Acan downregulation in parvalbumin GABAergic cells reduces spontaneous recovery of fear memories.

While persistence of fear memories is essential for survival, a failure to inhibit fear in response to harmless stimuli is a feature of anxiety disorders. Extinction training only temporarily suppresses fear memory recovery in adults, but it is highly effective in juvenile rodents. Maturation of GABAergic circuits, in particular of parvalbumin-positive (PV+) cells, restricts plasticity in the adult brain, thus reducing PV+ cell maturation could promote the suppression of fear memories following extinction training in adults. Epigenetic modifications such as histone acetylation control gene accessibility for transcription and help couple synaptic activity to changes in gene expression. Histone deacetylase 2 (Hdac2), in particular, restrains both structural and functional synaptic plasticity. However, whether and how Hdac2 controls the maturation of postnatal PV+ cells is not well understood. Here, we show that PV+- cell specific Hdac2 deletion limits spontaneous fear memory recovery in adult mice, while enhancing PV+ cell bouton remodeling and reducing perineuronal net aggregation around PV+ cells in prefrontal cortex and basolateral amygdala. Prefrontal cortex PV+ cells lacking Hdac2, show reduced expression of Acan, a critical perineuronal net component, which is rescued by Hdac2 re-expression. Pharmacological inhibition of Hdac2 before extinction training is sufficient to reduce both spontaneous fear memory recovery and Acan expression in wild-type adult mice, while these effects are occluded in PV+-cell specific Hdac2 conditional knockout mice. Finally, a brief knock-down of Acan expression mediated by intravenous siRNA delivery before extinction training but after fear memory acquisition is sufficient to reduce spontaneous fear recovery in wild-type mice. Altogether, these data suggest that controlled manipulation of PV+ cells by targeting Hdac2 activity, or the expression of its downstream effector Acan, promotes the long-term efficacy of extinction training in adults.

Riluzole restores memory and brain energy metabolism in AßPP-PS1 mouse model of Alzheimer's disease.

Alzheimer's disease (AD) is a neurodegenerative disorder leading to memory loss and impaired cognition. Despite several decades of research, AD therapeutic is not available. In this study, we have investigated the impact of a chronic intervention of riluzole on memory and neurometabolism in the AßPP-PS1 mouse model of AD. The 10-month-old AßPP-PS1 mice were administered 30 doses of riluzole (6 mg/kg, intragastrically) on an alternate day for two months. The memory was assessed using Morris Water Maze, while neurometabolism was evaluated by 1H-[13C]-NMR spectroscopy together with an intravenous infusion of [1,6-13C2]glucose. The normal saline-treated AßPP-PS1 mice exhibited a decrease in learning and memory that were restored to the control level following riluzole treatment. Most interestingly, the reduced 13C labeling of GluC4 and AspC3 from [1,6-13C]glucose in the AßPP-PS1 mice was restored to the control level following riluzole intervention. As a consequence, chronic riluzole treatment improved metabolic activity of glutamatergic neurons in AßPP-PS1 mice. Together these data suggest that riluzole may be useful for improving cognition in AD.

Impaired GABAergic and glutamatergic neurometabolic activity in aged mice brain as measured by 1 H-[13 C]-NMR spectroscopy.

Healthy aging is associated with a decline in cognitive function, and is a major risk factor for many neurodegenerative diseases. Although, there are several evidence that brain mitochondrial function is altered with aging its significance at the cellular level is elusive. In this study, we have investigated mitochondrial TCA cycle and neurotransmitter cycle fluxes associated with glutamatergic, GABAergic neurons and astroglia in the cerebral cortex and hippocampus of young (6 months) and aged (24 months) C57BL6 mice by using 1 H-[13 C]-NMR spectroscopy together with timed infusion of 13 C-labeled glucose and acetate. The ratio VCyc /VTCA was determined from a steady-state [2-13 C]acetate experiment. Metabolic fluxes were obtained by fitting a three-compartment metabolic model to 13 C turnover of amino acids from glucose. Levels of glutamate, aspartate and taurine were reduced in the cerebral cortex, while glutamine and choline were elevated in the hippocampus of aged mice. Interestingly, the rate of acetate oxidation increased in the cerebral cortex, while the flux of mitochondrial TCA cycle of glutamatergic neurons decreased in the cerebral cortex (P < .0001) and hippocampus (P = .025) of aged mice. The glutamate-glutamine neurotransmitter cycle flux was reduced in the cerebral cortex (P < .0001). The GABAergic TCA cycle flux was reduced in the cerebral cortex (P = .0008), while GABA-glutamine neurotransmitter cycling flux was also reduced in the cerebral cortex (P = .011) and hippocampus (P = .042) of aged brain. In conclusion, the reduction in excitatory and inhibitory neurotransmitter activity of glutamatergic and GABAergic neurons in the cerebral cortex and hippocampus correlates qualitatively with declined cognitive function in aged mice.

Chronic postnatal chemogenetic activation of forebrain excitatory neurons evokes persistent changes in mood behavior.

Early adversity is a risk factor for the development of adult psychopathology. Common across multiple rodent models of early adversity is increased signaling via forebrain Gq-coupled neurotransmitter receptors. We addressed whether enhanced Gq-mediated signaling in forebrain excitatory neurons during postnatal life can evoke persistent mood-related behavioral changes. Excitatory hM3Dq DREADD-mediated chemogenetic activation of forebrain excitatory neurons during postnatal life (P2-14), but not in juvenile or adult windows, increased anxiety-, despair-, and schizophrenia-like behavior in adulthood. This was accompanied by an enhanced metabolic rate of cortical and hippocampal glutamatergic and GABAergic neurons. Furthermore, we observed reduced activity and plasticity-associated marker expression, and perturbed excitatory/inhibitory currents in the hippocampus. These results indicate that Gq-signaling-mediated activation of forebrain excitatory neurons during the critical postnatal window is sufficient to program altered mood-related behavior, as well as functional changes in forebrain glutamate and GABA systems, recapitulating aspects of the consequences of early adversity.

Stress and adversity in early childhood can have long-lasting effects, predisposing people to mental illness and mood disorders in adult life. The weeks immediately before and after birth are critical for establishing key networks of neurons in the brain. Therefore, any disruption to these neural circuits during this time can be detrimental to emotional development. However, it is still unclear which cellular mechanisms cause these lasting changes in behavior. Studies in animals suggest that these long-term effects could result from abnormalities in a few signaling pathways in the brain. For example, it has been proposed that overstimulating the cells that activate circuits in the forebrain ­ also known as excitatory neurons ­ may contribute to the behavioral changes that persist into adulthood. To test this theory, Pati et al. used genetic engineering to modulate a signaling pathway in male mice, which is known to stimulate excitatory neurons in the forebrain. The experiments showed that prolonged activation of excitatory neurons in the first two weeks after birth resulted in anxious and despair-like behaviors as the animals aged. The mice also displayed discrepancies in how they responded to certain external sensory information, which is a hallmark of schizophrenia-like behavior. However, engineering the same changes in adolescent and adult mice had no effect on their mood-related behaviors. This animal study reinforces just how critical the first few weeks of life are for optimal brain development. It provides an insight into a possible mechanism of how disruption during this time could alter emotional behavior. The findings are also relevant to psychiatrists interested in the underlying causes of mental illness after early childhood adversity.

Delivery of BACE1 siRNA mediated by TARBP-BTP fusion protein reduces ß-amyloid deposits in a transgenic mouse model of Alzheimer's disease.

Systemic delivery of nucleic acids to the central nervous system (CNS) is a major challenge for the development of RNA interference-based therapeutics due to lack of stability, target specificity, non-permeability to the blood-brain barrier (BBB), and lack of suitable carriers. Using a designed bi-functional fusion protein TARBP-BTP in a complex with siRNA, we earlier demonstrated knockdown of target genes in the brain of both AßPP-PS1 (Alzheimer's disease, AD) and wild-type C57BL/6 mice. In this report, we further substantiate the approach through an extended use in AßPP-PS1 mice, which upon treatment with seven doses of ß-secretase AßPP cleaving Enzyme 1 (BACE1) TARBP-BTP:siRNA, led to target-specific effect in the mouse brain. Concomitant gene silencing of BACE1, and consequent reduction in plaque load in the cerebral cortex and hippocampus (greater than 60%) in mice treated with TARBP-BTP:siRNA complex, led to improvement in spatial learning and memory. The study validates the efficiency of TARBP-BTP fusion protein as an efficient mediator of RNAi, giving considerable scope for future intervention in neurodegenerative disorders through the use of short nucleic acids as gene specific inhibitors.

Increased astroglial activity and reduced neuronal function across brain in AßPP-PS1 mouse model of Alzheimer's disease.

Alzheimer's disease (AD) is the most common neurodegenerative disease associated with progressive loss of cognitive function, personality, and behavior. The present study evaluates neuronal and astroglial metabolic activity, and neurotransmitter cycle fluxes in AßPP-PS1 mouse model of AD by using 1H-[13C]-nuclear magnetic resonance (NMR) spectroscopy together with an infusion of either [1,6-13C2]glucose or [2-13C]acetate. The levels of N-acetyl-aspartate (NAA) and glutamate were found to be decreased in the cerebral cortex and hippocampus in AßPP-PS1 mice, when compared with wild type controls. The cerebral metabolic rate of acetate oxidation was increased in the hippocampus and cerebral cortex of AßPP-PS1 mice suggesting enhanced astroglial activity in AD. AßPP-PS1 mice exhibit severe reduction in glutamatergic and gamma-amino butyric acid (GABA)ergic neuronal metabolic activity and neurotransmitter cycling fluxes in the hippocampus, cerebral cortex, and striatum as compared with controls. These data suggest that metabolic activity of excitatory and inhibitory neurons is compromised across brain in AßPP-PS1 mouse model of AD.

Energetics of Excitatory and Inhibitory Neurotransmission in Aluminum Chloride Model of Alzheimer's Disease: Reversal of Behavioral and Metabolic Deficits by Rasa Sindoor.

Alzheimer's disease (AD) is an age-related neurodegenerative disorder, characterized by progressive loss of cognitive functions and memory. Excessive intake of aluminum chloride in drinking water is associated with amyloid plaques and neurofibrillary tangles in the brain, which are the hallmark of AD. We have evaluated brain energy metabolism in aluminum chloride (AlCl3) mouse model of AD. In addition, effectiveness of Rasa Sindoor (RS), a formulation used in Indian Ayurvedic medicine, for alleviation of symptoms of AD was evaluated. Mice were administered AlCl3 (40 mg/kg) intraperitoneally once a day for 60 days. The memory of mice was measured using Morris Water Maze test. The 13C labeling of brain amino acids was measured ex vivo in tissue extracts using 1H-[13C]-NMR spectroscopy with timed infusion of [1,6-13C2]glucose. The 13C turnover of brain amino acids was analyzed using a three-compartment metabolic model to derive the neurotransmitter cycling and TCA cycle rates associated with glutamatergic and GABAergic pathways. Exposure of AlCl3 led to reduction in memory of mice. The glutamatergic and GABAergic neurotransmitter cycling and glucose oxidation were found to be reduced in the cerebral cortex, hippocampus, and striatum following chronic AlCl3 treatment. The perturbation in metabolic rates was highest in the cerebral cortex. However, reduction in metabolic fluxes was higher in hippocampus and striatum following one month post AlCl3 treatment. Most interestingly, oral administration of RS (2 g/kg) restored memory as well as the energetics of neurotransmission in mice exposed to AlCl3. These data suggest therapeutic potential of RS to manage cognitive functions and memory in preclinical AD.

Amalaki Rasayana improved memory and neuronal metabolic activity in AbPP-PS1 mouse model of Alzheimer's disease.

Alzheimer's disease (AD) is the most common neurodegenerative disorder characterized by progressive loss of memory and cognitive function. The cerebral metabolic rate of glucose oxidation has been shown to be reduced in AD. The present study evaluated efficacy of dietary Amalaki Rasayana (AR), an Ayurvedic formulation used in Indian traditional system, in AbPP-PS1 mouse model of AD in ameliorating memory and neurometabolism, and compared with donepezil, a standard FDA approved drug for AD. The memory of mice was measured using Morris Water Maze analysis. The cerebral metabolism was followed by 13C labelling of brain amino acids in tissue extracts ex vivo using 1H-[13C]-NMR spectroscopy together with a short time infusion of [1,6-13C2]glucose to mice. The intervention with Amalaki Rasayana showed improved learning and memory in AbPP-PS1 mice. The 13C labelings of GluC4, GABAC2 and GlnC4 were reduced in AbPP-PS1 mice when compared with wild-type controls. Intervention of AR increased the 13C labelling of amino acids suggesting a significant enhancement in glutamatergic and GABAergic metabolic activity in AbPP-PS1 mice similar to that observed with donepezil treatment. These data suggest that AR has potential to improve memory and cognitive function in AD.

A designed recombinant fusion protein for targeted delivery of siRNA to the mouse brain.

RNA interference represents a novel therapeutic approach to modulate several neurodegenerative disease-related genes. However, exogenous delivery of siRNA restricts their transport into different tissues and specifically into the brain mainly due to its large size and the presence of the blood-brain barrier (BBB). To overcome these challenges, we developed here a strategy wherein a peptide known to target specific gangliosides was fused to a double-stranded RNA binding protein to deliver siRNA to the brain parenchyma. The designed fusion protein designated as TARBP-BTP consists of a double-stranded RNA-binding domain (dsRBD) of human Trans Activation response element (TAR) RNA Binding Protein (TARBP2) fused to a brain targeting peptide that binds to monosialoganglioside GM1. Conformation-specific binding of TARBP2 domain to siRNA led to the formation of homogenous serum-stable complex with targeting potential. Further, uptake of the complex in Neuro-2a, IMR32 and HepG2 cells analyzed by confocal microscopy and fluorescence activated cell sorting, revealed selective requirement of GM1 for entry. Remarkably, systemic delivery of the fluorescently labeled complex (TARBP-BTP:siRNA) in ΑßPP-PS1 mouse model of Alzheimer's disease (AD) led to distinctive localization in the cerebral hemisphere. Further, the delivery of siRNA mediated by TARBP-BTP led to significant knockdown of BACE1 in the brain, in both ΑßPP-PS1 mice and wild type C57BL/6. The study establishes the growing importance of fusion proteins in delivering therapeutic siRNA to brain tissues.