Hyperactive and impulsive behaviors of LMTK1 knockout mice.

Lemur tail kinase 1 (LMTK1), previously called Apoptosis-Associated Tyrosine Kinase (AATYK), remains an uncharacterized Ser/Thr protein kinase that is predominantly expressed in the brain. It is recently reported that LMTK1A, an isoform of LMTK1, binds to recycling endosomes through its palmitoylation and regulates endosomal trafficking by suppressing the activity of Rab11 small GTPase. In neurons, knockdown or knockout of LMTK1 results in longer axons, greater branching of dendrites and increased number of spines, suggesting that LMTK1 plays a role in neuronal circuit formation. However, its in vivo function remained to be investigated. Here, we examined the brain structures and behaviors of LMTK1 knockout (KO) mice. LMTK1 was expressed in most neurons throughout the brain. The overall brain structure appeared to be normal in LMTK1 KO mice, but the numbers of synapses were increased. LMTK1 KO mice had a slight impairment in memory formation and exhibited distinct psychiatric behaviors such as hyperactivity, impulsiveness and high motor coordination without social interaction deficits. Some of these abnormal behaviors represent core features of attention deficit hyperactive disorder (ADHD), suggesting the possible involvement of LMTK1 in the pathogenesis of ADHD.

Isoform-dependent subcellular localization of LMTK1A and LMTK1B and their roles in axon outgrowth and spine formation.

Lemur kinase 1 (LMTK1) is a membrane-bound Ser/Thr kinase that is expressed in neurons. There are two splicing variants of LMTK1 with different membrane binding modes, viz., cytosolic LMTK1A that binds to membranes through palmitoylation at the N-terminal cysteines and LMTK1B, an integral membrane protein with transmembrane sequences. We recently reported that LMTK1A regulates axon outgrowth and spine formation in neurons. However, data about LMTK1B are scarce. We analysed the expression and cellular localization of LMTK1B along with its role in axon and spine formation. We found that both LMTK1B and LMTK1A were expressed equally in the cerebral cortex and cerebellum of the mouse brain. Similar to LMTK1A, the wild type of LMTK1B was localized to Rab11-positive pericentrosomal compartment. The kinase negative (kn) mutant of LMTK1B was found to be associated with an increase in the tubular form of endoplasmic reticulum (ER), which was not the case with LMTK1A kn. Furthermore, unlike LMTK1A kn, LMTK1B kn did not stimulate the axon outgrowth and spine formation. These results suggest that while LMTK1A and LMTK1B share a common function in recycling endosomal trafficking at the pericentrosomal compartment, LMTK1B has an additional unique function in vesicle transport in the ER region.

The LMTK1-TBC1D9B-Rab11A Cascade Regulates Dendritic Spine Formation via Endosome Trafficking.

Dendritic spines are postsynaptic protrusions at excitatory synapses that are critical for proper neuronal synaptic transmission. While lipid and protein membrane components are necessary for spine formation, it is largely unknown how they are recruited to developing spines. Endosomal trafficking is one mechanism that may influence this development. We recently reported that Lemur kinase 1A (LMTK1A), a membrane-bound Ser/Thr kinase, regulates trafficking of endosomes in neurons. LMTK1 has been shown to be a p35 Cdk5 activator-binding protein and a substrate for Cdk5-p35; however, its neuronal function has not been sufficiently studied. Here, we investigate the role of LMTK1 in spine formation. Depletion of LMTK1 increases spine formation, maturation, and density in primary cultured neurons and in mouse brain of either sex. Additionally, expression of kinase-negative LMTK1 stimulates spine formation in primary neurons and in vivo LMTK1 controls spine formation through Rab11, a regulator of recycling endosome trafficking. We identify TBC1D9B, a Rab11A GTPase-activating protein (Rab11A GAP), as a LMTK1 binding protein, and find that TBC1D9B mediates LMTK1 activity on Rab11A. TBC1D9B inactivates Rab11A under the control of LMTK1A. Further, by analyzing the effect of decreased TBC1D9B expression in primary neurons, we demonstrate that TBC1D9B indeed regulates spine formation. This is the first demonstration of the biological function of TBC1D9B. Together, with the regulation of LMTK1 by Cdk5-p35, we propose the Cdk5-LMTK1-TBC1D9B-Rab11A cascade as a novel signaling mechanism regulating endosomal transport for synapse formation and function.SIGNIFICANCE STATEMENT Dendritic spines are postsynaptic specializations essential for synaptic transmission. However, it is not known how critical membrane components are recruited to spines for their formation. Endosomal trafficking is one such mechanism that may mediate this process. Here we investigate regulators of endosomal trafficking and their contribution to spine formation. We identify two novel factors, LMTK1 and TBC1D9B, which regulate spine formation upstream of Rab11A, a small GTPase. LMTK1 is a membrane bound Ser/Thr kinase regulated by Cdk5-p35, and TBC1D9B is a recently identified Rab11 GAP. LMTK1 controls the GAP activity of TBC1D9B on Rab11A, and TBC1D9B mediates the LMTK1 activity on Rab11A. We propose the Cdk5-LMTK1-TBC1D9B-Rab11A cascade as a novel mechanism controlling spine formation and function.