Neurobehavioral dysfunction in a mouse model of Down syndrome: upregulation of cystathionine ß-synthase, H2S overproduction, altered protein persulfidation, synaptic dysfunction, endoplasmic reticulum stress, and autophagy.

Down syndrome (DS) is a genetic condition where the person is born with an extra chromosome 21. DS is associated with accelerated aging; people with DS are prone to age-related neurological conditions including an early-onset Alzheimer's disease. Using the Dp(17)3Yey/ + mice, which overexpresses a portion of mouse chromosome 17, which encodes for the transsulfuration enzyme cystathionine ß-synthase (CBS), we investigated the functional role of the CBS/hydrogen sulfide (H2S) pathway in the pathogenesis of neurobehavioral dysfunction in DS. The data demonstrate that CBS is higher in the brain of the DS mice than in the brain of wild-type mice, with primary localization in astrocytes. DS mice exhibited impaired recognition memory and spatial learning, loss of synaptosomal function, endoplasmic reticulum stress, and autophagy. Treatment of mice with aminooxyacetate, a prototypical CBS inhibitor, improved neurobehavioral function, reduced the degree of reactive gliosis in the DS brain, increased the ability of the synaptosomes to generate ATP, and reduced endoplasmic reticulum stress. H2S levels in the brain of DS mice were higher than in wild-type mice, but, unexpectedly, protein persulfidation was decreased. Many of the above alterations were more pronounced in the female DS mice. There was a significant dysregulation of metabolism in the brain of DS mice, which affected amino acid, carbohydrate, lipid, endocannabinoid, and nucleotide metabolites; some of these alterations were reversed by treatment of the mice with the CBS inhibitor. Thus, the CBS/H2S pathway contributes to the pathogenesis of neurological dysfunction in DS in the current animal model.

Overproduction of hydrogen sulfide, generated by cystathionine ß-synthase, disrupts brain wave patterns and contributes to neurobehavioral dysfunction in a rat model of down syndrome.

Using a novel rat model of Down syndrome (DS), the functional role of the cystathionine-ß-synthase (CBS)/hydrogen sulfide (H2S) pathway was investigated on the pathogenesis of brain wave pattern alterations and neurobehavioral dysfunction. Increased expression of CBS and subsequent overproduction of H2S was observed in the brain of DS rats, with CBS primarily localizing to astrocytes and the vasculature. DS rats exhibited neurobehavioral defects, accompanied by a loss of gamma brain wave activity and a suppression of the expression of multiple pre- and postsynaptic proteins. Aminooxyacetate, a prototypical pharmacological inhibitor of CBS, increased the ability of the DS brain tissue to generate ATP in vitro and reversed the electrophysiological and neurobehavioral alterations in vivo. Thus, the CBS/H2S pathway contributes to the pathogenesis of neurological dysfunction in DS, most likely through dysregulation of cellular bioenergetics and gene expression.

Parvalbumin-Deficiency Accelerates the Age-Dependent ROS Production in Pvalb Neurons in vivo: Link to Neurodevelopmental Disorders.

In neurodevelopmental disorders (NDDs) including autism spectrum disorder (ASD) and schizophrenia, impairment/malfunctioning of a subpopulation of interneurons expressing the calcium-binding protein parvalbumin (PV) -here termed Pvalb neurons- has gradually emerged as a possible cause. These neurons may represent a hub or point-of-convergence in the etiology of NDD. Increased oxidative stress associated with mitochondria impairment in Pvalb neurons is discussed as an essential step in schizophrenia etiology. Since PV downregulation is a common finding in ASD and schizophrenia individuals and PV-deficient (PV-/-) mice show a strong ASD-like behavior phenotype, we investigated the putative link between PV expression, alterations in mitochondria and oxidative stress. In a longitudinal study with 1, 3, and 6-months old PV-/- and wild type mice, oxidative stress was investigated in 9 Pvalb neuron subpopulations in the hippocampus, striatum, somatosensory cortex, medial prefrontal cortex, thalamic reticular nucleus (TRN) and cerebellum. In Pvalb neuron somata in the striatum and TRN, we additionally determined mitochondria volume and distribution at these three time points. In all Pvalb neuron subpopulations, we observed an age-dependent increase in oxidative stress and the increase strongly correlated with PV expression levels, but not with mitochondria density in these Pvalb neurons. Moreover, oxidative stress was elevated in Pvalb neurons of PV-/- mice and the magnitude of the effect was again correlated with PV expression levels in the corresponding wild type Pvalb neuron subpopulations. The PV-dependent effect was insignificant at 1 month and relative differences between WT and PV-/- Pvalb neurons were largest at 3 months. Besides the increase in mitochondria volume in PV's absence in TRN and striatal PV-/- Pvalb neurons fully present already at 1 month, we observed a redistribution of mitochondria from the perinuclear region toward the plasma membrane at all time points. We suggest that in absence of PV, slow Ca2+ buffering normally exerted by PV is compensated by a (mal)adaptive, mostly sub-plasmalemmal increase in mitochondria resulting in increased oxidative stress observed in 3- and 6-months old mice. Since PV-/- mice display core ASD-like symptoms already at 1 month, oxidative stress in Pvalb neurons is not a likely cause for their ASD-related behavior observed at this age.

Absence of parvalbumin increases mitochondria volume and branching of dendrites in inhibitory Pvalb neurons in vivo: a point of convergence of autism spectrum disorder (ASD) risk gene phenotypes.

BACKGROUND: In fast firing, parvalbumin (PV)-expressing (Pvalb) interneurons, PV acts as an intracellular Ca2+ signal modulator with slow-onset kinetics. In Purkinje cells of PV-/- mice, adaptive/homeostatic mechanisms lead to an increase in mitochondria, organelles equally capable of delayed Ca2+ sequestering/buffering. An inverse regulation of PV and mitochondria likewise operates in cell model systems in vitro including myotubes, epithelial cells, and oligodendrocyte-like cells overexpressing PV. Whether such opposite regulation pertains to all Pvalb neurons is currently unknown. In oligodendrocyte-like cells, PV additionally decreases growth and branching of processes in a cell-autonomous manner. METHODS: The in vivo effects of absence of PV were investigated in inhibitory Pvalb neurons expressing EGFP, present in the somatosensory and medial prefrontal cortex, striatum, thalamic reticular nucleus, hippocampal regions DG, CA3, and CA1 and cerebellum of mice either wild-type or knockout (PV-/-) for the Pvalb gene. Changes in Pvalb neuron morphology and PV concentrations were determined using immunofluorescence, followed by 3D-reconstruction and quantitative image analyses. RESULTS: PV deficiency led to an increase in mitochondria volume and density in the soma; the magnitude of the effect was positively correlated with the estimated PV concentrations in the various Pvalb neuron subpopulations in wild-type neurons. The increase in dendrite length and branching, as well as thickness of proximal dendrites of selected PV-/- Pvalb neurons is likely the result of the observed increased density and length of mitochondria in these PV-/- Pvalb neuron dendrites. The increased branching and soma size directly linked to the absence of PV is assumed to contribute to the increased volume of the neocortex present in juvenile PV-/- mice. The extended dendritic branching is in line with the hypothesis of local hyperconnectivity in autism spectrum disorder (ASD) and ASD mouse models including PV-/- mice, which display all ASD core symptoms and several comorbidities including cortical macrocephaly at juvenile age. CONCLUSION: PV is involved in most proposed mechanisms implicated in ASD etiology: alterations in Ca2+ signaling affecting E/I balance, changes in mitochondria structure/function, and increased dendritic length and branching, possibly resulting in local hyperconnectivity, all in a likely cell autonomous way.

The Parvalbumin Hypothesis of Autism Spectrum Disorder.

The prevalence of autism spectrum disorder (ASD)-a type of neurodevelopmental disorder-is increasing and is around 2% in North America, Asia, and Europe. Besides the known genetic link, environmental, epigenetic, and metabolic factors have been implicated in ASD etiology. Although highly heterogeneous at the behavioral level, ASD comprises a set of core symptoms including impaired communication and social interaction skills as well as stereotyped and repetitive behaviors. This has led to the suggestion that a large part of the ASD phenotype is caused by changes in a few and common set of signaling pathways, the identification of which is a fundamental aim of autism research. Using advanced bioinformatics tools and the abundantly available genetic data, it is possible to classify the large number of ASD-associated genes according to cellular function and pathways. Cellular processes known to be impaired in ASD include gene regulation, synaptic transmission affecting the excitation/inhibition balance, neuronal Ca2+ signaling, development of short-/long-range connectivity (circuits and networks), and mitochondrial function. Such alterations often occur during early postnatal neurodevelopment. Among the neurons most affected in ASD as well as in schizophrenia are those expressing the Ca2+-binding protein parvalbumin (PV). These mainly inhibitory interneurons present in many different brain regions in humans and rodents are characterized by rapid, non-adaptive firing and have a high energy requirement. PV expression is often reduced at both messenger RNA (mRNA) and protein levels in human ASD brain samples and mouse ASD (and schizophrenia) models. Although the human PVALB gene is not a high-ranking susceptibility/risk gene for either disorder and is currently only listed in the SFARI Gene Archive, we propose and present supporting evidence for the Parvalbumin Hypothesis, which posits that decreased PV level is causally related to the etiology of ASD (and possibly schizophrenia).