Early Synaptic Defects in tulp1−/− Mice

The outer plexiform layer (OPL) of the vertebrate retina is where rod and cone photoreceptors make synaptic contact with second-order neurons of the inner nuclear layer (INL).1 Photoreceptors signal second-order neurons with the use of a graded synaptic output, whereby changes in stimulus intensity are conveyed by alterations in the tonic release of neurotransmitter at the active zone of the photoreceptor presynaptic membrane.1 In addition to this tonic release, the visual sensory system requires rapid transfer of information.2 At the photoreceptor synapse, hundreds of neurotransmitter-laden vesicles come in contact with the presynaptic membrane every second, liberating glutamate into the synaptic cleft. To support these requirements, photoreceptor terminals contain a highly specialized presynaptic apparatus, the ribbon synapse, which is thought to be a critical platform for the rapid transport, tethering, docking, and recycling of synaptic vesicles.1,3 Rod photoreceptor terminals typically contain a single ribbon, whereas larger cone terminals harbor multiple ribbons.1 Although many of the structural elements have been examined, the complete complement of their elements and how they interact to form functional synapses remain elusive. More insight into photoreceptor synaptic function is necessary for thorough understanding of the many disorders that affect vision.2,4 Retinitis pigmentosa (RP) refers to a large number of inherited retinal disorders characterized by photoreceptor degeneration. RP, which is genetically and phenotypically heterogeneous, affects more than 1 million persons worldwide.5,6 Mutations in TULP1 underlie an early-onset, severe form of autosomal recessive RP.7–10 TULP1 is a member of the Tubby-like protein family, which includes TULP2, TULP3, and TUB.11 TUB and TULP3 are widely distributed throughout the central nervous system; expression of TULP1 and TULP2 is restricted largely to rod and cone photoreceptors and the testis, respectively.11–16 Although the complete role of TULPs in the central nervous system remains unclear, TULP proteins are known to play important roles in neuronal development and function. This is emphasized by the association of TUB and TULP1 mutations with neurosensory disease phenotypes.7–10,12,17 Tulp1 is expressed exclusively in photoreceptors, localizing to the inner segment (IS), connecting cilium, perikarya, and terminals.18–20 Although the function of Tulp1 remains elusive, there is evidence that Tulp1 plays a role in intracellular trafficking in the IS and at the photoreceptor synapse.18,20–22 Tulp1 is a cytoplasmic protein that associates with cellular membranes and the actin cytoskeleton.21 In tulp1−/− mice, photoreceptor cells degenerate, rod and cone opsins are mislocalized, and rhodopsin-bearing extracellular vesicles accumulate around the ellipsoid region of the IS.18 These defects indicate that Tulp1 may be involved in actin cytoskeletal dynamics such as protein transport from the IS to the outer segment (OS) through the connecting cilium.18,20–22 We recently showed that Tulp1 colocalizes with and binds to the neuronal-specific protein Dynamin-1 at the IS and the terminals of photoreceptors.22 Dynamin-1 is a GTPase that binds actin and regulates endocytosis, vesicle development, and movement at the trans-Golgi network, the plasma membrane, and the synaptic membrane.22–24 Consistent with a role for Tulp1 at the photoreceptor synapse, the b-wave component of the electroretinogram (ERG) generated by depolarizing bipolar cells (DBCs) is markedly reduced in young tulp1−/− mice.22 These results suggest that Tulp1 plays an important role in synaptic function and motivated us to look more closely at the photoreceptor synapse. To accomplish this, we examined the architecture of the tulp1−/− synapse at postnatal day (P)16, an age at which synaptic development is complete in wild-type (wt) mice25 and precedes photoreceptor degeneration in tulp1−/− mice.18 To ascertain whether alterations are specific to the tulp1−/− retina and are not merely a consequence of the degenerative process, we made parallel studies of the retinal degeneration 10 (rd10) mouse, which experiences a comparable rate of photoreceptor degeneration because of a point mutation in exon 13 of the β-subunit of the rod cGMP phosphodiesterase gene.26 Except for the comparable rate of degeneration, no reports have been published of early synaptic abnormalities associated with this model; thus, it presents a fitting control strain for synaptic-specific comparisons. Although mouse models with synaptic-specific mutations (such as Cabp4−/−,27 Cacna1f nob2,28 and Bsn−/−29) exhibit decreased b-waves, altered bipolar dendrites, and presynaptic abnormalities, they do not exhibit photoreceptor degeneration or rhodopsin mislocalization comparable to that observed in tulp1−/− mice. In this study, we verified that Tulp1 is diffusely localized to the photoreceptor synapse and that its distribution overlaps that of several key synaptic proteins. Mice deficient in Tulp1 lack the tight spatial relationship between the ribbon-associated proteins, Bassoon and Piccolo, characteristic of wt animals. In addition, few intact ribbons are present in the OPL. We also report that dendrites of second-order neurons exhibit abnormalities at an early age in tulp1−/− mice. These structural defects are not apparent in rd10 animals. Although ERG a-waves of tulp1−/− mice are reduced in amplitude, they retain normal kinetics. In comparison, the tulp1−/− b-wave intensity-response function is desensitized, consistent with a functional defect at the photoreceptor output synapse. That similar synaptic and b-wave changes were not seen in rd10 mice indicates they are not generalized responses to photoreceptor degeneration. These results indicate that the absence of Tulp1 is associated with a synaptic malformation that precedes photoreceptor degeneration and most likely interferes with the proper development of postreceptoral neurons. Taken together, this appears to represent a phenotype not previously described. These data indicate that Tulp1 plays an important role in photoreceptor synapse development and in photoreceptor function and survival.