Dendritic Snares Add a New Twist to the Old Neuron Theory

PERSPECTIVE

Dendritic SNAREs add a new twist to the old neuron theory

Saak V. Ovsepian1 and J. Oliver Dolly1 International Centre for Neurotherapeutics, Dublin City University, Glasnevin, Dublin 9, Ireland

Edited by Thomas C. Südhof, Stanford University School of Medicine, Palo Alto, CA, and approved October 4, 2011 (received for review November 22, 2010)

Dendritic exocytosis underpins a broad range of integrative and homeostatic synaptic functions. Emerging data highlight the essential role of SNAREs in trafﬁcking and fusion of secretory organelles with release of peptides and neurotransmitters from dendrites. This Perspective analyzes recent evidence inferring axo-dendritic polarization of vesicular release machinery and pinpoints progress made with existing challenges in this rapidly progressing ﬁeld of dendritic research. Interpreting the relation of new molecular data to physiological results on secretion from dendrites would greatly advance our understanding of this facet of neuronal mechanisms.

dendritic release | neuronal polarization | retrograde transmission

lassically, a single neuron exocytosis at dendrites in compliance with its central hydrophilic layer containing receives multimodal informa- the view of highly conserved fusion ma- three conserved glutamines (Q) and one C tion as local electro-chemical chinery among different secretory path- arginine (R) (18). Accordingly, the con- signals through synaptic inputs ways, new findings indicate their notable tributing molecules are grouped into Qa-, converging onto its soma and dendrites. specialization, supporting the premise of Qb-, Qc-, Qbc-, and R-SNAREs (19, 20) After integration, these confined events molecular asymmetry as one of the key (Fig. 1A). In neurons, SNAREs driving are translated into action potentials at the affiliates of neuronal polarization. secretion are represented by a vesicle- axon initial segment, which rapidly prop- associated membrane protein (VAMP, known also as synaptobrevin, R-SNARE), agate to nerve terminals and trigger there SNARE Hypothesis and Secretory plasma membrane anchored-attached syn- quantal release of neurotransmitters. Re- Vesicle Fusion at Synapses cent electrophysiological and imaging data, taxin (Qa-SNARE), and SNAP (compris- however, dispute this rather naïve view of Membrane fusion constitutes one of the ing two SNARE motifs, Qbc-SNARE) fundamental biological processes governing neurons, highlighting unforeseen traits variants (21). Advantageously for research, targeting and merging of intracellular and dynamics of dendritic integration and these proteins constitute natural targets for organelles. Analysis of its mechanisms led communication mechanisms between Clostridial neurotoxins (CNTs, both botu- to the discovery of SNAREs as highly nerve cells. It emerges that neurons, in linum and tetanus toxins), which recognize conserved key nanoengines of various addition to transferring signals through and proteolytically inactivate SNAREs intracellular trafficking steps (11, 12). canonical chemical synapses at the axon with astounding efficiency and specificity, According to the contemporary model of causing blockade of synaptic transmission terminals, also interact via mixed electro- membrane fusion, SNAREs, along with (22–24) (Fig. 1B). Unstructured in mono- chemical and electrical synapses at so- several regulatory proteins, which are lo- meric state, SNAREs when associated matic and dendritic juxtapositions, as well calized in opposing membranes, attach and form highly stable ternary helical com- as through diffuse nonsynaptic volume propel the merger of membranes, using the – fi plexes (syntaxin and VAMP contributing transmission signaling (1 4). Signi cant free energy that is released during the evidence has also accrued suggesting es- one, whereas SNAP-25 provides two formation of a four-helix SNARE bundle. α sential roles for dendritic exocytosis in Although not undisputed, during the last -helices) before vesicular release (13, 18). these and numerous other integrative and 2 decades this model attracted scientific This multireaction process, which was ex- homeostatic processes. These include re- scrutiny from numerous angles and gained plicitly demonstrated at both the ensemble lease of transmitters, neurotrophins, and overwhelming support from functional, and single molecular levels, is initiated modulatory peptides from dendrites, along molecular, and genetic studies (13–15). by the opening of syntaxin and binding of with activity-dependent remodeling of In eukaryotic cells, the SNARE protein Munc18 to the syntaxin Habc N-terminal synaptic connections via regulated ex- family is represented by nine subfamilies, domain (25, 26), a step that promotes pression of receptors and ion channels with a total of 36 members identified in loose and reversible interaction between – (5 7). Although a great body of data humans (16). Originally, a strict separation SNARE motifs of syntaxin with SNAP-25 advocate the involvement of SNAREs in was assumed between these proteins re- and VAMP, leading to anchoring of the trafficking and vesicular fusion at den- siding on the “donor” and “acceptor” synaptic vesicle at the release site (Fig. C drites, nonvesicular release of retrograde membrane compartments, which led to 1 ). Upon binding of complexin, another messengers (e.g., endocannabinoids, their classification as vesicle membrane cytosolic regulatory protein, the transient arachidonic acid, nitric oxide, and carbon SNARE core scaffold gets primed for or target membrane SNAREs (v- and 2+ monoxide) from this location with their t-SNAREs) (17). This categorization, rapid activation by the Ca sensor syn- autocrine and paracrine effects on target however, proved to be inept when homo- aptotagmin (27); the latter enforces the and precursor neurons has additionally typic fusion events were considered and dissociation of complexin from the been documented (8–10). This Perspective especially in cases in which certain SNARE complex and puts into effect the reviews and analyzes key research streams SNAREs function in several transport on SNARE-dependent retrosynaptic steps, with varying partners. The more re- signaling in mammalian central neurons cent and widely accepted classification is, Author contributions: S.V.O. and J.O.D. wrote the paper. and highlights the outstanding issues therefore, based on reactive motifs con- The authors declare no conflict of interest. and challenges in this rapidly progressing tributed by each partner toward the for- This article is a PNAS direct submission. fi fi topic. Despite the well-de ned signi - mation of ultrastable coiled-coil SNARE 1To whom correspondence may be addressed. E-mail: saak. cance of SNAREs for trafficking and complexes during membrane fusion, with [email protected] or [email protected].

www.pnas.org/cgi/doi/10.1073/pnas.1017235108 PNAS | November 29, 2011 | vol. 108 | no. 48 | 19113–19120 Downloaded by guest on September 24, 2021 A C SV docking N-terminal SNARE TM domains motif domain Munc18 ATP Complexin Qa-SNARE Releasable 2 Qb-SNARE vesicle SV priming Qc-SNARE

Qbc-SNARE 1 3

R-SNARE SV fusion

2+ B Syntaxin 1A 4 Ca ATP SNAP, NSF Synaptobrevin 2 BoNT/C BoNT/A D BoNT/E Synaptobrevin 2 BoNT/F SNAP-25 Syntaxin 1A BoNT/D COOH

Extracellular milieu TeNT or BoNT/B Secretory organelle

BoNT/G SNAP-25

Fig. 1. SNAREs and vesicular secretion from neurons. (A) Types of SNAREs with their gross structures schematized. The C-terminal of SNAREs corresponds to the transmembrane (TM) domains; the central portion contains the SNARE motif, and the N-terminal of Qa-SNAREs (syntaxins in neurons) possesses antiparallel three-helix bundles. In Qbc SNAREs (SNAP-23 and SNAP-25 in neurons), duplicated SNARE motifs are connected by a linker that is frequently palmitoylated (zig- zag lines) and anchors the protein to the membrane. During vesicular fusion at nerve terminals Qbc SNAREs (SNAP-23/25 variants) contribute two α-helices to the four-helical core complex, with the other two provided by Qa (syntaxin) and R (VAMP) SNAREs. Dashed borders highlight domains that are missing in some subfamily members. Modified with permission from ref. 18. (B) SNAREs constitute molecular targets of Clostridial neurotoxins, which recognize and cleave these fusogenic proteins at specific scissile bonds. Neuronal SNAREs serve as substrate for the protease light chains of tetanus toxin and botulinum neurotoxins: arrows indicate the cleavage sites of individual SNAREs by the different neurotoxins (BoNT, A-G serotypes of botulinum neurotoxin; TeNT, tetanus toxin). Plasma and vesicle membranes are indicated in gray (Left and Right, respectively). (C) Schematic of the SNARE-dependent synaptic vesicle cycle. Conforma- tional shift from a closed to an open state of the N-terminal motif of syntaxin (Qa SNARE, red) conditioned by SM protein Munc18 initiates the loose interaction of syntaxin with SNAP-25 (Qbc SNARE, green) and VAMP (R-SNARE, blue), leading to ATP-dependent docking of SVs at the active zone (1, 2). This complex gets primed by complexin and Munc18 for activation by synaptotagmin and Ca2+ (3). Upon Ca2+ influx, synaptotagmin dissociates the complexin from the fusion complex and reacts with membrane phospholipids and the SNAREs, driving membrane fusion (4) and release of vesicular contents into the synaptic cleft. This step is followed by dissociation of the SNARE complex by NSF using the hydrolysis of ATP as energy source, liberating SNAREs for further reuse. (D)A crystal structure of SNARE core complex with four parallel α-helices associated in the ternary complex. The C-terminal of the helices, which points toward the vesicle and surface membranes, are oriented to the right. Modified with permission from ref. 136.

zippering of four α-helical parallel bundles, the cargo proteins and peptides form ag- Release of both “classic” transmitters resulting in vesicle fusion with the plasma gregates, resulting in condensation of the and peptides from dendrites seems to be membrane and neurosecretion (28) granule matrix and membrane budding primarily mediated via regulated exo- (Fig. 1 C and D). The bulk of recent data with the formation of early secretory cytosis, except under basal conditions their emerging from research on dendritic exo- granules, which mature by fusion with constitutive release has been documented cytosis, in general consistent with the other granules while being transported (33–35) (Fig. 2B). Tannic acid fixation of model outlined above, suggests subtle var- through the trans-Golgi networks toward dendrites within the supraoptic nucleus iations in the machinery and mechanisms their release sites. After fusion with the revealed the morphology of LDCVs, with regulating vesicular release. plasmalema, vesicle membrane compo- evidence of “omega” fusion profiles at the nents of LDCVs are retrieved and re- plasma membrane, clearly indicating the Dendritic Secretory Organelles cycled to the Golgi, where they get refilled vesicular secretion of peptides from den- In somato-dendrites and axon terminals of with new cargo, thereby generating vesi- drites (36) (Fig. 2A). In several other nerve cells, secretory organelles are rep- cles de novo in each cycle. SSVs also bud neuron types, smaller dendritic vesicular resented primarily by small synaptic vesi- from Golgi as immature vesicles but have elements have been observed, which may fi cles (SSVs), lled with low molecular morphology and protein composition dif- correspond to SSVs (31, 36). Despite the existence of discrete populations of se- weight transmitters, and large dense core ferent from those of mature SSVs. These vesicles (LDCVs), containing peptides, cretory organelles (rapid releasable and also reach release sites through active modulators, biological amines, or neuro- reserve pools) in dendrites and their ac- trophins (29, 30) (Fig. 2A). Although both transport and there undergo maturation. tivity-dependent segregation have been LDCVs and SSVs are generated from the However, in contrast to LDCVs, SSVs are proposed long ago (37), mechanisms Golgi, they exhibit significant differences refilled and extensively recycled locally of their targeting to the various pools in their life cycles and destiny. In LDCVs before their degradation (31, 32). and plasma membrane are essentially

19114 | www.pnas.org/cgi/doi/10.1073/pnas.1017235108 Ovsepian and Dolly Downloaded by guest on September 24, 2021 A B comes from nigral dopaminergic cells and TC PG hypothalamic neurons. Dopamine has been observed in a variety of secretory organelles, including LDCV, SSVs, and tuberovesicular structures resembling smooth endoplasmic reticular stores (44). The advantageous anatomical arrange- ments of dopaminergic neurons with seg- regated dendrites, in substantia nigra pars Axon reticulate, from somatic and axonal compartments not only made it possible to perform selective quantitative analysis of the dendritic release but provides a model for real-time monitoring of this physiolog-

100pA ical process in vivo (45, 46). Nevertheless, 200ms BMC identification of subcellular storage com- Dendrite partments and precise molecular events leading to dendritic secretion of dopamine C remained a subject of longstanding con- Control Nimodepine Bay K troversy (44, 47–49) until a dual, quantal- reverse carrier-mediated secretion hypothesis was proposed (45). According to this model, dopamine can be released through both vesicular and carrier-medi- Dendrite ated secretory mechanisms. Importantly, potent inhibition of dopamine secretion from somato-dendritic compartments of these neurons by CNTs in vitro and in vivo confirmed the involvement of SNAREs 500ms 100pA (50, 51). These functional data received Fig. 2. (A) Electron micrographs of rat hypothalamic area showing a dendritic shaft packed with LDCVs. support from more recent immunocyto- The presence of synaptic contacts with axon terminal identifies the process as a dendrite. Arrowhead chemical studies, which highlighted the (Lower) indicates released dendritic secretory granule content into the interstitial space. Modified with presence of the main CNTs-sensitive permission from refs. 36 and 137. (Scale bar, 1.0 μm.) (B) Tufted cell (TC) dendro-dendritic inhibition SNARE variants, including syntaxin3, mediated by periglomerular (PG) neurons. In the presence of tetrodotoxin, a voltage step is followed by VAMP2, and SNAP-25, in dendrites of a long-lasting barrage of inhibitory postsynaptic currents that were abolished by the GABAA receptor nigral dopaminergic neurons (52). antagonist bicuculine (BMC). (C)L-typeCa2+ currents, which are normally not coupled to exocytosis, are Similar experiments also provided con- – required for TC PG dendro-dendritic inhibition. Dendro-dendritic inhibition is reduced by nimodepine clusive evidence for dendritic vesicular se- 2+ fi (L-type Ca channel blocker) and recovered by activation of the same channel with Bay K. Modi ed with cretion from hypothalamic neurons. In permission from ref. 34. dendrites of magnocellular neurons, for instance, a steep Ca2+-dependence of oxy- unknown. Unlike axon terminals, which be developed directly from dendritic ER tocin release with clear structural attributes of vesicular storage and secretion from rely on a somatic supply of LDCVs, sub- elements, bypassing the Golgi apparatus fi stantial data suggest local mRNA trans- (38, 41). Although this straight route of LDCVs has been rmly established (36, 53). The presence of SNAP-25 and syntaxin lation and peptides synthesis in dendrites, secretory organelles to plasma membrane fi with neurotransmitter secretion endo- in dendrites has so far received little were also veri ed at this location (54). Importantly, probing dendritic oxytocin plasmic reticular (ER) sites visualized functional evidence, with targeting proteins release with nanomolar concentrations of throughout the dendritic shafts of hippo- remaining essentially unknown, if true it tetanus toxin revealed an essential role for campal and hypothalamic neurons (31, would represent a unique, noncanonical intracellular vesicle flow pathway that VAMP in the secretion of this peptide- 38, 39). The lack of functional data on hormone (55). With earlier studies dem- the principles and organization of dendritic could explain how membrane proteins and α peptides get locally trafficked in dendrites onstrating the presence of -SNAP mRNA endomembranes renders current un- and possible local translation of SNAP-25 derstanding of postsynaptic membrane dy- lacking Golgi outposts (42). In addition to secretory vesicles, several other exocytotic in dendrites of hypothalamic neurons (56, namics descriptive and incomplete (40). 57), blockade of oxytocin release by CNTs fi organelles, including early endosomes, re- The signi cance of locally made vs. so- substantiated the role of SNAREs as key matically produced neurotransmitters and cycling endosomes, late endosomes, and lysosomes, have been identified in den- mediators of dendritic peptide secretion peptides for dendritic secretion is currently here. Moreover, the data from several also a matter of debate and intense re- drites of various neuron types, extensively reviewed recently (42, 43). other brain areas, including hippocampus search. Equally, it remains unclear why (58, 59), olfactory bulb (60), cerebellum only a fraction of dendrites contain Golgi (61), and neocortex (62, 63), are consistent secretory outspots, raising the fundamental Secretion from Dendrites of Central with the requirement of SNARE proteins question of the significance of local trans- Neurons Relies on SNAREs in dendritic neurosecretion and suggest the lation in the absence of secretory pathways There is considerable evidence for the in- pervasion of this mechanism throughout therein. Interestingly, the presence of volvement of SNAREs in secretion of the brain. It should be highlighted, how- smooth ER-derived organelles associated both SSVs and LDCVs from dendrites of ever, that most of the data on SNARE- with small vesicular structures raises the central neurons. The bulk of the data on dependent dendritic secretion rely exclu- possibility that some exocytotic vesicles can SNARE-dependence of LDCV release sively on CNT-induced inhibition of

Ovsepian and Dolly PNAS | November 29, 2011 | vol. 108 | no. 48 | 19115 Downloaded by guest on September 24, 2021 synaptic transmission, which is indirect ev- onstrated in the cytoplasm of both the existence of such regulated Ca2+-induced idence for SNARE involvement. In light soma and large dendrites of these cells dendritic secretion (79, 80), both the source of significant functional overlap and re- (77). Evidence from nigral dopaminergic and routes of Ca2+ entry, as well as the dundancy between different SNARE var- neurons suggests functional polarization Ca2+ sensitivity of fusion machinery in iants (64, 65) and recently recognized of VAMP isoforms, with VAMP7 (TI- dendrites, seem to differ significantly from reliance of constitutive and regulated re- VAMP) [but not botulinum neurotoxin those at axon terminals. Unlike release lease on different SNAREs (64, 66–68), type B (BoNT/B)-sensitive VAMP variants] at axon endings that relies on action the data described above should be in- contributing to regulated vesicular secre- potential-induced nondecremented de- terpreted with a great degree of caution tion from dendrites (50, 53) despite the polarization, Ca2+ elevations in dendrites and warrant further in-depth research. abundance of VAMP2 in both compart- can be graded and may be driven by back ments. Intriguingly, the controversy over propagating action potentials or prolonged Evidence for Specialization of Exocytotic the presence of nonfunctional SNAREs in dendritic Ca2+ spikes. Additionally, they can Machinery for Dendritic Secretion dendrites of hippocampal neurons has be mediated through activation of Ca2+- In biological systems, membrane fusion been partially resolved by the identifica- permeable receptor-channel complexes must be carefully controlled to exclude tion of VAMP2 on the pool of vesicles or by release of Ca2+ from internal stores vesicle delivery to incorrect acceptor com- that bypass this neuronal compartment on (37, 81, 82). Interestingly, a number of in- partments that may prove detrimental. the transcytotic journey to final functional vestigations suggest that different voltage- Therefore, intracellular pathways of secre- destination at axon terminals (78). It regated Ca2+ channels operate at these two tory organelles follow highly regulated mains to be established whether VAMP2 poles of neurons, with L- and R-types routes, to ensure targeted delivery to their redundant in dendrites of nigral dopami- dominating in dendrites, in contrast to axon fusion end-points. These journeys seem to nergic neurons (52) can be involved terminals that depend primarily on P/Q- be controlled primarily by Ras-related with LDCVs targeted for release at and N-type channels to mediate Ca2+ in- GTPases, Rab proteins instrumental in axon terminals. flux (83, 84) (Fig. 2B and see below). An ensuring the specificity of contacts between illustrative example of such polarization is 2+ fusion partners (18, 20). Although special- Ca Triggering of Dendritic Vesicular provided by dentate gyrus granule cells, ization of certain SNARE variants for in- Secretion where blockade of L-type Ca2+ current dividual fusion reactions and for guiding At a canonical synapse, secretory vesicles in inhibits the dendritic release of dynorphin vesicular traffic has been recognized, the preparation for fusion dock at release sites at the perforant pathway synapses but has extent to which SNAREs contribute to and get primed for Ca2+-induced fusion no effect on secretion of the same sub- these regulated flows of exocytotic organ- with the plasma membrane (65) (Fig. 1C). stance from the axon terminals of these elles and their routing to different com- Upon arrival of action potentials, voltage- neurons (85). Differences have also been partments remains a subject of great gated Ca2+ channels open and allow influx documented in the kinetics of free cytosolic interest and ongoing debate; this is pri- of a pulse of Ca2+, thereby raising the in- [Ca2+], with fast dendritic Ca2+ transients marily due to the capacity of some tracellular [Ca2+] so that synchronous ve- displaying more diffuse and protracted SNAREs to assemble promiscuously sicular release gets triggered. Although characteristics. The latter seem to depend with different variants (69, 70). For in- there is convincing evidence to support the more heavily on activation of store- stance, in yeast ER–Golgi traffic, Sec22 and Ykt6 can substitute for each other as R-SNAREs. Similarly, in mammalian cells, A Pre-synapse B SNAP-25 and SNAP-23, as well as VAMP2 Actin and cellubrevin, are capable of replacing each other to varying degrees in regulated Bassoon Piccolo exocytosis (64, 71). Nevertheless, specific Actin SNAREs have been assigned to most of SV

the fusion steps and organelle types (18), Rab3 ELKS ELKS Piccolo RIM Munc13 with illustrative examples of such speciali- Liprin-α Bassoon RIMs zation in neuronal dendrites represented SNAREs VDCC Munc18 by syntaxin 4 and SNAP-23 (72, 73). Munc13 Highly enriched in dendritic spines, these Rab3 Liprin-a SNAREs are known to mediate dendrite- RIM-BP SAP-97 SV speciﬁc fusion reactions, reviewed in detail PSD95 recently (42). SNAREs Synaptotagmin Adaptation of biochemical and physiological mechanisms of secretion from axon Actin

terminals and dendrites has also been VAMP SNAP-25 proposed on the basis of data obtained from LAR Neurexin other models adopted for studying the Syntaxin VDCC targeting of secretory vesicles to these two Post-synapse Synaptic cleft poles of neurons (74, 75). In hypothalamic neurons, for example, dendritic LDCVs Fig. 3. Molecular architecture of the active zone. (A) Schematic of a canonical synapse with presynaptic seem to differ in their content from those release site enriched with secretory vesicles (SV, blue), active zone proteins (light brown), SNAREs/Ca2+ 2+ destined for release from axon terminals sensor (red), scaffolding/cytoskeletal proteins (orange), and voltage-gated Ca channels (yellow). The and have been proposed to rely on distinct adjacent postsynaptic receptive element comprises PSD95, SAP-97 (pink), receptors (green), scaffolding/ cytoskeletal proteins (orange), and SNAREs (red). (B) Protein interaction network targeting secretory exocytotic mechanisms (76). In the same vesicles and driving their regulated fusion at the active zone. In cooperation with Rab3-GTPase and way, in hippocampal neurons the mem- anchoring/cytoskeletal proteins [leucocyte common antigen-related protein (LAR), neurexins, actin], brane-associated SNAP-25 explicitly shown active zone proteins that include members of ﬁve protein families (RIMs, Bassoon and Piccolo, ELKS, and in axons was hardly detectable in dendrites, liprins-α) assist docking and priming of synaptic vesicles for SNARE-mediated fusion. VDCC, voltage- even though its presence has been dem- dependent Ca2+ channel.

19116 | www.pnas.org/cgi/doi/10.1073/pnas.1017235108 Ovsepian and Dolly Downloaded by guest on September 24, 2021 operating Ca2+ channels, with a metabo- drites for diffuse neurosecretion with ret- tropic mechanism contributing significantly rograde modulator signaling. A range of to the process (82, 86). In concert with diffuse metabotropic effects caused by slower kinetics of dendritic Ca2+ spikes, dendritic release provides direct experi- fl Dendrite a greater component of diffuse ef ux of mental support for this inference. SSVs [Ca2+] from internal stores would contrib- LDCVs ute appreciably toward global [Ca2+]ele- Sites for Dendritic Secretion: Active Zone vs. Ectopic Release? vations, which in turn should favor diffuse ER release of mediators or secretion of peptide Requirement of active zone-like special- and hormone-like substances from den- izations for exocytosis from dendrites is one DDS drites (87–89). Interestingly, although the of the long-standing research queries of L-, R-type 2+ VDCC relation between [Ca ] and secretion of neurobiology. These restricted protein-rich VAMP7 both classic transmitters and peptides (62, areas of presynaptic plasma membrane NMDA SNAP-23 90) seems to be steeper at axon terminals, provide a molecular scaffold for docking SOCC neurosecretion from dendrites exhibits and priming of synaptic vesicles, with their Syntaxin3,4 greater sensitivity to smaller elevations in controlled fusion and release of neuro- SNAP-25 [Ca2+] and exhibits higher tolerance to re- transmitters triggered by action potential- duction of extracellular [Ca2+] (84, 91, 92). induced Ca2+ elevations (Fig. 3 A and B). Hence, in contrast to micromolar [Ca2+] In nerve cells, vesicular secretion at active being required for release of classic trans- zones and distant from membrane release mitters at axon terminals (93, 94), the ab- specializations (ectopic release) is well solute minimum [Ca2+] elevations required documented (100). Although the dendro- for dendritic release range between 50 and dendritic juxtapositions, with closely asso- TGN 200 nM (62, 95). These concentrations ciated secretory organelles, were among match closely the intracellular [Ca2+]re- the first synaptic elements recognized in the quired for spontaneous exocytosis of neu- brain at an ultrastructural level (101), there Soma rotransmitters at nerve endings without is so far no evidence for the presence of notable effects on evoked release proper- electron-dense active zone tufts in den- ties (67, 96). The higher efficiency of Ca2+ drites. Notably, together with the presence in dendrites has been speculated to be due of dense uniform vesicular pools, reminis- to distinct synaptotagmin variants or con- cent of specialized release sites at canonical tribution of an additional high-affinity Ca2+ synapses (101–105), diffusely distributed VAMP1,2 P/Q-, N-type sensor linking voltage-activated Ca2+ influx secretory organelles have also been dem- SNAP-25 VDCC to the dendritic secretory machinery (92). onstrated in dendrites of central neurons, Syntaxin1,2 SOCC This notion received support from experi- without preference for aggregations at ments showing that despite the fact that the membrane juxtapositions (36, 106, 107). Axon NMDA receptor-channel complex at den- Manifestly, vesicular clustering at axon LDCVs drites in olfactory bulb granule cells is not terminal is typically associated with an directly coupled to vesicular release, small electron-dense matrix, referred to as pre- Ca2+ elevations mediated through NMDA synaptic grids (108), which have not been SSVs receptor activation can be sufficient to identified at dendrites of central neurons 1 2 trigger dendritic secretion of GABA (79). (31, 36, 106). Likewise, the molecular It is noteworthy that synaptotagmin 12 (97) composition and architecture of dendro- and double C2 domain 2b (doc2b) (98) dendritic synapses remains poorly studied PSD ADS seem to ensure greater [Ca2+] sensitivity of (42), despite the latter being well recog- the fusion machinery mediating spontane- nized anatomically and physiologically. The ous release at nerve terminals and, perhaps, presence of postsynaptic scaffolding mole- could contribute toward dendritic secre- cules, such as PSD-93 and PSD-95, at Fig. 4. Polarized exocytotic machinery of central tion. Intriguingly, the direct contribution of dendro-dendritic juxtapositions of granule/ neurons: a schematic. With both LDCVs and SSVs synaptotagmin 10 to insulin-like growth mitral cells suggests their homology with (light brown and white circles, respectively) iden- factor 1 (IGF-1) release from somato-den- conventional postsynaptic elements (109). tified in dendrites and axon terminals, Ca2+ ele- drites of olfactory bulb neurons has been However, no data are available on Rab3a vations that triggers dendritic secretion are fi mediated by activation of L- and R-type voltage- identi ed recently (99). Localized on IGF-1 interacting molecules (RIMs), proteins rich 2+ containing vesicles, Ca2+ binding by syn- in E, L, K, and S (ELKS), Velis, Liprin-α or dependent Ca channels (VDCC), NMDA receptor- channel complex, and store-operating Ca2+ chan- aptotagmin 10 causes these vesicles to un- Munc13, Munc18, membrane-associated nels (SOCC). This contrasts with axon terminals, dergo exocytosis with spatial and temporal SNAREs, and other hallmark active zone 2+ 2+ where Ca elevations are mediated by P/Q-, N- characteristics distinct from Ca -de- proteins here and at other potential den- type VDCC as well as SOCC mechanisms. Principal pendent exocytosis controlled by synapto- dro-dendritic junctions. Likewise, in mag- SNAREs identified at dendrites of neurons are 2+ tagmin 1 (99). These and perhaps other nocellular hypothalamic neurons, Ca - VAMP7, SNAP-23, syntaxin3 and -4, and SNAP-25; yet-unidentified sensor proteins also con- dependent relocation of LDCVs from the at axon terminals SNAREs are represented by tribute toward greater responsiveness of deeper cytoplasm to the submembrane VAMP1 and -2, SNAP-25, and syntaxin1 and 2. dendritic secretory machinery to small ele- compartments has been demonstrated, yet PSD, postsynaptic density. DDS, dendro-dendritic 2+ vations of cytoplasmic [Ca ]. Thus, the with no ultrastructural evidence for release synapse; ADS, axo-dendritic synapse. Note that neurosecretion at axon terminal can be caused by higher responsiveness of dendritic fusion site specializations nearby (107). In the “ ”“ 2+ reversible hemifusion ( kiss-and-stay, kiss-and- machinery to local and global Ca ele- absence of active zones, the sensitivity of run”) (1) or complete collapse of synaptic vesicles vations and the activation of graded vesic- dendritic LDCV neurosecretion to CNTs with the plasmalemma (2). Recycling of synaptic ular release of transmitters or peptides are (107, 110) indicates ectopic SNARE-de- vesicles after their complete fusion involves cla- consistent with the specialization of den- pendent (v-SNAREs) release of peptides thrin-dependent endocytosis (coated vesicles).

Ovsepian and Dolly PNAS | November 29, 2011 | vol. 108 | no. 48 | 19117 Downloaded by guest on September 24, 2021 from the dendrites of these neurons, similar “for us, the cause of...polarization...is likely to unveil recipes for potential rem- to that described in adrenal chromaffin uniquely in the relationship which exists edies to normalize brain functions that cells (111, 112). Data obtained from nigral between the neurons...where the seat is have been disrupted by numerous dis- dopaminergic cells indicate that although the entry of excitation” (122). Indeed, as orders and diseases. Emerging data sug- their axon terminals in the striatum are established later, these entry sites corre- gest that release of mediators and peptides enriched with vesicular aggregates and ac- spond to chemical synapses, specialized from dendrites exhibiting instructive and tive zone type specializations with closely for vectorial transmission of signals via homeostatic effects offers neurons enor- positioned vesicular pools (44, 113), their vesicular release of neurotransmitters mous integrative power and diversity. In- dendrites in the substantia nigra are devoid (123, 124), a process blocked by CNTs. deed, with the capacity for graded tuning of such aggregations or any release spe- Accumulating data indicate congruity of of afferent inputs through release of cializations (114, 115). Note that as in hy- SNARE-dependent exocytotic mecha- modulators and transmitters, dendrites pothalamic neurons, dopamine secretion at nisms and machinery at axon terminals effectively filter and sort inputs and adjust both corpus striatum and substantia nigra is and dendrites. However, as in other po- their strength. Although recent research strongly inhibited by CNTs, which is con- larized cells (125), some SNARE in- greatly advanced our understanding of sistent with its reliance on SNAREs (50). hibitors have revealed differential effects dendritic physiology, it has also raised Interestingly, the ectopic dopamine release at these two neuronal poles, implying axo- a number of fundamental questions with from dendrites of nigral neurons agrees dendritic specialization of membrane fu- important implications for the SNARE with diffuse immunoreactivity of SNAP-25 sion nanoengines and release mechanisms hypothesis and neuronal doctrine. Recog- therein (52, 54), contrasting with the (Fig. 4). Interestingly, although fusigenic nition of SNAREs and other regulatory punctuated presence of this SNARE vari- asymmetry in epithelial cells seems to be proteins specialized for retrograde signal- ant at presynaptic axon terminals of other established primarily by differential dis- ing at dendrites queries the canonical central neurons (116, 117). Finally, analysis tribution of individual t-SNAREs (126, synapse-based view of redundancy of some of the distribution of vesicular glutamate 127) as well as their regulation by different SNARE proteins and predicts decorous transporters-3 (VGluT3), an SSV protein variants of Sec/Munc SM proteins (128, sects for these molecules in the “functional expressed preferentially in dendrites of 129), in neurons both t- and v-SNAREs catalog” of neurons. Likewise, vesicular cortical neurons (118, 119), also revealed seem to contribute to such polarization secretion from dendrites in the absence of its dispersed immunoreactivity at both (50, 73). Though explicit, these original active zones along with apparently higher subsynaptic areas as well as along the pri- data require further experimental valida- [Ca2+] sensitivity of dendritic exocytosis mary and secondary apical and basal den- tion with the use of advanced optical tools call for further refinement of the current drites (119), with no indication of active for single-molecule studies combined with canonical synapse-based neurotransmis- zone-like specializations therein. Thus, al- genetic approaches and electrophysiology. sion hypothesis. Ironically, the emerging though the distribution of secretory organ- Ad interim, it is tempting to speculate that view on asymmetric secretory mechanisms elles in dendrites of some central neurons polarization of secretory machinery and at axo-dendritic poles of neurons, though suggests exocytotic specializations, the bulk mechanisms facilitated the remarkable complementing Ramon-y-Cajal’s doctrine of the data are consistent with their diffuse extension of dendritic functions during of dynamic polarization of neurons, distribution with fusion outside of active evolution far beyond mere information also revitalizes the reticularist view of zones, posing the fundamental question of transmission and wiring of individual neuronal assemblies, led by Ramon-y- regulatory mechanisms and the physiologi- neurons into functional networks. This Cajal’s key opponent Camillo Golgi, cal significance of extrasynaptic retrograde would provide dendrites with astounding who championed the intimate reciprocal signaling. Unquestionably, recent advances flexibility and the capacity for integrating interaction between nerve cells (135). in optical imaging combined with computer and processing tremendous volumes of Clearly, along with the deep physiological models simulating transmission between information (130, 131). Indeed, along division between dendrites and axons, neurons, which made possible analysis of with the significant retrograde influence the data outlined above strongly suggest presynaptic dynamics at the level of the on synaptic inputs and dendritic cable their common functional and evolutionary individual vesicle (100, 120), should greatly properties through regulation of voltage- missions—channeling and modulation of assist future studies in these and numerous gated K+ and other currents, dendritic information flow between neurons. Not- other fascinating questions raised by secretion endows nerve cells with the withstanding all successes and failures, dendritic research. capacity to support a broad array of in- the stakes remain high for defining the tegrative and homeostatic functions meaning and significance of axo-dendritic Functional Polarization of Neurons and (132–134). Thus, in addition to shared polarization of release mechanisms for SNAREs traits and functions with those recognized information processing at the level of the The highly asymmetric morphology of for axon terminals, SNARE-dependent single neuron and overall integrative nerve cells led Ramon-y-Cajal to put for- dendritic secretion seems to be specialized brain functions. ward the principle of functional polariza- for functions unique to this neuronal tion of neurons, which postulated compartment. ACKNOWLEDGMENTS. We thank Drs. Gary unidirectional flow of information from Lawrence, Valerie B. O’Leary, and Neal Lemon for Concluding Remarks and Future insightful discussions and comments on dendrites to axons. Together with the an- this manuscript. We apologize for not citing all atomical, physiological, and biochemical Directions relevant articles on the topic because of space discreteness of nerve cells, the polarization Disentangling the precise means used by constraints. This work was supported by the principle rests as one of the cornerstones of nerve cells for information processing and Program for Research in Third Level Institu- fi tions Cycle 4 grant from the Irish Higher neuronal doctrine, af rming nerve cells transmission would afford important clues Educational Authority for the Neuroscience as basic structural and functional modules to one of the most enduring mysteries of section of “Targeted-driven therapeutics and of the brain (121). He explained that our times—the human brain. It also is theranostics.”

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