The word "Motherless" in the title refers to Lionel's status as an orphan. It would be fair to say that the title could just as easily have been Fatherless Brooklyn. As this is a bildungsroman, or coming-of-age novel, the fact that Lionel is an orphan plays a huge role in his life.
One of the main themes of this novel is Lionel's quest to discover his true identity. As most people's identity is constructed, to a large degree, on the relationship with their parents, the fact that Lionel is "motherless" is extremely significant. In fact, Lionel's search for Frank's killer becomes synonymous with his search for his own identity. It is only with Frank's death that Lionel, despite his motherless status, is able to truly discover who he is. By becoming a true detective and avenging Minna, Lionel finds his place in the world.
The fact that Lionel suffers from Tourette's Syndrome makes his status of "motherless" even more problematic. Lionel's way of expressing himself and his emotions is, to say the least, unique, and having a mother to understand him could have made a vast difference to him.
By using the word "motherless" in the title of this great novel, Jonathan Lethem draws immediate focus to one of the biggest challenges and upheavals in the life of our protagonist.