Tsc1 haploinsufficiency is sufficient to increase dendritic patterning and Filamin A levels

Abstract

Most individuals with tuberous sclerosis complex (TSC) are born with a mutant allele of either TSC1 or TSC2 and a mosaic of psychological and cognitive defects. Tsc1 loss of heterozygosity contributes to severe dendritic abnormalities that are rescued by normalizing the levels of the actin-cross linking protein, Filamin A (FLNA). However, it is unclear whether dendrites and FLNA levels are abnormal in an heterozygote Tsc1 condition. Here, we examined dendritic morphology and FLNA levels in the olfactory bulb of Tsc1 wild type and heterozygote mice. Using in vivo neonatal electroporation to label newborn neurons followed by sholl analysis, we found that Tsc1 haploinsufficiency is associated with increased dendritic complexity and total dendritic length as well as increased FLNA levels. Since reducing FLNA levels has been shown to decrease Tsc1(+/-) dendritic complexity, these data suggest that increased FLNA levels in Tsc1(+/-) mice contribute to abnormal dendritic patterning in the Tsc1 heterozygote condition of individuals with TSC.

Keywords: Circuit; Dendrite; Filamin A; Network; Neurogenesis; Tuberous sclerosis complex; mTOR.

Figure 1. Constitutive and conditional Tsc1^+/− neurons display increased dendritic patterning compared to Tsc1^+/+ neurons

(A) Diagram illustrating electroporation in P0 neonates. Most transfected newborn neurons reach their final location in the olfactory bulb (OB) by P14. (B) Image and reconstruction of the basal dendrite of an OB granule cell expressing tdTomato with superimposed concentric circles for sholl analysis. (C) # crossings as a function of distance for the basal dendrites of tdTomato+ neurons at P14 in Tsc1^+/+ (black), Tsc1^+/− (violet) and Tsc1^fl/+ (green) mice. Neurons were electroporated with pCAG-Cre leading to Tsc1 heterozygosity in Tsc1^fl/+ mice and tdTomato expression due to crossing with R26R-Stop-tdTomato mice. *: P<0.05, **: P<0.01, purple for comparisons between Tsc1 wild type and heterozygote mice, and green between wild type and Tsc1^fl/− mice using two way ANOVA followed by Bonferroni multiple comparisons. (D) Total dendritic length corresponding to the conditions in C.

Figure 2. FLNA levels are increased in Tsc1^+/− nice and correlate with TSC1 levels

(A) Immunoblots for FLNA, TSC1, and ERK1/2 from P14 OB of Tsc1^+/+ (black) and Tsc1^+/− (grey). (B) Normalized % changes for FLNA/ERK. (C) Plot of FLNA versus TSC1 levels.