Abstract

   Microglial abnormalities may contribute to neurodevelopmental
   disorders. PTEN is implicated as a susceptibility gene for autism
   spectrum disorders and its germline ablation in mice causes behavioral
   abnormalities. Here we find postnatal PTEN deletion in microglia causes
   deficits in sociability and novel object recognition test. Mutant mice
   harbor markedly more activated microglia that manifest enhanced
   phagocytosis. Interestingly, two-week postponement of microglia PTEN
   ablation leads to no social interaction defects, even though mutant
   microglia remain abnormal in adult animals. Disturbed neurodevelopment
   caused by early PTEN deletion in microglia is characterized by
   insufficient VGLUT1 protein in synaptosomes, likely a consequence of
   enhanced removal by microglia. In correlation, in vitro acute slice
   recordings demonstrate weakened synaptic inputs to layer 5 pyramidal
   neurons in the developing cortex. Therefore, microglial PTEN safeguards
   integrity of neural substrates underlying sociability in a
   developmentally determined manner.

   Keywords: microglia, PTEN, phagocytosis, VGLUT1, autism spectrum
   disorders, cerebral cortex, auditory cortex, layer 5

Introduction

   Microglia are tissue-resident macrophages in the central nervous
   system. Their progenitors develop in the yolk sac, migrate into the
   embryonic brain and by local proliferation populate the entire brain
   throughout life ([47]1–[48]4). Microglia participate in synaptic
   pruning, remodeling and maturation, particularly during postnatal
   neurodevelopment ([49]5–[50]7). Ablation of CX[3]CR1, a chemokine
   receptor that is expressed by microglia and mediates microglia-neuron
   interactions ([51]8), leads to impaired synapse maturation, decreased
   functional brain connectivity, and autism-like abnormal behaviors
   ([52]9, [53]10). Mice lacking the Triggering Receptor Expressed On
   Myeloid Cells 2 (TREM2), which in the brain is mainly expressed by
   microglia, exhibit a loss of microglia and abnormally increased spine
   densities selectively in the hippocampal CA1 region, and these mice
   suffer from behavioral defects ([54]11). On the other hand, increasing
   evidence also indicates that, in adult brain, microglia actively probe
   neuronal states and play important roles in damage clearance, immune
   protection, trophic support and neuroplasticity ([55]12–[56]16).
   Phenotypic and functional abnormalities of microglia are intimately
   associated with neurodegenerative diseases. Prominently, in rodent
   models of neurodegenerative diseases including the Alzheimer’s disease
   (AD) and Amyotrophic lateral sclerosis (ALS), microglia display a
   stereotypic disease-associated microglia (DAM) phenotype ([57]17),
   which appears to be an inflammatory state driven by a TREM2-dependent
   pathway ([58]18). While the disease-associated microglia phenotype may
   or may not be a cause of pathogenesis, it is clear that changes to
   microglia, particularly those genetically determined, could lead to
   behavioral phenotypes either via a neurodevelopmental route in infants
   or children or via a functional route in adults.

   The autism spectrum disorder (ASD) is a heterogenous group of
   neurodevelopmental disorders that are characterized by childhood-onset
   impairment of communication and social skills combined with the
   presence of repetitive behaviors. Genetic predisposition play a
   significant role, with ~1000 genes by some estimate potentially
   contributing to ASD susceptibility ([59]19). Certain monogenic
   mutations greatly increase the risk of ASD, such as those affecting
   FMR1 in fragile X syndrome ([60]20, [61]21), MECP2 in Rett syndrome
   ([62]22), and TSC1 and TSC2 in Tuberous Sclerosis Complex ([63]23). A
   commonly dysregulated pathway in these syndromic ASDs is the PI3 kinase
   and mTOR pathway ([64]24). Dysregulation of PI3K and mTOR activities in
   neurons affects migration, axon formation and targeting, dendritic
   growth and spine development, potentially contributing to ASD
   pathogenesis directly ([65]24). Phosphatase and Tensin Homolog (PTEN)
   is a tumor suppressor and a main lipid phosphatase that negatively
   regulates PI3K/Akt/mTOR pathways, which are vital to normal functions
   of essentially all cell types ([66]25). Mutations of PTEN are found in
   ASD patients, particularly those with macrocephaly ([67]26–[68]30). In
   mice, Nse-cre-driven Pten deletion in a subset of neurons in the cortex
   and hippocampus leads to macrocephaly and deficits in social
   interactions reminiscent of ASD ([69]31, [70]32), indicating a neuronal
   root of ASD pathogenesis due to loss of PTEN function. Given the fact
   that PTEN is universally expressed, other cell types may also
   contribute. Indeed, studies of PTEN mutations that cause cytoplasmic-
   or nuclear-predominant localization have revealed abnormalities of not
   only neurons but also glial cells, including microglia, in association
   with ASD-like behavioral phenotypes. In particular, dysregulated
   subcellular PTEN localization cause aberrant microglia activation with
   increased phagocytic activities ([71]33, [72]34). Consistent with the
   idea that abnormal microglia functions may be involved in ASD
   pathogenesis due to PTEN mutations ([73]33, [74]35–[75]37), microglial
   overexpression of eIF4E leads to elevated protein synthesis and
   functional disturbance of microglia, altered synapse density, and
   ASD-like behaviors in mice ([76]37). Our current study is designed to
   specifically probe how PTEN in microglia may regulate microglia-neuron
   crosstalk and whether microglial PTEN perturbation could affect
   neurodevelopment and animal behaviors. We report postnatal PTEN
   deletion in microglia immediately after birth leads to accumulation of
   activated microglia, accompanied by reduced synapse density and
   markedly changed firing patterns of cortical layer 5 (L5) pyramidal
   neurons, leading to impaired sociability later in life. Postponement of
   genetic deletion by 14 days completely avoided neuronal and behavioral
   impairment, demonstrating a developmental period during which intact
   microglia are essential for normal neural substrates underlying
   sociability.

Results

Postnatal Pten ablation in microglia leads to deficits in sociability and
novelty exploration

   To investigate a potential role for PTEN in microglia in shaping animal
   behaviors, we created mice with inducible Pten deletion in a
   microglia-specific manner by breeding Cx3cr1 ^CreERT2/+ mice with Pten
   ^fl/fl mice. To specifically examine PTEN-controlled microglial
   functions during postnatal neurodevelopment, as detailed in Methods and
   illustrated in [77]Figure 1A , we injected tamoxifen into the stomach
   of newborn Cx3cr1 ^CreERT2/+ Pten ^fl/fl and littermate control Cx3cr1
   ^CreERT2/+ Pten ^+/+ mice daily from postnatal day 0 (P0) to day 2
   (P2). In this system, microglia and other cells in the brain are intact
   throughout embryonic development. To verify the efficiency of Pten
   deletion by this procedure, microglia were isolated at different time
   points to test PTEN protein expression. As compared to cells from
   littermate control mice, microglia from Cx3cr1 ^CreERT2/+ Pten ^fl/fl
   mice largely lost PTEN protein as early as one week after the tamoxifen
   injection and remained so for as long as we tested ([78] Figures S1A, B
   ). Taking advantage of the EYFP expression from the knock-in Cx3cr1
   ^CreERT2 allele, we used anti-GFP antibody to stain for EYFP on tissue
   sections and verified that the targeted cells were essentially all
   Iba-1^+ microglia but not astrocytes (S100b^+), oligodendrocytes
   (Olig2^+), or neurons (NeuN^+) ([79] Figures S1C, D ). For convenience,
   we subsequently call tamoxifen-treated Cx3cr1 ^CreERT2/+ Pten ^fl/fl
   and Cx3cr1 ^CreERT2/+ Pten ^+/+ mice mPTEN^KO(0) and mPTEN^WT(0),
   respectively.

Figure 1.

   Figure 1
   [80]Open in a new tab

   Postnatal Pten ablation in microglia leads to deficits in sociability
   and novelty exploration (A) Experimental scheme. (B) Numbers of marbles
   buried in 30 min during the marble-burying test (mPTEN^WT(0), n=14;
   mPTEN^KO(0), n=14). (C) Time spent exploring a novel versus a familiar
   object by mPTEN^WT(0) (n=14) or mPTEN^KO(0) (n=15) mice in the novel
   object recognition test. (D) Time spent interacting with an inanimate
   object or a mouse by mPTEN^WT(0) (n=17) or mPTEN^KO(0) (n=17) mice in
   the three-chamber interaction test. (E) Time spent interacting with a
   familiar or a stranger mouse by mPTEN^WT(0) (n=17) or mPTEN^KO(0)
   (n=17) mice in the three-chamber test. (F) Locomotor activities and
   thigmotaxis in the open-field test. Total distance traveled (m), total
   time of being immobile (sec), total time of being in the center (sec),
   number of fecal boli left and thigmotaxis by individual mPTEN^WT(0)
   (n=15) and mPTEN^KO(0) (n=15) mice. *P < 0.05, **P < 0.01, ****P <
   0.0001, by unpaired (open field and marble-burying) or paired (novel
   objection recognition and three-chamber interaction) t tests.

   To examine behavioral consequences of postnatal PTEN deletion in
   microglia, we first compared mPTEN^KO(0) and mPTEN^WT(0) mice (>8 weeks
   old) in a marble-burying test, which could reveal signs of ASD-like
   repetitive behaviors and potentially increased anxieties ([81]38). As
   shown in [82]Figure 1B , the two groups of mice were comparable. On the
   other hand, when tested in a novel object recognition test (NORT),
   mPTEN^KO(0) mice failed to exhibit a preference for exploring the novel
   object, unlike their mPTEN^WT(0) counterparts ([83] Figure 1C ). In
   social interaction tests, whereas mPTEN^WT(0) mice showed a clear
   preference for interacting with a companion mouse than with an
   inanimate object ([84] Figure 1D ) and for interacting with a stranger
   than with a familiar mouse ([85] Figure 1E ), the mPTEN^KO(0) mice did
   not show such preferences ([86] Figures 1D, E ). The failure of