AAV2 mediated retrograde transduction of corticospinal motor neurons reveals initial and selective apical dendrite degeneration in ALS.

Corticospinal motor neurons (CSMN) are the cortical component of motor neuron circuitry, which controls voluntary movement and degenerates in diseases such as amyotrophic lateral sclerosis, primary lateral sclerosis and hereditary spastic paraplegia. By using dual labeling combined with molecular marker analysis, we identified AAV2-2 mediated retrograde transduction as an effective approach to selectively target CSMN without affecting other neuron populations both in wild-type and hSOD1(G93A) transgenic ALS mice. This approach reveals very precise details of cytoarchitectural defects within vulnerable neurons in vivo. We report that CSMN vulnerability is marked by selective degeneration of apical dendrites especially in layer II/III of the hSOD1(G93A) mouse motor cortex, where cortical input to CSMN function is vastly modulated. While our findings confirm the presence of astrogliosis and microglia activation, they do not lend support to their direct role for the initiation of CSMN vulnerability. This study enables development of targeted gene replacement strategies to CSMN in the cerebral cortex, and reveals CSMN cortical modulation defects as a potential cause of neuronal vulnerability in ALS.

High serum androstenedione levels correlate with impaired memory in the surgically menopausal rat: a replication and new findings.

After natural menopause in women, androstenedione becomes the primary hormone secreted by the residual follicle-depleted ovaries. In two independent studies, in rodents that had undergone ovarian follicular depletion, we found that higher endogenous serum androstenedione levels correlated with increased working memory errors. This led to the hypothesis that higher androstenedione levels impair memory. The current study directly tested this hypothesis, examining the cognitive effects of exogenous androstenedione administration in rodents. Middle-aged ovariectomised rats received vehicle or one of two doses of androstenedione. Rats were tested on a spatial working and reference memory maze battery including the water-radial arm maze, Morris water maze (MM) and delay match-to-sample task. Androstenedione at the highest dose impaired reference memory as well as the ability to maintain performance as memory demand was elevated. This was true for both high temporal demand memory retention of one item of spatial information, as well as the ability to handle multiple items of spatial working memory information. We measured glutamic acid decarboxylase (GAD) protein in multiple brain regions to determine whether the gamma-aminobutyric acid (GABA) system relates to androstenedione-induced memory impairments. Results showed that higher entorhinal cortex GAD levels were correlated with worse MM performance, irrespective of androstenedione treatment. These findings suggest that androstenedione, the main hormone produced by the follicle-depleted ovary, is detrimental to working memory, reference memory and memory retention. Furthermore, while spatial reference memory performance might be related to the GABAergic system, it does not appear to be altered with androstenedione administration, at least at the doses used in the current study.

Endothelial Cell Fate Determination: A Top Notch Job in Vascular Decision-Making.

As vascular networks form, endothelial cells (ECs) undergo cell fate decisions that determine whether they become tip or stalk cells of the developing vascular plexus or mature into arterial, venous, or lymphatic endothelium. EC fate decisions are coordinated with neighboring cells to initiate sprouting, maintain endothelial barrier, or ensure appropriate specialization of vessels. We describe mechanisms that control EC fate at specific steps in these processes, with an emphasis on the role of the Notch signaling pathway. Specific EC fate determination steps that are highlighted are tip/stalk selection during sprouting angiogenesis, venous-arterial specification, arteriogenesis, lymphatic vessel specification, and lymphatic valve formation.

Endothelial Notch signaling directly regulates the small GTPase RND1 to facilitate Notch suppression of endothelial migration.

To control sprouting angiogenesis, endothelial Notch signaling suppresses tip cell formation, migration, and proliferation while promoting barrier formation. Each of these responses may be regulated by distinct Notch-regulated effectors. Notch activity is highly dynamic in sprouting endothelial cells, while constitutive Notch signaling drives homeostatic endothelial polarization, indicating the need for both rapid and constitutive Notch targets. In contrast to previous screens that focus on genes regulated by constitutively active Notch, we characterized the dynamic response to Notch. We examined transcriptional changes from 1.5 to 6 h after Notch signal activation via ligand-specific or EGTA induction in cultured primary human endothelial cells and neonatal mouse brain. In each combination of endothelial type and Notch manipulation, transcriptomic analysis identified distinct but overlapping sets of rapidly regulated genes and revealed many novel Notch target genes. Among the novel Notch-regulated signaling pathways identified were effectors in GPCR signaling, notably, the constitutively active GTPase RND1. In endothelial cells, RND1 was shown to be a novel direct Notch transcriptional target and required for Notch control of sprouting angiogenesis, endothelial migration, and Ras activity. We conclude that RND1 is directly regulated by endothelial Notch signaling in a rapid fashion in order to suppress endothelial migration.

Homology modeling of FFA2 identifies novel agonists that potentiate insulin secretion.

Critical aspects of maintaining glucose homeostasis in the face of chronic insulin resistance and type 2 diabetes (T2D) are increased insulin secretion and adaptive expansion of beta cell mass. Nutrient and hormone sensing G protein-coupled receptors are important mediators of these properties. A growing body of evidence now suggests that the G protein-coupled receptor, free fatty acid receptor 2 (FFA2), is capable of contributing to the maintenance of glucose homeostasis by acting at the pancreatic beta cell as well as at other metabolically active tissues. We have previously demonstrated that Gαq/11-biased agonism of FFA2 can potentiate glucose stimulated insulin secretion (GSIS) as well as promote beta cell proliferation. However, the currently available Gαq/11-biased agonists for FFA2 exhibit low potency, making them difficult to examine in vivo. This study sought to identify Gαq/11-biased FFA2-selective agonists with potent GSIS-stimulating effects. To do this, we generated an FFA2 homology model that was used to screen a library of 10 million drug-like compounds. Although FFA2 and the related short chain fatty acid receptor FFA3 share 52% sequence similarity, our virtual screen identified over 50 compounds with predicted selectivity and increased potency for FFA2 over FFA3. Subsequent in vitro calcium mobilization assays and GSIS assays resulted in the identification of a compound that can potentiate GSIS via activation of Gαq/11 with 100-fold increased potency compared with previously described Gαq/11-biased FFA2 agonists. These methods and findings provide a foundation for future discovery efforts to identify biased FFA2 agonists as potential T2D therapeutics.

Loss of Free Fatty Acid Receptor 2 leads to impaired islet mass and beta cell survival.

The regulation of pancreatic ß cell mass is a critical factor to help maintain normoglycemia during insulin resistance. Nutrient-sensing G protein-coupled receptors (GPCR) contribute to aspects of ß cell function, including regulation of ß cell mass. Nutrients such as free fatty acids (FFAs) contribute to precise regulation of ß cell mass by signaling through cognate GPCRs, and considerable evidence suggests that circulating FFAs promote ß cell expansion by direct and indirect mechanisms. Free Fatty Acid Receptor 2 (FFA2) is a ß cell-expressed GPCR that is activated by short chain fatty acids, particularly acetate. Recent studies of FFA2 suggest that it may act as a regulator of ß cell function. Here, we set out to explore what role FFA2 may play in regulation of ß cell mass. Interestingly, Ffar2(-/-) mice exhibit diminished ß cell mass at birth and throughout adulthood, and increased ß cell death at adolescent time points, suggesting a role for FFA2 in establishment and maintenance of ß cell mass. Additionally, activation of FFA2 with Gαq/11-biased agonists substantially increased ß cell proliferation in in vitro and ex vivo proliferation assays. Collectively, these data suggest that FFA2 may be a novel therapeutic target to stimulate ß cell growth and proliferation.

An Acetate-Specific GPCR, FFAR2, Regulates Insulin Secretion.

G protein-coupled receptors have been well described to contribute to the regulation of glucose-stimulated insulin secretion (GSIS). The short-chain fatty acid-sensing G protein-coupled receptor, free fatty acid receptor 2 (FFAR2), is expressed in pancreatic ß-cells, and in rodents, its expression is altered during insulin resistance. Thus, we explored the role of FFAR2 in regulating GSIS. First, assessing the phenotype of wild-type and Ffar2(-/-) mice in vivo, we observed no differences with regard to glucose homeostasis on normal or high-fat diet, with a marginally significant defect in insulin secretion in Ffar2(-/-) mice during hyperglycemic clamps. In ex vivo insulin secretion studies, we observed diminished GSIS from Ffar2(-/-) islets relative to wild-type islets under high-glucose conditions. Further, in the presence of acetate, the primary endogenous ligand for FFAR2, we observed FFAR2-dependent potentiation of GSIS, whereas FFAR2-specific agonists resulted in either potentiation or inhibition of GSIS, which we found to result from selective signaling through either Gαq/11 or Gαi/o, respectively. Lastly, in ex vivo insulin secretion studies of human islets, we observed that acetate and FFAR2 agonists elicited different signaling properties at human FFAR2 than at mouse FFAR2. Taken together, our studies reveal that FFAR2 signaling occurs by divergent G protein pathways that can selectively potentiate or inhibit GSIS in mouse islets. Further, we have identified important differences in the response of mouse and human FFAR2 to selective agonists, and we suggest that these differences warrant consideration in the continued investigation of FFAR2 as a novel type 2 diabetes target.

Cognitive-impairing effects of medroxyprogesterone acetate in the rat: independent and interactive effects across time.

RATIONALE: The synthetic progestin medroxyprogesterone acetate (MPA), widely used in hormone therapy (HT) and as the contraceptive Depo Provera, is implicated in detrimental cognitive effects in women. Recent evidence in aged ovariectomized (Ovx) rodents shows that short-term MPA treatment impairs cognition and alters the GABAergic system. OBJECTIVES: Using rats, we evaluated the long-lasting cognitive and GABAergic effects of MPA administered in young adulthood (Early-MPA), modeling contraception, and how this early exposure interacts with later MPA treatment (Late-MPA), modeling HT. METHODS: Early-MPA treatment involved weekly anti-ovulatory MPA injections (3.5 mg) from 4 to 8 months of age in ovary-intact rats. At 10 months old, rats were Ovx and weekly MPA injections were re-initiated and continued throughout testing for Late-MPA treatment. RESULTS: On the water radial-arm maze, all MPA-treated groups showed working memory impairment compared to Controls (p < 0.05); Early + Late-MPA rats were impaired on multiple dimensions of working memory (p < 0.05). On the Morris maze, Late-MPA rats showed greater overnight forgetting compared to Controls (p < 0.05). At study conclusion, MPA was detected in serum in all MPA-treated groups except Early-MPA, confirming treatment and clearance from serum in Early-MPA rats. In animals with detectable serum MPA, higher MPA levels were associated with less dorsal-hippocampal glutamic acid decarboxylase, the synthesizing enzyme for GABA (p = 0.0059). CONCLUSIONS: Findings suggest that MPA treatment leads to long-lasting cognitive impairments in the rodent, even in the absence of circulating MPA in animals given prior MPA treatment, which may relate to the GABAergic system. Further research defining the parameters of the negative impact of this widely used progestin on brain and cognition is warranted.