx of Held, to directly study the functions of RIM proteins. Removal of all RIM1/2 isoforms strongly reduced the presynaptic Ca²+ channel density, revealing a role of RIM proteins in Ca²+ channel targeting. Removal of RIMs also reduced the readily releasable pool, paralleled by a similar reduction of the number of docked vesicles, and the Ca²+ channel-vesicle coupling was decreased. Thus, RIM proteins co-ordinately regulate key functions for fast transmitter release, enabling a high presynaptic Ca²+ channel density and vesicle docking at the active zone.

Phosphorylation of RIM1α by PKA Triggers Presynaptic Long-Term Potentiation at Cerebellar Parallel Fiber Synapses

Presynaptic activation of protein kinase A (PKA) induces LTP in cerebellar parallel fiber synapses. Presynaptic LTP is known to require the active zone protein RIM1alpha, but the underlying induction mechanism remains unclear. We now show that PKA directly phosphorylates RIM1alpha at two sites. Using paired recordings from cultured cerebellar granule and Purkinje neurons, we demonstrate that LTP is absent in neurons from RIM1alpha KO mice but is rescued by presynaptic expression of RIM1alpha. Mutant RIM1alpha lacking the N-terminal phosphorylation site is unable to rescue LTP in RIM1alpha knockout neurons but selectively suppresses LTP in wild-type neurons. Our findings suggest that PKA-mediated phosphorylation of the active zone protein RIM1alpha at a single N-terminal site induces presynaptic LTP.

Endocannabinoid-Mediated Long-Term Plasticity Requires cAMP/PKA Signaling and RIM1α

Endocannabinoids (eCBs) have emerged as key activity-dependent signals that, by activating presynaptic cannabinoid receptors (i.e., CB1) coupled to G(i/o) protein, can mediate short-term and long-term synaptic depression (LTD). While the presynaptic mechanisms underlying eCB-dependent short-term depression have been identified, the molecular events linking CB1 receptors to LTD are unknown. Here we show in the hippocampus that long-term, but not short-term, eCB-dependent depression of inhibitory transmission requires presynaptic cAMP/PKA signaling. We further identify the active zone protein RIM1alpha as a key mediator of both CB1 receptor effects on the release machinery and eCB-dependent LTD in the hippocampus. Moreover, we show that eCB-dependent LTD in the amygdala and hippocampus shares major mechanistic features. These findings reveal the signaling pathway by which CB1 receptors mediate long-term effects of eCBs in two crucial brain structures. Furthermore, our results highlight a conserved mechanism of presynaptic plasticity in the brain.

RIM Proteins Activate Vesicle Priming by Reversing Autoinhibitory Homodimerization of Munc13

At a synapse, the presynaptic active zone mediates synaptic vesicle exocytosis. RIM proteins are active zone scaffolding molecules that--among others--mediate vesicle priming and directly or indirectly interact with most other essential presynaptic proteins. In particular, the Zn²+ finger domain of RIMs binds to the C₂A domain of the priming factor Munc13, which forms a homodimer in the absence of RIM but a heterodimer with it. Here, we show that RIMs mediate vesicle priming not by coupling Munc13 to other active zone proteins as thought but by directly activating Munc13. Specifically, we found that the isolated Zn²+ finger domain of RIMs autonomously promoted vesicle priming by binding to Munc13, thereby relieving Munc13 homodimerization. Strikingly, constitutively monomeric mutants of Munc13 rescued priming in RIM-deficient synapses, whereas wild-type Munc13 did not. Both mutant and wild-type Munc13, however, rescued priming in Munc13-deficient synapses. Thus, homodimerization of Munc13 inhibits its priming function, and RIMs activate priming by disrupting Munc13 homodimerization.

Molecular Mechanisms for Synchronous, Asynchronous, and Spontaneous Neurotransmitter Release

Most neuronal communication relies upon the synchronous release of neurotransmitters, which occurs through synaptic vesicle exocytosis triggered by action potential invasion of a presynaptic bouton. However, neurotransmitters are also released asynchronously with a longer, variable delay following an action potential or spontaneously in the absence of action potentials. A compelling body of research has identified roles and mechanisms for synchronous release, but asynchronous release and spontaneous release are less well understood. In this review, we analyze how the mechanisms of the three release modes overlap and what molecular pathways underlie asynchronous and spontaneous release. We conclude that the modes of release have key fusion processes in common but may differ in the source of and necessity for Ca(2+) to trigger release and in the identity of the Ca(2+) sensor for release.

Redundant functions of RIM1α and RIM2α in Ca2+-triggered neurotransmitter release

α‐RIMs (RIM1α and RIM2α) are multidomain active zone proteins of presynaptic terminals. α‐RIMs bind to Rab3 on synaptic vesicles and to Munc13 on the active zone via their N‐terminal region, and interact with other synaptic proteins via their central and C‐terminal regions. Although RIM1α has been well characterized, nothing is known about the function of RIM2α. We now show that RIM1α and RIM2α are expressed in overlapping but distinct patterns throughout the brain. To examine and compare their functions, we generated knockout mice lacking RIM2α, and crossed them with previously produced RIM1α knockout mice. We found that deletion of either RIM1α or RIM2α is not lethal, but ablation of both α‐RIMs causes postnatal death. This lethality is not due to a loss of synapse structure or a developmental change, but to a defect in neurotransmitter release. Synapses without α‐RIMs still contain active zones and release neurotransmitters, but are unable to mediate normal Ca2+‐triggered release. Our data thus demonstrate that α‐RIMs are not essential for synapse formation or synaptic exocytosis, but are required for normal Ca2+‐triggering of exocytosis.

Efficient Lymphoreticular Prion Propagation Requires PrP c in Stromal and Hematopoietic Cells

ABSTRACT In most prion diseases, infectivity accumulates in lymphoreticular organs early after infection. Defects in hematopoietic compartments, such as impaired B-cell maturation, or in stromal compartments, such as abrogation of follicular dendritic cells, can delay or prevent lymphoreticular prion colonization. However, the nature of the compartment in which prion replication takes place is controversial, and it is unclear whether this compartment coincides with that expressing the normal prion protein (PrPc). Here we studied the distribution of infectivity in splenic fractions of wild-type and fetal liver chimeric mice carrying the gene that encodes PrPc (Prnp) solely on hematopoietic or on stromal cells. We fractionated spleens at various times after intraperitoneal challenge with prions and assayed infectivity by bioassay. Upon high-dose challenge, chimeras carrying PrPcon hematopoietic cells accumulated prions in stroma and in purified splenocytes. In contrast, after low-dose challenge ablation ofPrnp in either compartment prevented splenic accumulation of infectivity, indicating that optimal prion replication requires PrPc expression by both stromal and hematopoietic compartments.

ELKS2α/CAST Deletion Selectively Increases Neurotransmitter Release at Inhibitory Synapses

The presynaptic active zone is composed of a protein network that contains ELKS2alpha (a.k.a. CAST) as a central component. Here we demonstrate that in mice, deletion of ELKS2alpha caused a large increase in inhibitory, but not excitatory, neurotransmitter release, and potentiated the size, but not the properties, of the readily-releasable pool of vesicles at inhibitory synapses. Quantitative electron microscopy revealed that the ELKS2alpha deletion did not change the number of docked vesicles or other ultrastructural parameters of synapses, except for a small decrease in synaptic vesicle numbers. The ELKS2alpha deletion did, however, alter the excitatory/inhibitory balance and exploratory behaviors, possibly as a result of the increased synaptic inhibition. Thus, as opposed to previous studies indicating that ELKS2alpha is essential for mediating neurotransmitter release, our results suggest that ELKS2alpha normally restricts release and limits the size of the readily-releasable pool of synaptic vesicles at the active zone of inhibitory synapses.