Michael Helwig

Circulating Ghrelin Levels and Central Ghrelin Receptor Expression are Elevated in Response to Food Deprivation in a Seasonal Mammal (Phodopus sungorus)

Ghrelin is an endogenous ligand for the growth hormone secretagogue receptor (GHSR). However, the functional interaction of ligand and receptor is not very well understood. We demonstrate that GHSR mRNA is up‐regulated after food deprivation (48 h) in the hypothalamic arcuate nucleus and ventromedial nucleus of the seasonal Siberian hamster, Phodopus sungorus. This increase is accompanied by a two‐fold elevation of circulating ghrelin concentration. Chronic changes in feeding state imposed by food restriction over a period of 12 weeks during long day‐length induced increased GHSR gene expression, whereas food restriction for 6 weeks had no effect. Phodopus sungorus reveals remarkable seasonal changes in body weight, fat mass and circulating leptin levels. Ghrelin is generally regarded as having opposing effects on appetite and body weight with respect to those exhibited by leptin. However, our study revealed that seasonal adaptations were not accompanied by changes in either GHSR gene expression or circulating ghrelin concentration. Therefore, we suggest that ghrelin only plays a minor role in modulating long‐term seasonal body weight cycles. Our findings imply that ghrelin predominantly acts as a short‐term regulator of feeding.

The Neuroendocrine Protein 7B2 Suppresses the Aggregation of Neurodegenerative Disease-related Proteins

Background: The neuroendocrine protein 7B2 blocks the aggregation of certain secreted proteins. Results: 7B2 co-localizes with protein aggregates in Parkinson and Alzheimer disease brains; blocks the fibrillation of Aβ1–40, Aβ1–42, and α-synuclein; and blocks Aβ1–42-induced Neuro-2A cell death. Conclusion: 7B2 inhibits the cytotoxicity of Aβ1–42 by modulation of oligomer formation. Significance: 7B2 is a novel anti-aggregation secretory chaperone associated with neurodegenerative disease. Neurodegenerative diseases such as Alzheimer (AD) and Parkinson (PD) are characterized by abnormal aggregation of misfolded β-sheet-rich proteins, including amyloid-β (Aβ)-derived peptides and tau in AD and α-synuclein in PD. Correct folding and assembly of these proteins are controlled by ubiquitously expressed molecular chaperones; however, our understanding of neuron-specific chaperones and their involvement in the pathogenesis of neurodegenerative diseases is limited. We here describe novel chaperone-like functions for the secretory protein 7B2, which is widely expressed in neuronal and endocrine tissues. In in vitro experiments, 7B2 efficiently prevented fibrillation and formation of Aβ1–42, Aβ1–40, and α-synuclein aggregates at a molar ratio of 1:10. In cell culture experiments, inclusion of recombinant 7B2, either in the medium of Neuro-2A cells or intracellularly via adenoviral 7B2 overexpression, blocked the neurocytotoxic effect of Aβ1–42 and significantly increased cell viability. Conversely, knockdown of 7B2 by RNAi increased Aβ1–42-induced cytotoxicity. In the brains of APP/PSEN1 mice, a model of AD amyloidosis, immunoreactive 7B2 co-localized with aggregation-prone proteins and their respective aggregates. Furthermore, in the hippocampus and substantia nigra of human AD- and PD-affected brains, 7B2 was highly co-localized with Aβ plaques and α-synuclein deposits, strongly suggesting physiological association. Our data provide insight into novel functions of 7B2 and establish this neural protein as an anti-aggregation chaperone associated with neurodegenerative disease.

Seasonal leptin resistance is associated with impaired signalling via JAK2-STAT3 but not ERK, possibly mediated by reduced hypothalamic GRB2 protein

The Siberian hamster, Phodopus sungorus, undergoes a striking seasonal cycle of leptin sensitivity and body weight regulation, but the molecular mechanism and relevance to human leptin insensitivity are unknown. Here we show that nuclear translocation of phospho-STAT3 in the hypothalamus is rapidly stimulated by leptin to a greater extent in hamsters held in short-day length (SD) as compared to long-day length (LD). Intriguingly, effects of leptin on STAT3 appeared to be in part limited to nuclear translocation of phospho-STAT3 associated with the cell surface rather than phosphorylation of STAT3. The number of phospho-ERK cells within the hypothalamus was unaffected by either photoperiod or leptin. However, proximal to ERK phosphorylation, hypothalamic SH2-containing tyrosine phosphatase (SHP2) and the small growth factor receptor-binding protein (GRB2), which act as competitive negative modulators on binding of SOCS3 to leptin receptor (LRb)-associated Tyr985, were increased in SD compared to LD. Our findings suggest that activation of STAT3 by leptin may be dependent on interaction of stimulatory SHP2/GRB2 as well as inhibitory SOCS3 on the level of competitive binding to LRb-associated Tyr985. This hypothetical mechanism may represent the molecular identity of seasonally induced adjustments in leptin sensitivity and may be applied to investigating leptin sensitivity in other rodent models.

Dynamic Modulation of Prohormone Convertase 2 (PC2)-mediated Precursor Processing by 7B2 Protein PREFERENTIAL EFFECT ON GLUCAGON SYNTHESIS

The small neuroendocrine protein 7B2 is required for the production of active prohormone convertase 2 (PC2), an enzyme involved in the synthesis of peptide hormones, such as glucagon and proopiomelanocortin-derived α-melanocyte-stimulating hormone. However, whether 7B2 can dynamically modulate peptide production through regulation of PC2 activity remains unclear. Infection of the pancreatic alpha cell line α-TC6 with 7B2-encoding adenovirus efficiently increased production of glucagon, whereas siRNA-mediated knockdown of 7B2 significantly decreased stored glucagon. Furthermore, rescue of 7B2 expression in primary pituitary cultures prepared from 7B2 null mice restored melanocyte-stimulating hormone production, substantiating the role of 7B2 as a regulatory factor in peptide biosynthesis. In anterior pituitary and pancreatic beta cell lines, however, overexpression of 7B2 affected neither production nor secretion of peptides despite increased release of active PC2. In direct contrast, 7B2 overexpression decreased the secretion and increased the activity of PC2 within α-TC6 cells; the increased intracellular concentration of active PC2 within these cells may therefore account for the enhanced production of glucagon. In line with these findings, we found elevated circulating glucagon levels in 7B2-overexpressing cast/cast mice in vivo. Surprisingly, when proopiomelanocortin and proglucagon were co-expressed in either pituitary or pancreatic alpha cell lines, proglucagon processing was preferentially decreased when 7B2 was knocked down. Taken together, these results suggest that proglucagon cleavage has a greater dependence on PC2 activity than other precursors and moreover that 7B2-dependent routing of PC2 to secretory granules is cell line-specific. The manipulation of 7B2 could therefore represent an effective way to selectively regulate synthesis of certain PC2-dependent peptides.

A novel function for proSAAS as an amyloid anti‐aggregant in Alzheimer's disease

Neurodegenerative diseases such as Alzheimers disease (AD) are characterized by an abnormal aggregation of misfolded beta‐sheet rich proteins such as β‐amyloid (Aβ). Various ubiquitously expressed molecular chaperones control the correct folding of cellular proteins and prevent the accumulation of harmful species. We here describe a novel anti‐aggregant chaperone function for the neuroendocrine protein proSAAS, an abundant secretory polypeptide that is widely expressed within neural and endocrine tissues and which has previously been associated with neurodegenerative disease in various proteomics studies. In the brains of 12‐month‐old APdE9 mice, and in the cortex of a human AD‐affected brain, proSAAS immunoreactivity was highly colocalized with amyloid pathology. Immunoreactive proSAAS co‐immunoprecipitated with Aβ immunoreactivity in lysates from APdE9 mouse brains. In vitro, proSAAS efficiently prevented the fibrillation of Aβ1–42 at molar ratios of 1 : 10, and this anti‐aggregation effect was dose dependent. Structure‐function studies showed that residues 97–180 were sufficient for the anti‐aggregation function against Aβ. Finally, inclusion of recombinant proSAAS in the medium of Neuro2a cells, as well as lentiviral‐mediated proSAAS over‐expression, blocked the neurocytotoxic effect of Aβ1–42 in Neuro2a cells. Taken together, our results suggest that proSAAS may play a role in Alzheimers disease pathology.

Regulation of Neuropeptide Processing Enzymes by Catecholamines in Endocrine Cells

Treatment of cultured bovine adrenal chromaffin cells with the catecholamine transport blocker reserpine was shown previously to increase enkephalin levels severalfold. To explore the biochemical mechanism of this effect, we examined the effect of reserpine treatment on the activities of three different peptide precursor processing enzymes: carboxypeptidase E (CPE) and the prohormone convertases (PCs) PC1/3 and PC2. Reserpine treatment increased both CPE and PC activity in extracts of cultured chromaffin cells; total protein levels were unaltered for any enzyme. Further analysis showed that the increase in CPE activity was due to an elevated Vmax, with no change in the Km for substrate hydrolysis or the levels of CPE mRNA. Reserpine activation of endogenous processing enzymes was also observed in extracts prepared from PC12 cells stably expressing PC1/3 or PC2. In vitro experiments using purified enzymes showed that catecholamines inhibited CPE, PC1/3, and PC2, with dopamine quinone the most potent inhibitor (IC50 values of ∼50–500 μM); dopamine, norepinephrine, and epinephrine exhibited inhibition in the micromolar range. The inhibition of purified CPE with catecholamines was time-dependent and, for dopamine quinone, dilution-independent, suggesting covalent modification of the protein by the catecholamine. Because the catecholamine concentrations found to be inhibitory to PC1/3, PC2, and CPE are well within the physiological range found in chromaffin granules, we conclude that catecholaminergic transmitter systems have the potential to exert considerable dynamic influence over peptidergic transmitter synthesis by altering the activity of peptide processing enzymes.

Photoperiod-Dependent Regulation of Carboxypeptidase E Affects the Selective Processing of Neuropeptides in the Seasonal Siberian Hamster (Phodopus sungorus)

The production of bioactive peptides from biologically inactive precursors involves extensive post‐translational processing, including enzymatic cleavage by proteolytic peptidases. Endoproteolytic prohormone‐convertases initially cleave the precursors of many neuropeptides at specific amino acid sequences to generate intermediates with basic amino acid extensions on their C‐termini. Subsequently, the related exopeptidases, carboxypeptidases D and E (CPD and CPE), are responsible for removing these amino acids before the peptides achieve biological activity. We investigated the effect of photoperiod on the processing of the neuropeptide precursor pro‐opiomelanocortin (POMC) and its derived neuropeptides, α‐ melanocyte‐stimulating hormone (MSH) and β‐endorphin (END), within the hypothalamus of the seasonal Siberian hamster (Phodopus sungorus). We thus compared hypothalamic distribution of CPD, CPE, α‐MSH and β‐END using immunohistochemistry and measured the enzyme activity of CPE and concentrations of C‐terminally cleaved α‐MSH in short‐day (SD; 8 : 16 h light/dark) and long‐day (LD; 16 : 8 h light/dark) acclimatised hamsters. Increased immunoreactivity (‐IR) of CPE, as well as higher CPE activity, was observed in SD. This increase was accompanied by more β‐END‐IR cells and substantially higher levels of C‐ terminally cleaved α‐MSH, as determined by radioimmunoassay. Our results suggest that exoproteolytic cleavage of POMC‐derived neuropeptides is tightly regulated by photoperiod in the Siberian hamster. Higher levels of biological active α‐MSH‐ and β‐END in SD are consistent with the hypothesis that post‐translational processing is a key event in the regulation of seasonal energy balance.

The neural chaperone proSAAS blocks α-synuclein fibrillation and neurotoxicity

Significance Aberrant protein homeostasis (proteostasis) is increasingly recognized as a major causal factor in the development of protein aggregates, including aggregation of α-synuclein in Parkinson’s disease. Cellular proteins known as chaperones control proteostatic processes and are strongly associated with many different neurodegenerative diseases. We show here that an abundant brain- and endocrine-specific secretory chaperone known as proSAAS (named after four residues in the amino terminal region) exhibits potent antiaggregant effects on α-synuclein aggregation, and we identify a region of the protein that is necessary for this antiaggregant effect. We further show that proSAAS expression is able to stop α-synuclein from killing dopaminergic cells in a nigral cell model of Parkinson’s disease. This work demonstrates an important role for the proSAAS protein in blocking aggregation in neurodegeneration. Emerging evidence strongly suggests that chaperone proteins are cytoprotective in neurodegenerative proteinopathies involving protein aggregation; for example, in the accumulation of aggregated α-synuclein into the Lewy bodies present in Parkinson’s disease. Of the various chaperones known to be associated with neurodegenerative disease, the small secretory chaperone known as proSAAS (named after four residues in the amino terminal region) has many attractive properties. We show here that proSAAS, widely expressed in neurons throughout the brain, is associated with aggregated synuclein deposits in the substantia nigra of patients with Parkinson’s disease. Recombinant proSAAS potently inhibits the fibrillation of α-synuclein in an in vitro assay; residues 158–180, containing a largely conserved element, are critical to this bioactivity. ProSAAS also exhibits a neuroprotective function; proSAAS-encoding lentivirus blocks α-synuclein-induced cytotoxicity in primary cultures of nigral dopaminergic neurons, and recombinant proSAAS blocks α-synuclein–induced cytotoxicity in SH-SY5Y cells. Four independent proteomics studies have previously identified proSAAS as a potential cerebrospinal fluid biomarker in various neurodegenerative diseases. Coupled with prior work showing that proSAAS blocks β-amyloid aggregation into fibrils, this study supports the idea that neuronal proSAAS plays an important role in proteostatic processes. ProSAAS thus represents a possible therapeutic target in neurodegenerative disease.

Seasonal regulation of cocaine- and amphetamine-regulated transcript in the arcuate nucleus of Djungarian hamster (Phodopus sungorus)

The hypothalamic neuropeptidergic system involved in the photoperiodic control of energy metabolism in seasonal mammals, is poorly understood. In the present study we examined whether distribution and number of the hypothalamic neuronal cell populations containing cocaine- and amphetamine-regulated transcript (CART) are influenced by different photoperiod and ambient temperature, or by food status in the Djungarian hamster (Phodopus sungorus). Hamsters bred and raised in long day photoperiod at room temperature (16 h light/8h dark at 23 degrees C; LD) were transferred to short day photoperiod and moderate cold (8h light/16 h dark at 16 degrees C; SD). After a 4 weeks acclimation period, uterus and body weight were decreased in SD as compared to controls maintained in LD. The number of CART-immunoreactive cells within the arcuate nucleus (ARC) was significantly higher in SD hamsters compared to LD control. This increase was restricted to the rostro to mid portion of the ARC, specifically in the hypothalamic retrochiasmatic area close to the rostral ARC and in the hypothalamic region lateral to the ARC and ventral to the ventromedial hypothalamic nuclei. In similar hypothalamic regions, food deprivation for 48 h significantly decreased the number of CART-immunoreactive cells in SD hamsters. Shortening of photoperiod combined with lowering of ambient temperature and food deprivation had no effect on the number of CART-immunoreactive cells in the lateral hypothalamic area. These findings suggest that photoperiod and ambient temperature influence energy metabolism potentially by alterations of the CART neuronal system in the rostral portion of the ARC in Djungarian hamsters.