Dissertation

The Clustered Gamma-Protocadherins Regulate Cortical Interneuron Programmed Cell Death

Mancia Leon, Walter Rene

2022

Overview

In the field of neurodevelopmental biology, what regulates the production of cells and how the final number of cells is determined in the central nervous system (CNS) remains one of the long-standing questions. Indeed, what ultimately determines the brain size and how the final number of neurons is computed in the CNS remains unknown. What makes this question more intriguing is that in the CNS, a supernumerary number of neurons is produced in development. Excess neurons are eliminated postnatally through PCD (PCD). Hence PCD is a developmental feature necessary to establish the final number of neurons in each region of the brain. Yet, how PCD is regulated in the CNS and what molecules are involved in its regulation remains controversial and largely unresolved.In my thesis, I introduce a set of fifty-eight cell-adhesion molecules named clustered protocadherins (Pcdhs) and show evidence that these molecules are key to establishing the final inhibitory neuronal population in the cerebral cortex through the regulation of PCD. The 58 Pcdh genes are tamdebly arranged into three smaller gene clusters: alpha (Pcdhα), beta (Pcdhβ) and gamma (Pcdhγ). Given the genomic complexity of Pcdhs, I first use a series of whole cluster genetic deletions of the Pcdhs gene locus to probe the function of Pcdhα, Pcdhβ or Pcdhγ in the regulation of cIN cell death in mice. Using these cluster deletion mice, we show that Pcdhγ genes, but not Pcdhα or Pcdhβ genes, are required for the survival of approximately 50% of cINs through a BAX-dependent mechanism. I then probe whether cINs compete for survival using PCHDγs by employing a co-transplantation assay I developed. My data suggest that indeed Pcdhγ-deficient and wild-type (WT) cINs of the same age compete for survival in a mechanism that involves Pcdhγ. Surprisingly, three-dimensional reconstructions and patch-clamp recordings indicate that the Pcdhγ mutant cells have similar morphology, excitability and receive similar numbers of inhibitory and excitatory synaptic inputs compared to wild type cINs.Next, I investigated which Pcdhγ genes are key to the regulation of cIN survival. The Pcdhγ gene cluster encodes 22 unique isoforms, which are subclassified as A-type, B-type or C-type isoforms. Importantly, deletion of the C-type isoforms (Pcdhγc3, Pcdhγc4 and Pcdhγc5), but not of the A-type or B-type Pcdhγs, results in neonatal lethality. Hence, to compare in the same host microenvironment the survival of cINs carrying WT or mutant Pcdhγ and to bypass neonatal lethality, I developed a co-transplantation assay using two different reporter systems (GFP and RFP). Using this assay and a series of constitutive Pcdhγ isoform deletions mice, I found that the A- and B-type Pcdhγ have no significant role in cIN PCD. However, the removal of Pcdhγ C-type isoforms (Pcdhγc3, Pcdhγc4 and Pcdhγc5), and in particular the sole removal of Pcdhγc4, resulted in increased cIN cell death. Together these transplantation experiments suggested that survival of cINs largely depends on the expression of Pcdhγc4, but not expression of Pcdhγc3 or Pcdhγc5, in cINs.To complement observations above, I developed lentiviral constructs to overexpress Pcdhγc4 or Pcdhγc5 in cIN precursors that lack the entire Pcdhγ gene set. I found that the expression of Pcdhγc4, but not that of Pcdhγc5, significantly rescued a fraction of cINs destined to die.Lastly and in collaboration with Andrea Hasentaub and Michael Striker laboratories, we probed the activity of cINs carrying or lacking a full set of Pcdhγ genes using two-photon calcium imaging across the period of cell death (data not included).In summary, my thesis work identifies for the first time a cell-adhesion molecule involved in the regulation of PCD in cINs and show evidence that among all Pcdh genes, Pcdhγc4 is key for determining the final number of cINs for the cortex.

• Publisher: ProQuest Dissertations & Theses
• Subject: Developmental biology / Neurosciences
• ISBN: 9798351418742