The Drosophila KIF1A Homolog unc-104 Is Important for Site-Specific Synapse Maturation

Abstract

Mutations in the kinesin-3 family member KIF1A have been associated with hereditary spastic paraplegia (HSP), hereditary and sensory autonomic neuropathy type 2 (HSAN2) and non-syndromic intellectual disability (ID). Both autosomal recessive and autosomal dominant forms of inheritance have been reported. Loss of KIF1A or its homolog unc-104 causes early postnatal or embryonic lethality in mice and Drosophila, respectively. In this study, we use a previously described hypomorphic allele of unc-104, unc-104bris, to investigate the impact of partial loss-of-function of kinesin-3 on synapse maturation at the Drosophila neuromuscular junction (NMJ). Unc-104bris mutants exhibit structural defects where a subset of synapses at the NMJ lack all investigated active zone (AZ) proteins, suggesting a complete failure in the formation of the cytomatrix at the active zone (CAZ) at these sites. Modulating synaptic Bruchpilot (Brp) levels by ectopic overexpression or RNAi-mediated knockdown suggests that the loss of AZ components such as Ca2+ channels and Liprin-α is caused by impaired kinesin-3 based transport rather than due to the absence of the key AZ organizer protein, Brp. In addition to defects in CAZ assembly, unc-104bris mutants display further defects such as depletion of dense core and synaptic vesicle (SV) markers from the NMJ. Notably, the level of Rab3, which is important for the allocation of AZ proteins to individual release sites, was severely reduced at unc-104bris mutant NMJs. Overexpression of Rab3 partially ameliorates synaptic phenotypes of unc-104bris larvae, suggesting that lack of presynaptic Rab3 contributes to defects in synapse maturation.

The KIF1A homolog Unc-104 is important for spontaneous release, postsynaptic density maturation and perisynaptic scaffold organization

“…Loss of SVs from the NMJ as well as alterations in the size of the readily releasable pool are possible causes for the decreased mEJP frequency at unc-104 bris mutant NMJs. While there is direct ultrastructural evidence for the loss of SVs from boutons in unc-104 null mutant embryos6, we did not observe any reduction in the amount of SVs at central synapses in unc-104 bris mutant larvae23. Interestingly, while presynaptic over-expression of the UAS- unc-104-mcherry transgene in wild-type background did not cause any changes in EJP or mEJP amplitudes, it resulted in an increase in mEJP frequency (Fig.…”

“…1c,f). Kinesin-3 is the major transporter of SVs, and previous reports show several SV markers are severely reduced at unc-104 mutant NMJs623. Loss of SVs from the NMJ as well as alterations in the size of the readily releasable pool are possible causes for the decreased mEJP frequency at unc-104 bris mutant NMJs.…”

“…At wild-type NMJs only the youngest synapses, in sum approximately 5%, lack the AZ organizing protein Brp24. In contrast, at the NMJs of unc-104 bris mutant larvae about 25% of the postsynaptic densities (PSDs) are unapposed by Brp2023. In order to examine the functional consequences of impaired kinesin-3 function, we performed intracellular current clamp recordings at 3 rd instar larval NMJs.…”

“…Multiple presynaptic structural defects have so far been identified in the context of loss of unc-104 function62023. However, the importance of Unc-104 for postsynaptic development is largely unexplored.…”

“…The recessive point mutation unc-104 bris at the forkhead associated (FHA) domain causes morphological changes at the neuromuscular junction (NMJ) such as increased NMJ length and synaptic bouton number as well as reduced bouton size20. In addition, unc-104 bris have increased proportion of postsynaptic glutamate receptor fields that are unapposed by presynaptic active zone (AZ) scaffolding protein Bruchpilot (Brp)20212223. Furthermore, we have recently shown that these Brp-negative synapses lack all major AZ components investigated, such as Liprin-α, Cacophony and SRPK79D, suggesting a crucial role of unc-104 in presynaptic maturation23.…”