PTEN In Neurons Research Articles

Spatially Precise Genetic Engineering at the Electrode-Tissue Interface.

The interface between electrodes and neural tissues plays a pivotal role in determining the efficacy and fidelity of neural activity recording and modulation. While considerable efforts have been made to improve the electrode-tissue interface, the majority of studies have primarily concentrated on the development of biocompatible neural electrodes through abiotic materials and structural engineering. In this study, an approach is presented that seamlessly integrates abiotic and biotic engineering principles into the electrode-tissue interface. Specifically, ultraflexible neural electrodes with short hairpin RNAs (shRNAs) designed to silence the expression of endogenous genes within neural tissues are combined. The system facilitates shRNA-mediated knockdown of phosphatase and tensin homolog deleted on chromosome 10 (PTEN) and polypyrimidine tract-binding protein 1 (PTBP1), two essential genes associated in neural survival/growth and neurogenesis, within specific cell populations located at the electrode-tissue interface. Additionally, it is demonstrated that the downregulation of PTEN in neurons can result in an enlargement of neuronal cell bodies at the electrode-tissue interface. Furthermore, the system enables long-term monitoring of neuronal activities following PTEN knockdown in a mouse model of Parkinson's disease and traumatic brain injury. The system provides a versatile approach for genetically engineering the electrode-tissue interface with unparalleled precision, paving the way for the development of regenerative electronics and next-generation brain-machine interfaces.

PTEN Regulates Dendritic Arborization by Decreasing Microtubule Polymerization Rate.

Phosphatase and tensin homolog (PTEN) is a major negative regulator of the phosphatidylinositol-3-kinase (PI3K)/Akt/mechanistic target of rapamycin (mTOR) pathway. Loss-of-function mutations in PTEN have been found in a subset of patients with macrocephaly and autism spectrum disorder (ASD). PTEN loss in neurons leads to somal hypertrophy, aberrant migration, dendritic overgrowth, increased spine density, and hyperactivity of neuronal circuits. These neuronal overgrowth phenotypes are present on Pten knock-out (KO) and reconstitution with autism-associated point mutations. The mechanism underlying dendritic overgrowth in Pten deficient neurons is unclear. In this study, we examined how Pten loss impacts microtubule (MT) dynamics in both sexes using retroviral infection and transfection strategies to manipulate PTEN expression and tag the plus-end MT binding protein, end-binding protein 3 (EB3). We found Pten KO neurons sprout more new processes over time compared with wild-type (WT) neurons. We also found an increase in MT polymerization rate in Pten KO dendritic growth cones. Reducing MT polymerization rate to the WT level was sufficient to reduce dendritic overgrowth in Pten KO neurons in vitro and in vivo Finally, we found that rescue of dendritic overgrowth via inhibition of MT polymerization was sufficient to improve the performance of Pten KO mice in a spatial memory task. Taken together, our data suggests that one factor underlying PTEN loss dependent dendritic overgrowth is increased MT polymerization. This opens the possibility for an intersectional approach targeting MT polymerization and mTOR with low doses of inhibitors to achieve therapeutic gains with minimal side effects in pathologies associated with loss of neuronal PTEN function.SIGNIFICANCE STATEMENT Loss of Pten function because of genetic deletion or expression of mutations associated with autism spectrum disorder (ASD), results in overgrowth of neurons including increased total dendritic length and branching. We have discovered that this overgrowth is accompanied by increased rate of microtubule (MT) polymerization. The increased polymerization rate is insensitive to acute inhibition of mechanistic target of rapamycin (mTOR)C1 or protein synthesis. Direct pharmacological inhibition of MT polymerization can slow the polymerization rate in Pten knock-out (KO) neurons to rates seen in wild-type (WT) neurons. Correction of the MT polymerization rate rescues increased total dendritic arborization and spatial memory. Our studies suggest that phosphatase and tensin homolog (PTEN) inhibits dendritic growth through parallel regulation of protein synthesis and cytoskeletal polymerization.

MiR-29a and the PTEN–GSK3β axis are involved in aluminum-induced damage to primary hippocampal neuronal networks

We previously reported that aluminum (Al) can cause a range of neurotoxic injuries including progressive irreversible synaptic structural damage and synaptic dysfunction, and eventually neuronal deaths. Mechanism of Al-induced electrophysiological and neuronal connectivity changes in neurons may indicate damage to the neuronal network. Here, mouse primary hippocampal neurons were cultured on micro-electrode array (MEA)- and high-content analysis (HCA)-related plates, showing that Al exposure significantly inhibited hippocampal neuronal electrical spike activity and neurite outgrowth characterized by a reduction in neurite branching and a decrease in the average total neurite length in relation to both Al dose and time of incubation. In recent years, miR-29a/ phosphatase and tensin homolog (PTEN) have been found to play pivotal roles in the morphogenesis of neurons, it has been confirmed in vitro and in vivo that the PTEN–Glycogen synthase kinase-3β (GSK-3β) axis regulates neurite outgrowth. The present study demonstrated that increases in Al exposure and dose gradually reduce miR-29a expression. Up-regulation of miR-29a in the hippocampal neurons by lentivirus transfection reversed the decrease in electrical spike activity and the reduction in both neurite branching and length induced by Al. Moreover, miR-29a suppressed the expression of PTEN and increased the level of phosphorylated Protein Kinase B (p-AKT) and p-GSK-3β which were inhibited by the Al treatment. This suggests that miR-29a is critically involved in the functional and structural neuronal damage induced by Al and is a potential target for Al neurotoxicity. Moreover, the reduction of neurite length and branching induced by Al exposure was regulated by miR-29a and its target neuronal PTEN–GSK3β signaling pathway, which also represents a possible mechanism of Al-induced the inhibition of the electrical activity. Collectively, Al-induced damage to the neuronal network occurred through miR-29a-mediated alterations of the PTEN–GSK3β signaling pathway.

Harnessing PTEN's Growth Potential in Neuronal Development and Disease.

PTEN is a powerful regulator of neuronal growth. It globally suppresses axon extension and branching during both nervous system development and regeneration, by antagonizing growth-promoting PI3K/PI(3,4,5)P3 signaling. We recently identified that the transmembrane protein PRG2/LPPR3 functions as a modulator of PTEN function during axon morphogenesis. Our work demonstrates that through inhibition of PTEN activity, PRG2 stabilizes membrane PI(3,4,5)P3. In turn, PRG2 deficiency attenuates the formation of branches in a PTEN-dependent manner, albeit without affecting the overall growth capacity of extending axons. Thus, PRG2 is poised to temporally and locally relieve growth suppression mediated by PTEN in neurons and, in effect, to redirect growth specifically to axonal branches. In this commentary, we discuss potential implications and unresolved questions regarding the regulation of axonal PTEN in neurons. Given their widespread implication during neuronal development and regeneration, identification of mechanisms that confer spatiotemporal control of PTEN may unveil new approaches to reprogram PI3K signaling in neurodevelopmental disorders and regeneration research.

Intermittent fasting uncovers and rescues cognitive phenotypes in PTEN neuronal haploinsufficient mice

Phosphatase and tensin homolog (PTEN) is an important protein with key modulatory functions in cell growth and survival. PTEN is crucial during embryogenesis and plays a key role in the central nervous system (CNS), where it directly modulates neuronal development and synaptic plasticity. Loss of PTEN signaling function is associated with cognitive deficits and synaptic plasticity impairment. Accordingly, Pten mutations have a strong link with autism spectrum disorder. In this study, neuronal Pten haploinsufficient male mice were subjected to a long-term environmental intervention – intermittent fasting (IF) – and then evaluated for alterations in exploratory, anxiety and learning and memory behaviors. Although no significant effects on spatial memory were observed, mutant mice showed impaired contextual fear memory in the passive avoidance test – an outcome that was effectively rescued by IF. In this study, we demonstrated that IF modulation, in addition to its rescue of the memory deficit, was also required to uncover behavioral phenotypes otherwise hidden in this neuronal Pten haploinsufficiency model.

Behavioral abnormalities in mice lacking mesenchyme-specific Pten

Phosphatase and tensin homolog (Pten) is a negative regulator of cell proliferation and growth. Using a Cre-recombinase approach with Lox sequences flanking the fibroblast-specific protein 1 (Fsp1 aka S100A4; a mesenchymal marker), we probed sites of expression using a β-galactosidase Rosa26LoxP reporter allele; the transgene driving deletion of Pten (exons 4–5) was found throughout the brain parenchyma and pituitary, suggesting that deletion of Pten in Fsp1-positive cells may influence behavior. Because CNS-specific deletion of Pten influences social and anxiety-like behaviors and S100A4 is expressed in astrocytes, we predicted that loss of Pten in Fsp1-expressing cells would result in deficits in social interaction and increased anxiety. We further predicted that environmental enrichment would compensate for genetic deficits in these behaviors. We conducted a battery of behavioral assays on Fsp1-Cre;PtenLoxP/LoxP male and female homozygous knockouts (Pten−/−) and compared their behavior to PtenLoxP/LoxP (Pten+/+) conspecifics. Despite extensive physical differences (including reduced hippocampal size) and deficits in sensorimotor function, Pten−/− mice behaved remarkably similar to control mice on nearly all behavioral tasks. These results suggest that the social and anxiety-like phenotypes observed in CNS-specific Pten−/− mice may depend on neuronal Pten, as lack of Pten in Fsp1-expressing cells of the CNS had little effect on these behaviors.

Neuronal RARβ Signaling Modulates PTEN Activity Directly in Neurons and via Exosome Transfer in Astrocytes to Prevent Glial Scar Formation and Induce Spinal Cord Regeneration.

Spinal cord injuries (SCIs) often result in permanent damage in the adult due to the very limited capacity of axonal regeneration. Intrinsic neuronal programs and the formation of a glial scar are the main obstacles. Here, we identify a single target, neuronal retinoic acid receptor β (RARβ), which modulates these two aspects of the postinjury physiological response. Activation of RARβ in the neuron inactivates phosphatase and tensin homolog and induces its transfer into the astrocytes in small vesicles, where it prevents scar formation. This may open new therapeutic avenues for SCIs.

Sensorimotor Cortex Injection of Adeno-Associated Viral Vector Mediates Knockout of PTEN in Neurons of the Brain and Spinal Cord of Mice

Cre/loxP-mediated inactivation of phosphatase and tensin homolog (PTEN) is proposed to be a promising therapeutic agent for promoting CNS and PNS regeneration. And adeno-associated virus (AAV) vector has been developed as an attractive gene delivery system with proven safety. In the present study, we investigated Cre/loxP-mediated knockout of PTEN in the sensorimotor cortex, hippocampus, and spinal cord in PTEN floxed mice by immunohistological analysis of PI3K/AKT/mTOR expression in neurons of the sensorimotor cortex, hippocampus, and spinal cord after sensorimotor cortex injection of AAV-Cre. Two weeks after injection of AAV-Cre, the sensorimotor cortex, hippocampus, and spinal cord were dissected and examined the expression of downstream molecules pAKT and pS6 of PI3K/AKT signaling pathway. The results showed that remote delivery of AAV-Cre through sensorimotor cortex injection mediated PTEN knockout in neurons of the sensorimotor cortex, hippocampus, and spinal cord. We propose sensorimotor cortex injection of AAV may provide a potential strategy of gene therapy for the CNS diseases.

PTEN degradation after ischemic stroke: A double-edged sword

Tumor suppressor phosphatase and tensin homolog (PTEN) is highly expressed in neurons and PTEN inhibition has been reported to be neuroprotective against ischemic stroke in experimental models. On the other hand, PTEN deletion has been shown to lead to cognitive impairment. In the current study, we examined the expression and functions of PTEN in an ischemic stroke rodent model. We found rapid S-nitrosylation and degradation of PTEN after cerebral ischemia/reperfusion injury. PTEN degradation leads to activation of Akt. PTEN partial deletion or PTEN inhibition increased the expression of GABAA receptor (GABAAR) γ2 subunit and enhanced GABAA receptor current. After cerebral ischemia, increased expression of GABAAR γ2 subunit was observed in the ischemia region and the penumbra area. We also observed PTEN loss in astrocytes after cerebral ischemia. Astrocytic PTEN partial knockout increased astrocyte activation and exacerbated ischemic damage. We speculated that ischemic stroke induced neuronal PTEN degradation, hence enhanced GABAA receptor-medicated neuronal activity inhibition which could attenuate excitotoxicity and provide neuroprotection during the acute phase after stroke, while inhibiting long-term functional recovery and contributing to vascular cognitive impairment after stroke. On the other hand, ischemic stroke induced astrocytic PTEN loss and enhanced ischemic damage and astrogliosis. Taken together, our study indicates that ischemic stroke induces rapid PTEN degradation in both neurons and astrocytes which play both protective and detrimental action in a spatiotemporal- and cell-type-dependent manner. Our study provides critical insight for targeting PTEN signaling pathway for stroke treatment.

Nuclear trafficking of Pten after brain injury leads to neuron survival not death

There is controversy whether accumulation of the tumor suppressor PTEN protein in the cell nucleus under stress conditions such as trauma and stroke causes cell death. A number of in vitro studies have reported enhanced apoptosis in neurons possessing nuclear PTEN, with the interpretation that its nuclear phosphatase activity leads to reduction of the survival protein phospho-Akt. However, there have been no in vivo studies to show that nuclear PTEN in neurons under stress is detrimental. Using a mouse model of injury, we demonstrate here that brain trauma altered the nucleo-cytoplasmic distribution of Pten, resulting in increased nuclear Pten but only in surviving neurons near the lesion. This event was driven by Ndfip1, an adaptor and activator of protein ubiquitination by Nedd4 E3 ligases. Neurons next to the lesion with nuclear PTEN were invariably negative for TUNEL, a marker for cell death. These neurons also showed increased Ndfip1 which we previously showed to be associated with neuron survival. Biochemical assays revealed that overall levels of Pten in the affected cortex were unchanged after trauma, suggesting that Pten abundance globally had not increased but rather Pten subcellular location in affected neurons had changed. Following experimental injury, the number of neurons with nuclear Pten was reduced in heterozygous mice (Ndfip1+/−) although lesion volumes were increased. We conclude that nuclear trafficking of Pten following injury leads to neuron survival not death.

Regulation of axonal growth and neuromuscular junction formation by neuronal phosphatase and tensin homologue signaling

During the development of the vertebrate neuromuscular junction (NMJ), motor axon tips stop growing after contacting muscle and transform into presynaptic terminals that secrete the neurotransmitter acetylcholine and activate postsynaptic ACh receptors (AChRs) to trigger muscle contraction. The neuron-intrinsic signaling that retards axonal growth to facilitate stable nerve-muscle interaction and synaptogenesis is poorly understood. In this paper, we report a novel function of presynaptic signaling by phosphatase and tensin homologue (PTEN) in mediating a growth-to-synaptogenesis transition in neurons. In Xenopus nerve-muscle cocultures, axonal growth speed was halved after contact with muscle, when compared with before contact, but when cultures were exposed to the PTEN blocker bisperoxo (1,10-phenanthroline) oxovanadate, axons touching muscle grew ~50% faster than their counterparts in control cultures. Suppression of neuronal PTEN expression using morpholinos or the forced expression of catalytically inactive PTEN in neurons also resulted in faster than normal axonal advance after contact with muscle cells. Significantly, interference with PTEN by each of these methods also led to reduced AChR clustering at innervation sites in muscle, indicating that disruption of neuronal PTEN signaling inhibited NMJ assembly. We thus propose that PTEN-dependent slowing of axonal growth enables the establishment of stable nerve-muscle contacts that develop into NMJs.

A role for Pten in paediatric intestinal dysmotility disorders

The enteric nervous system (ENS) is a network of neurons and glia that lies within the gut wall. It is responsible for the normal regulation of gut motility and secretory activities. Hirschsprung's disease (HD) is a congenital defect of the ENS, characterised by an absence of ganglia in the distal colon. Intestinal neuronal dysplasia (IND) is a condition that clinically resembles HD, characterised by hyperganglionosis, giant and ectopic ganglia, resulting in intestinal dysmotility. Intestinal ganglioneuromatosis is characterised by hyperplasia and hypertrophy of enteric neuronal cells and causes chronic intestinal pseudo-obstruction (CIPO). Phosphatase and tensin homolog deleted on chromosome 10 (Pten) is a phosphatase that is critical for controlling cell growth, proliferation and cell death. A recent study of Pten knockout mice showed evidence of ganglioneuromatosis in the ENS suggesting a role for this protein in ENS development. Ganglioneuromatosis patients have also been shown to have a decreased level of Pten expression in the colon. The aim of our study was to investigate Pten expression in the ENS of HD and IND patients compared to normal controls. Resected tissue from 10 HD and 10 IND type B patients was fixed and embedded in paraffin wax. Normal control colon tissue was obtained from ten patients who underwent a colostomy closure for imperforate anus. Sections were cut and immunohistochemistry was carried out using a Pten antibody. Results were analysed by light microscopy. Staining showed that Pten was strongly expressed in ganglia of both the submucosal and myenteric plexus of normal and HD specimens from the ganglionic colon. Pten expression was significantly reduced in the giant ganglia in IND patients in both the myenteric and submucosal plexuses compared to the normal controls. Specimens from the aganglionic region of HD did not show Pten expression. To the best of our knowledge, this is the first study demonstrating a marked reduction of Pten expression in the myenteric and submucous plexuses of IND patients. Neuronal Pten deficiency in IND may disrupt the chemical pathway associated with the proliferation and development of neuronal cells forming mature ganglia and thus cause motility dysfunction.

Decrease in PTEN and increase in Akt expression and neuron size in aged rat spinal cord

PTEN is a tumor suppressor gene known to play an important role in the regulation of cell size. In this study we compared PTEN expression in the spinal cord of young (5 months old) vs. aged (32 months old) female rats and correlated them with alterations in neuron size and morphology in the same animals. Total and phosphorylated PTEN (pPTEN) as well as its downstream target phosphorylated Akt (pAkt) were assessed by Western blotting. Spinal cord neurons were morphometrically characterized. Total PTEN, pPTEN and total Akt expression were significantly higher in young rats than in aged animals. Expression of pAkt was stronger in aged animals. A significant increase in neuronal size was observed in large motoneurons of aged as compared with young rats. Our data show that in the spinal cord of rats, neuronal PTEN expression diminishes with advanced age while neuronal size increases. These results suggest that in the spinal cord, an age-related reduction in PTEN and increase of pAkt expression may be involved in the progressive enlargement of neurons.

Regulation of PTEN in neurons by myosin-based transport mechanisms

Regulation of PTEN in neurons by myosin-based transport mechanisms