Oestrogen treatment restores dentate gyrus development in premature newborns by IGF1 regulation.

Prematurely-born infants cared for in the neonatal units suffer from memory and learning deficits. Prematurity diminishes neurogenesis and synaptogenesis in the hippocampal dentate gyrus (DG). This dysmaturation of neurons is attributed to elevated PSD95, NMDR2A, and IGF1 levels. Since oestrogen treatment plays key roles in the development and plasticity of DG, we hypothesized that 17ß-estradiol (E2) treatment would ameliorate neurogenesis and synaptogenesis in the DG, reversing cognitive deficits in premature newborns. Additionally, E2-induced recovery would be mediated by IGF1 signalling. These hypotheses were tested in a rabbit model of prematurity and nonmaternal care, in which premature kits were gavage-fed and reared by laboratory personnel. We compared E2- and vehicle-treated preterm kits for morphological, molecular, and behavioural parameters. We also treated kits with oestrogen degrader, RAD1901, and assessed IGF1 signalling. We found that E2 treatment increased the number of Tbr2+ and DCX+ neuronal progenitors and increased the density of glutamatergic synapses in the DG. E2 treatment restored PSD95 and NMDAR2A levels and cognitive function in preterm kits. Transcriptomic analyses showed that E2 treatment contributed to recovery by influencing interactions between IGF1R and neurodegenerative, as well as glutamatergic genes. ERα expression was reduced on completion of E2 treatment at D7, followed by D30 elevation. E2-induced fluctuation in ERα levels was associated with a reciprocal elevation in IGF1/2 expression at D7 and reduction at D30. ERα degradation by RAD1901 treatment enhanced IGF1 levels, suggesting ERα inhibits IGF1 expression. E2 treatment alleviates the prematurity-induced maldevelopment of DG and cognitive dysfunctions by regulating ERα and IGF1 levels.

Elevated insulin growth factor-1 in dentate gyrus induces cognitive deficits in pre-term newborns.

Prematurely born infants are deprived of maternal hormones and cared for in the stressful environment of Neonatal Intensive Care Units (NICUs). They suffer from long-lasting deficits in learning and memory. Here, we show that prematurity and associated neonatal stress disrupt dentate gyrus (DG) development and induce long-term cognitive deficits and that these effects are mediated by insulin growth factor-1 (IGF1). Nonmaternal care of premature rabbits increased the number of granule cells and interneurons and reduced neurogenesis, suggesting accelerated premature maturation of DG. However, the density of glutamatergic synapses, mature dendritic spines, and synaptic transmission were reduced in preterm kits compared with full-term controls, indicating that premature synaptic maturation was abnormal. These findings were consistent with cognitive deficits observed in premature rabbits and appeared to be driven by transcriptomic changes in the granule cells. Preterm kits displayed reduced weight, elevated serum cortisol and growth hormone, and higher IGF1 expression in the liver and DG relative to full-term controls. Importantly, blocking IGF-1 receptor in premature kits restored cognitive deficits, increased the density of glutamatergic puncta, and rescued NR2B and PSD95 levels in the DG. Hence, IGF1 inhibition alleviates prematurity-induced cognitive dysfunction and synaptic changes in the DG through modulation of NR2B and PSD95. The study identifies a novel strategy to potentially rescue DG maldevelopment and cognitive dysfunction in premature infants under stress in NICUs.

Shh activation restores interneurons and cognitive function in newborns with intraventricular haemorrhage.

Premature infants with germinal matrix haemorrhage-intraventricular haemorrhage (GMH-IVH) suffer from neurobehavioural deficits as they enter childhood and adolescence. Yet the underlying mechanisms remain unclear. Impaired development and function of interneurons contribute to neuropsychiatric disorders. Therefore, we hypothesized that the occurrence of IVH would reduce interneuron neurogenesis in the medial ganglionic eminence and diminish the population of parvalbumin+ and somatostatin+ cortical interneurons. Because Sonic Hedgehog promotes the production of cortical interneurons, we also postulated that the activation of Sonic Hedgehog signalling might restore neurogenesis, cortical interneuron population, and neurobehavioural function in premature newborns with IVH. These hypotheses were tested in a preterm rabbit model of IVH and autopsy samples from human preterm infants. We compared premature newborns with and without IVH for intraneuronal progenitors, cortical interneurons, transcription factors regulating neurogenesis, single-cell transcriptome of medial ganglionic eminence and neurobehavioural functions. We treated premature rabbit kits with adenovirus expressing Sonic Hedgehog (Ad-Shh) or green fluorescence protein gene to determine the effect of Sonic Hedgehog activation on the interneuron production, cortical interneuron population and neurobehaviour. We discovered that IVH reduced the number of Nkx2.1+ and Dlx2+ progenitors in the medial ganglionic eminence of both humans and rabbits by attenuating their proliferation and inducing apoptosis. Moreover, IVH decreased the population of parvalbumin+ and somatostatin+ neurons in the frontal cortex of both preterm infants and kits relative to controls. Sonic Hedgehog expression and the downstream transcription factors, including Nkx2.1, Mash1, Lhx6 and Sox6, were also reduced in kits with IVH. Consistent with these findings, single-cell transcriptomic analyses of medial ganglionic eminence identified a distinct subpopulation of cells exhibiting perturbation in genes regulating neurogenesis, ciliogenesis, mitochondrial function and MAPK signalling in rabbits with IVH. More importantly, restoration of Sonic Hedgehog level by Ad-Shh treatment ameliorated neurogenesis, cortical interneuron population and neurobehavioural function in kits with IVH. Additionally, Sonic Hedgehog activation alleviated IVH-induced inflammation and several transcriptomic changes in the medial ganglionic eminence. Taken together, IVH reduced intraneuronal production and cortical interneuron population by downregulating Sonic Hedgehog signalling in both preterm rabbits and humans. Notably, activation of Sonic Hedgehog signalling restored interneuron neurogenesis, cortical interneurons and cognitive function in rabbit kits with IVH. These findings highlight disruption in cortical interneurons in IVH and identify a novel therapeutic strategy to restore cortical interneurons and cognitive function in infants with IVH. These studies can accelerate the development of new therapies to enhance the neurodevelopmental outcome of survivors with IVH.

Cerebral gray matter injuries in infants with intraventricular hemorrhage.

While intraventricular hemorrhage (IVH) predominantly damages the periventricular white matter, it induces substantial injury to the cerebral gray matter. IVH destroys the germinal matrix, suppresses neurogenesis, and disrupts corticogenesis, thereby reducing the number of neurons in the upper cortical layer and volume of the cerebral gray matter. The pathogenesis of gray matter injury is attributed to IVH-induced oxidative stress, inflammation, and mass effect damaging the germinal matrix as well as to post-hemorrhagic ventricular dilation (PHVD). The IVH-induced cerebral gray matter injury and PHVD contribute to cognitive deficits and neurobehavioral disorders. Neuroimaging has enhanced our understanding of cerebral gray matter injury and is a valuable predictor of neurodevelopmental outcomes. Evidence from therapies tested in preclinical models and clinical trials suggests that strategies to promote neurogenesis, reduce cerebral inflammation and oxidative stress, and remove blood clots from the ventricles might enhance the outcome of these infants. This review offers an integrated view of new insights into the mechanisms underlying gray matter injury in premature infants with IVH and highlights the imminent therapies to restore neurodevelopmental dysfunction in IVH survivors.

PPAR-γ activation enhances myelination and neurological recovery in premature rabbits with intraventricular hemorrhage.

Intraventricular hemorrhage (IVH) results in periventricular inflammation, hypomyelination of the white matter, and hydrocephalus in premature infants. No effective therapy exists to prevent these disorders. Peroxisome proliferator activated receptor-γ (PPAR-γ) agonists reduce inflammation, alleviate free radical generation, and enhance microglial phagocytosis, promoting clearance of debris and red blood cells. We hypothesized that activation of PPAR-γ would enhance myelination, reduce hydrocephalus, and promote neurological recovery in newborns with IVH. These hypotheses were tested in a preterm rabbit model of IVH; autopsy brain samples from premature infants with and without IVH were analyzed. We found that IVH augmented PPAR-γ expression in microglia of both preterm human infants and rabbit kits. The treatment with PPAR-γ agonist or PPAR-γ overexpression by adenovirus delivery further elevated PPAR-γ levels in microglia, reduced proinflammatory cytokines, increased microglial phagocytosis, and improved oligodendrocyte progenitor cell (OPC) maturation in kits with IVH. Transcriptomic analyses of OPCs identified previously unrecognized PPAR-γ-induced genes for purinergic signaling, cyclic adenosine monophosphate generation, and antioxidant production, which would reprogram these progenitors toward promoting myelination. RNA-sequencing analyses of microglia revealed PPAR-γ-triggered down-regulation of several proinflammatory genes and transcripts having roles in Parkinson's disease and amyotrophic lateral sclerosis, contributing to neurological recovery in kits with IVH. Accordingly, PPAR-γ activation enhanced myelination and neurological function in kits with IVH. This also enhanced microglial phagocytosis of red blood cells but did not reduce hydrocephalus. Treatment with PPAR-γ agonist might enhance myelination and neurological recovery in premature infants with IVH.

Reduced Hippocampal Dendrite Branching, Spine Density and Neurocognitive Function in Premature Rabbits, and Reversal with Estrogen or TrkB Agonist Treatment.

Preterm-born children suffer from neurological and behavioral disorders. Herein, we hypothesized that premature birth and non-maternal care of preterm newborns might disrupt neurobehavioral function, hippocampal dendritic arborization, and dendritic spine density. Additionally, we assessed whether 17ß-estradiol (E2) replacement or the TrkB receptor agonist, 7,8-dihydroxyflavone (DHF), would reverse compromised dendritic development and cognitive function in preterm newborns. These hypotheses were tested by comparing preterm (E28.5) rabbit kits cared and gavage-fed by laboratory personnel and term-kits reared and breast-fed by their mother doe at an equivalent postconceptional age. Neurobehavioral tests showed that both premature-birth and formula-feeding with non-maternal care led to increased anxiety behavior, poor social interaction, and lack of novelty preference compared with term-kits. Dendritic branching and number of total or mushroom dendritic spines were reduced in the CA1 field of preterm-kits compared with term controls. While CDC42 and Rac1/2/3 expression levels were lower, RhoA-activity was higher in preterm-kits compared with term controls. Both E2 and DHF treatment reversed prematurity-induced reduction in spine density, reduced total RhoA-GTPase levels, and enhanced cognitive function. Hence, prematurity and non-maternal care result in cognitive deficits, and reduced dendritic arbors and spines in CA1. E2 replacement or DHF treatment might reverse changes in dendritic spines and improve neurodevelopment in premature infants.

Novel Mouse Tauopathy Model for Repetitive Mild Traumatic Brain Injury: Evaluation of Long-Term Effects on Cognition and Biomarker Levels After Therapeutic Inhibition of Tau Phosphorylation.

Traumatic brain injury (TBI) is a risk factor for a group of neurodegenerative diseases termed tauopathies, which includes Alzheimer's disease and chronic traumatic encephalopathy (CTE). Although TBI is stratified by impact severity as either mild (m), moderate or severe, mTBI is the most common and the most difficult to diagnose. Tauopathies are pathologically related by the accumulation of hyperphosphorylated tau (P-tau) and increased total tau (T-tau). Here we describe: (i) a novel human tau-expressing transgenic mouse model, TghTau/PS1, to study repetitive mild closed head injury (rmCHI), (ii) quantitative comparison of T-tau and P-tau from brain and plasma in TghTau/PS1 mice over a 12 month period following rmCHI (and sham), (iii) the usefulness of P-tau as an early- and late-stage blood-based biochemical biomarker for rmCHI, (iii) the influence of kinase-targeted therapeutic intervention on rmCHI-associated cognitive deficits using a combination of lithium chloride (LiCl) and R-roscovitine (ros), and (iv) correlation of behavioral and cognitive changes with concentrations of the brain and blood-based T-tau and P-tau. Compared to sham-treated mice, behavior changes and cognitive deficits of rmCHI-treated TghTau/PS1 mice correlated with increases in both cortex and plasma T-tau and P-tau levels over 12 months. In addition, T-tau, but more predominantly P-tau, levels were significantly reduced in the cortex and plasma by LiCl + ros approaching the biomarker levels in sham and drug-treated sham mice (the drugs had only modest effects on the T-tau and P-tau levels in sham mice) throughout the 12 month study period. Furthermore, although we also observed a reversal of the abnormal behavior and cognitive deficits in the drug-treated rmCHI mice (compared to the untreated rmCHI mice) throughout the time course, these drug-treated effects were most pronounced up until 10 and 12 months where the abnormal behavior and cognition deficits began to gradually increase. These studies describe: (a) a translational relevant animal model for TBI-linked tauopathies, and (b) utilization of T-tau and P-tau as rmCHI biomarkers in plasma to monitor novel therapeutic strategies and treatment regimens for these neurodegenerative diseases.

PrPC expression and calpain activity independently mediate the effects of closed head injury in mice.

The normal cellular prion protein (PrPC) is a sialoglycoprotein with a glycophosphatidylinositol anchor and expressed in highest levels within the CNS particularly at neuronal synapses. This membrane-bound protein is involved with many cell functions including cell signaling and neuroprotection. Calpains are calcium-activated cysteine proteases that typically undergo controlled activation. PrPC is a calpain substrate and is neurotoxic if it undergoes aberrant processing with cytosol accumulation. Following traumatic brain injury (TBI), there is an abnormal influx of Ca+2 and overactivation of calpains resulting in neuronal dysfunction and cell death. We investigated whether PrPC expression and calpain activity have an effect on, or are affected by, TBI. PrPC expression in the hippocampus, cortex and cerebellum of WT and Tga20 (PrPC overexpression) mice were unchanged after closed head injury (CHI). Further, PrPC in WT and Tga20 mice was resistant to TBI-induced calpain proteolysis. CHI-induced calpain activation resulted in breakdown products (BDPs) of αII-spectrin (SBDPs) and GFAP (GBDP-44K) in all brain regions and mouse lines. CHI caused significant increases in SBDP145, GFAP and GBDP-44K when compared to sham. With few exceptions, the calpain inhibitor, SNJ-1945, reduced SBDP145 and GBDP-44K levels. Behavioral studies suggested that PrPC and calpain independently affect learning and memory. Overall, we conclude that: (i) there is SNJ-1945-sensitive calpain activation in both neuron and glial cells following CHI, (ii) closed head trauma is not affected by, nor does it have an influence on, PrPC expression, and (iii) PrPC expression plays a minor role, if any, in CHI-induced calpain activation in vivo.

Enhancing NAD+ Salvage Pathway Reverts the Toxicity of Primary Astrocytes Expressing Amyotrophic Lateral Sclerosis-linked Mutant Superoxide Dismutase 1 (SOD1).

Nicotinamide adenine dinucleotide (NAD(+)) participates in redox reactions and NAD(+)-dependent signaling pathways. Although the redox reactions are critical for efficient mitochondrial metabolism, they are not accompanied by any net consumption of the nucleotide. On the contrary, NAD(+)-dependent signaling processes lead to its degradation. Three distinct families of enzymes consume NAD(+) as substrate: poly(ADP-ribose) polymerases, ADP-ribosyl cyclases (CD38 and CD157), and sirtuins (SIRT1-7). Because all of the above enzymes generate nicotinamide as a byproduct, mammalian cells have evolved an NAD(+) salvage pathway capable of resynthesizing NAD(+) from nicotinamide. Overexpression of the rate-limiting enzyme in this pathway, nicotinamide phosphoribosyltransferase, increases total and mitochondrial NAD(+) levels in astrocytes. Moreover, targeting nicotinamide phosphoribosyltransferase to the mitochondria also enhances NAD(+) salvage pathway in astrocytes. Supplementation with the NAD(+) precursors nicotinamide mononucleotide and nicotinamide riboside also increases NAD(+) levels in astrocytes. Amyotrophic lateral sclerosis (ALS) is caused by the progressive degeneration of motor neurons in the spinal cord, brain stem, and motor cortex. Superoxide dismutase 1 (SOD1) mutations account for up to 20% of familial ALS and 1-2% of apparently sporadic ALS cases. Primary astrocytes isolated from mutant human superoxide dismutase 1-overexpressing mice as well as human post-mortem ALS spinal cord-derived astrocytes induce motor neuron death in co-culture. Increasing total and mitochondrial NAD(+) content in ALS astrocytes increases oxidative stress resistance and reverts their toxicity toward co-cultured motor neurons. Taken together, our results suggest that enhancing the NAD(+) salvage pathway in astrocytes could be a potential therapeutic target to prevent astrocyte-mediated motor neuron death in ALS.

Changes in Protein Expression and Lysine Acetylation Induced by Decreased Glutathione Levels in Astrocytes.

Astrocytes and neurons form a highly specialized functional unit, and the loss or gain of astrocytic functions can influence the initiation and progression of different neurodegenerative diseases. Neurons depend on the antioxidant protection provided by neighboring astrocytes. Glutathione (γ-l-glutamyl-l-cysteinyl-glycine) is a major component of the antioxidant system that defends cells against the toxic effects of reactive oxygen/nitrogen species. A decline in glutathione levels has been observed in aging and neurodegenerative diseases, and it aggravates the pathology in an amyotrophic lateral sclerosis-mouse model. Using a SILAC-based quantitative proteomic approach, we analyzed changes in global protein expression and lysine acetylation in primary astrocyte cultures obtained from wild-type mice or those deficient in the glutamate-cysteine ligase modifier subunit (GCLM). GCLM knockout astrocytes display an ∼80% reduction in total glutathione levels. We identified potential molecular targets and novel sites of acetylation that are affected by the chronic decrease in glutathione levels and observed a response mediated by Nrf2 activation. In addition, sequence analysis of peptides displaying increased acetylation in GCLM knockout astrocytes revealed an enrichment of cysteine residues in the vicinity of the acetylation site, which suggests potential crosstalk between lysine-acetylation and cysteine modification. Regulation of several metabolic and antioxidant pathways was observed at the level of protein expression and lysine acetylation, revealing a coordinated response involving transcriptional and posttranslational regulation.