Diverged morphology changes of astrocytic and neuronal primary cilia under reactive insults.

Primary cilia are centriole-derived sensory organelles that are present in most mammalian cells, including astrocytes and neurons. Evidence is emerging that astrocyte and neuronal primary cilia demonstrate a dichotomy in the mature mouse brain. However, it is unknown how astrocytic and neuronal primary cilia change their morphology and ciliary proteins when exposed to reactive insults including epilepsy and traumatic brain injury. We used a double transgenic mouse strain (Arl13b-mCherry; Centrin2-GFP), in which we found spontaneous seizures, and a cortical injury model to examine the morphological changes of astrocytic and neuronal primary cilia under reactive conditions. Transgenic overexpression of Arl13b drastically increases the length of astrocytic and neuronal primary cilia in the hippocampus, as well as the cilia lengths of cultured astrocytes and neurons. Spontaneous seizures shorten Arl13b-positive astrocytic cilia and AC3-positive neuronal cilia in the hippocampus. In a cortical injury model, Arl13b is not detectable in primary cilia, but Arl13b protein relocates to the cell body and has robust expression in the proximity of injured tissues. In contrast, the number of AC3-positive cilia near injured tissues remains unchanged, but their lengths become shorter. These results on astrocytic cilia implicate Arl13b in regulating astrocyte proliferation and tissue regeneration, while the shortening of AC3-positive cilia suggests adaptive changes of neuronal primary cilia under excitotoxicity.

Comparative Phosphoproteomic Profiling of Type III Adenylyl Cyclase Knockout and Control, Male, and Female Mice.

Type III adenylyl cyclase (AC3, ADCY3) is predominantly enriched in neuronal primary cilia throughout the central nervous system (CNS). Genome-wide association studies in humans have associated ADCY3 with major depressive disorder and autistic spectrum disorder, both of which exhibit sexual dimorphism. To date, it is unclear how AC3 affects protein phosphorylation and signal networks in central neurons, and what causes the sexual dimorphism of autism. We employed a mass spectrometry (MS)-based phosphoproteomic approach to quantitatively profile differences in phosphorylation between inducible AC3 knockout (KO) and wild type (WT), male and female mice. In total, we identified 4,655 phosphopeptides from 1,756 proteins, among which 565 phosphopeptides from 322 proteins were repetitively detected in all samples. Over 46% phosphopeptides were identified in at least three out of eight biological replicas. Comparison of AC3 KO and WT datasets revealed that phosphopeptides with motifs matching proline-directed kinases' recognition sites had a lower abundance in the KO dataset than in WTs. We detected 14 phosphopeptides restricted to WT dataset (i.e., Rabl6, Spast and Ppp1r14a) and 35 exclusively in KOs (i.e., Sptan1, Arhgap20, Arhgap44, and Pde1b). Moreover, 95 phosphopeptides (out of 90 proteins) were identified only in female dataset and 26 only in males. Label-free MS spectrum quantification using Skyline further identified phosphopeptides that had higher abundance in each sample group. In total, 204 proteins had sex-biased phosphorylation and 167 of them had increased expression in females relative to males. Interestingly, among the 204 gender-biased phosphoproteins, 31% were found to be associated with autism, including Dlg1, Dlgap2, Syn1, Syngap1, Ctnna1, Ctnnd1, Ctnnd2, Pkp4, and Arvcf. Therefore, this study also provides the first phosphoproteomics evidence suggesting that gender-biased post-translational phosphorylation may be implicated in the sexual dimorphism of autism.

Neuronal and astrocytic primary cilia in the mature brain.

Primary cilia are tiny microtubule-based signaling devices that regulate a variety of physiological functions, including metabolism and cell division. Defects in primary cilia lead to a myriad of diseases in humans such as obesity and cancers. In the mature brain, both neurons and astrocytes contain a single primary cilium. Although neuronal primary cilia are not directly involved in synaptic communication, their pathophysiological impacts on obesity and mental disorders are well recognized. In contrast, research on astrocytic primary cilia lags far behind. Currently, little is known about their functions and molecular pathways in the mature brain. Unlike neurons, postnatal astrocytes retain the capacity of cell division and can become reactive and proliferate in response to various brain insults such as epilepsy, ischemia, traumatic brain injury, and neurodegenerative ß-amyloid plaques. Since primary cilia derive from the mother centrioles, astrocyte proliferation must occur in coordination with the dismantling and ciliogenesis of astrocyte cilia. In this regard, the functions, signal pathways, and structural dynamics of neuronal and astrocytic primary cilia are fundamentally different. Here we discuss and compare the current understanding of neuronal and astrocytic primary cilia.