CSF hyperdynamics in rats mimicking the obesity and androgen excess characteristic of patients with idiopathic intracranial hypertension.

BACKGROUND: Idiopathic intracranial hypertension (IIH) is a syndrome exhibiting elevated intracranial pressure (ICP), visual disturbances, and severe headache. IIH primarily affects young obese women, though it can occur in individuals of any age, BMI, and sex. IIH is characterized by systemic metabolic dysregulation with a profile of increased androgen hormones. However, the contribution of obesity/hormonal perturbations to cerebrospinal fluid (CSF) dynamics remains unresolved. METHODS: We employed obese female Zucker rats and adjuvant testosterone to reveal IIH causal drivers. ICP and CSF dynamics were determined with in vivo experimentation and magnetic resonance imaging, testosterone levels assessed with mass spectrometry, and choroid plexus function revealed with transcriptomics. RESULTS: Obese rats had undisturbed CSF testosterone levels and no changes in ICP or CSF dynamics. Adjuvant testosterone treatment of obese rats elevated the CSF secretion rate, although with no effect on the ICP, due to elevated CSF drainage capacity of these rats. CONCLUSIONS: Obesity in itself therefore does not suffice to recapitulate the IIH symptoms in rats, but modulation of CSF dynamics appears with adjuvant testosterone treatment, which mimics the androgen excess observed in female IIH patients. Obesity-induced androgen dysregulation may thus contribute to the disease mechanism of IIH and could potentially serve as a future therapeutic target.

Modelling idiopathic intracranial hypertension in rats: contributions of high fat diet and testosterone to intracranial pressure and cerebrospinal fluid production.

BACKGROUND: Idiopathic intracranial hypertension (IIH) is a condition characterized by increased intracranial pressure (ICP), impaired vision, and headache. Most cases of IIH occur in obese women of childbearing age, though age, BMI, and female sex do not encompass all aspects of IIH pathophysiology. Systemic metabolic dysregulation has been identified in IIH with a profile of androgen excess. However, the mechanistic coupling between obesity/hormonal perturbations and cerebrospinal fluid dynamics remains unresolved. METHODS: Female Wistar rats were either fed a high fat diet (HFD) for 21 weeks or exposed to adjuvant testosterone treatment for 28 days to recapitulate IIH causal drivers. Cerebrospinal fluid (CSF) and blood testosterone levels were determined with mass spectrometry, ICP and CSF dynamics with in vivo experimentation, and the choroid plexus function revealed with transcriptomics and ex vivo isotope-based flux assays. RESULTS: HFD-fed rats presented with increased ICP (65%), which was accompanied by increased CSF outflow resistance (50%) without altered CSF secretion rate or choroid plexus gene expression. Chronic adjuvant testosterone treatment of lean rats caused elevated ICP (55%) and CSF secretion rate (85%), in association with increased activity of the choroid plexus Na+,K+,2Cl- cotransporter, NKCC1. CONCLUSIONS: HFD-induced ICP elevation in experimental rats occurred with decreased CSF drainage capacity. Adjuvant testosterone, mimicking the androgen excess observed in female IIH patients, elevated the CSF secretion rate and thus ICP. Obesity-induced androgen dysregulation may thus contribute to the disease mechanism of IIH.

Transcriptional profiling of transport mechanisms and regulatory pathways in rat choroid plexus.

BACKGROUND: Dysregulation of brain fluid homeostasis associates with brain pathologies in which fluid accumulation leads to elevated intracranial pressure. Surgical intervention remains standard care, since specific and efficient pharmacological treatment options are limited for pathologies with disturbed brain fluid homeostasis. Such lack of therapeutic targets originates, in part, from the incomplete map of the molecular mechanisms underlying cerebrospinal fluid (CSF) secretion by the choroid plexus. METHODS: The transcriptomic profile of rat choroid plexus was generated by RNA Sequencing (RNAseq) of whole tissue and epithelial cells captured by fluorescence-activated cell sorting (FACS), and compared to proximal tubules. The bioinformatic analysis comprised mapping to reference genome followed by filtering for type, location, and association with alias and protein function. The transporters and associated regulatory modules were arranged in discovery tables according to their transcriptional abundance and tied together in association network analysis. RESULTS: The transcriptomic profile of choroid plexus displays high similarity between sex and species (human, rat, and mouse) and lesser similarity to another high-capacity fluid-transporting epithelium, the proximal tubules. The discovery tables provide lists of transport mechanisms that could participate in CSF secretion and suggest regulatory candidates. CONCLUSIONS: With quantification of the transport protein transcript abundance in choroid plexus and their potentially linked regulatory modules, we envision a molecular tool to devise rational hypotheses regarding future delineation of choroidal transport proteins involved in CSF secretion and their regulation. Our vision is to obtain future pharmaceutical targets towards modulation of CSF production in pathologies involving disturbed brain water dynamics.

Acetazolamide modulates intracranial pressure directly by its action on the cerebrospinal fluid secretion apparatus.

BACKGROUND: Elevated intracranial pressure (ICP) is observed in many neurological pathologies, e.g. hydrocephalus and stroke. This condition is routinely relieved with neurosurgical approaches, since effective and targeted pharmacological tools are still lacking. The carbonic anhydrase inhibitor, acetazolamide (AZE), may be employed to treat elevated ICP. However, its effectiveness is questioned, its location of action unresolved, and its tolerability low. Here, we determined the efficacy and mode of action of AZE in the rat . METHODS: We employed in vivo approaches including ICP and cerebrospinal fluid secretion measurements in anaesthetized rats and telemetric monitoring of ICP and blood pressure in awake rats in combination with ex vivo choroidal radioisotope flux assays and transcriptomic analysis. RESULTS: AZE effectively reduced the ICP, irrespective of the mode of drug administration and level of anaesthesia. The effect appeared to occur via a direct action on the choroid plexus and an associated decrease in cerebrospinal fluid secretion, and not indirectly via the systemic action of AZE on renal and vascular processes. Upon a single administration, the reduced ICP endured for approximately 10 h post-AZE delivery with no long-term changes of brain water content or choroidal transporter expression. However, a persistent reduction of ICP was secured with repeated AZE administrations throughout the day. CONCLUSIONS: AZE lowers ICP directly via its ability to reduce the choroid plexus CSF secretion, irrespective of mode of drug administration.

Evidence of oxidative stress and mitochondrial dysfunction in spinocerebellar ataxia type 2 (SCA2) patient fibroblasts: Effect of coenzyme Q10 supplementation on these parameters.

Spinocerebellar ataxia type 2 (SCA2) is a rare neurodegenerative disorder caused by a CAG repeat expansion in the ataxin-2 gene. We show increased oxidative stress, abnormalities in the antioxidant system, changes in complexes involved in oxidative phosphorylation and changes in mitochondrial morphology in SCA2 patient fibroblasts compared to controls, and we show that treatment with CoQ10 can partially reverse these changes. Together, our results suggest that oxidative stress and mitochondrial dysfunction may be contributory factors to the pathophysiology of SCA2 and that therapeutic strategies involving manipulation of the antioxidant system could prove to be of clinical benefit.

Identification of a small-molecule ligand that activates the neuropeptide receptor GPR171 and increases food intake.

Several neuropeptide systems in the hypothalamus, including neuropeptide Y and agouti-related protein (AgRP), control food intake. Peptides derived from proSAAS, a precursor implicated in the regulation of body weight, also control food intake. GPR171 is a heterotrimeric guanine nucleotide-binding protein (G protein)-coupled receptor (GPCR) for BigLEN (b-LEN), a peptide derived from proSAAS. To facilitate studies exploring the physiological role of GPR171, we sought to identify small-molecule ligands for this receptor by performing a virtual screen of a compound library for interaction with a homology model of GPR171. We identified MS0015203 as an agonist of GPR171 and demonstrated the selectivity of MS0015203 for GPR171 by testing the binding of this compound to 80 other membrane proteins, including family A GPCRs. Reducing the expression of GPR171 by shRNA (short hairpin RNA)-mediated knockdown blunted the cellular and tissue response to MS0015203. Peripheral injection of MS0015203 into mice increased food intake and body weight, and these responses were significantly attenuated in mice with decreased expression of GPR171 in the hypothalamus. Together, these results suggest that MS0015203 is a useful tool to probe the pharmacological and functional properties of GPR171 and that ligands targeting GPR171 may eventually lead to therapeutics for food-related disorders.

ProSAAS-derived peptides are differentially processed and sorted in mouse brain and AtT-20 cells.

ProSAAS is the precursor for some of the most abundant peptides found in mouse brain and other tissues, including peptides named SAAS, PEN, and LEN. Both SAAS and LEN are found in big and little forms due to differential processing. Initial processing of proSAAS is mediated by furin (and/or furin-like enzymes) and carboxypeptidase D, while the smaller forms are generated by secretory granule prohormone convertases and carboxypeptidase E. In mouse hypothalamus, PEN and big LEN colocalize with neuropeptide Y. In the present study, little LEN and SAAS were detected in mouse hypothalamus but not in cell bodies of neuropeptide Y-expressing neurons. PEN and big LEN show substantial colocalization in hypothalamus, but big LEN and little LEN do not. An antiserum to SAAS that detects both big and little forms of this peptide did not show substantial colocalization with PEN or big LEN. To further study this, the AtT-20 cells mouse pituitary corticotrophic cell line was transfected with rat proSAAS and the distribution of peptides examined. As found in mouse hypothalamus, only some of the proSAAS-derived peptides colocalized with each other in AtT-20 cells. The two sites within proSAAS that are known to be efficiently cleaved by furin were altered by site-directed mutagenesis to convert the P4 Arg into Lys; this change converts the sequences from furin consensus sites into prohormone convertase consensus sites. Upon expression of the mutated form of proSAAS in AtT-20 cells, there was significantly more colocalization of proSAAS-derived peptides PEN and SAAS. Taken together, these results indicate that proSAAS is initially cleaved in the Golgi or trans-Golgi network by furin and/or furin-like enzymes and the resulting fragments are sorted into distinct vesicles and further processed by additional enzymes into the mature peptides.

GPR171 is a hypothalamic G protein-coupled receptor for BigLEN, a neuropeptide involved in feeding.

Multiple peptide systems, including neuropeptide Y, leptin, ghrelin, and others, are involved with the control of food intake and body weight. The peptide LENSSPQAPARRLLPP (BigLEN) has been proposed to act through an unknown receptor to regulate body weight. In the present study, we used a combination of ligand-binding and receptor-activity assays to characterize a Gαi/o protein-coupled receptor activated by BigLEN in the mouse hypothalamus and Neuro2A cells. We then selected orphan G protein-coupled receptors expressed in the hypothalamus and Neuro2A cells and tested each for activation by BigLEN. G protein-coupled receptor 171 (GPR171) is activated by BigLEN, but not by the C terminally truncated peptide LittleLEN. The four C-terminal amino acids of BigLEN are sufficient to bind and activate GPR171. Overexpression of GPR171 leads to an increase, and knockdown leads to a decrease, in binding and signaling by BigLEN and the C-terminal peptide. In the hypothalamus GPR171 expression complements the expression of BigLEN, and its level and activity are elevated in mice lacking BigLEN. In mice, shRNA-mediated knockdown of hypothalamic GPR171 leads to a decrease in BigLEN signaling and results in changes in food intake and metabolism. The combination of GPR171 shRNA together with neutralization of BigLEN peptide by antibody absorption nearly eliminates acute feeding in food-deprived mice. Taken together, these results demonstrate that GPR171 is the BigLEN receptor and that the BigLEN-GPR171 system plays an important role in regulating responses associated with feeding and metabolism in mice.

ProSAAS-derived peptides are colocalized with neuropeptide Y and function as neuropeptides in the regulation of food intake.

ProSAAS is the precursor of a number of peptides that have been proposed to function as neuropeptides. Because proSAAS mRNA is highly expressed in the arcuate nucleus of the hypothalamus, we examined the cellular localization of several proSAAS-derived peptides in the mouse hypothalamus and found that they generally colocalized with neuropeptide Y (NPY), but not α-melanocyte stimulating hormone. However, unlike proNPY mRNA, which is upregulated by food deprivation in the mediobasal hypothalamus, neither proSAAS mRNA nor proSAAS-derived peptides were significantly altered by 1-2 days of food deprivation in wild-type mice. Furthermore, while proSAAS mRNA levels in the mediobasal hypothalamus were significantly lower in Cpe(fat/fat) mice as compared to wild-type littermates, proNPY mRNA levels in the mediobasal hypothalamus and in other subregions of the hypothalamus were not significantly different between wild-type and Cpe(fat/fat) mice. Intracerebroventricular injections of antibodies to two proSAAS-derived peptides (big LEN and PEN) significantly reduced food intake in fasted mice, while injections of antibodies to two other proSAAS-derived peptides (little LEN and little SAAS) did not. Whole-cell patch clamp recordings of parvocellular neurons in the hypothalamic paraventricular nucleus, a target of arcuate NPY projections, showed that big LEN produced a rapid and reversible inhibition of synaptic glutamate release that was spike independent and abolished by blocking postsynaptic G protein activity, suggesting the involvement of a postsynaptic G protein-coupled receptor and the release of a retrograde synaptic messenger. Taken together with previous studies, these findings support a role for proSAAS-derived peptides such as big LEN as neuropeptides regulating food intake.

Analysis of peptides in prohormone convertase 1/3 null mouse brain using quantitative peptidomics.

Neuropeptides are produced from larger precursors by limited proteolysis, first by endopeptidases and then by carboxypeptidases. Major endopeptidases required for these cleavages include prohormone convertase (PC) 1/3 and PC2. In this study, quantitative peptidomics analysis was used to characterize the specific role PC1/3 plays in this process. Peptides isolated from hypothalamus, amygdala, and striatum of PC1/3 null mice were compared with those from heterozygous and wild-type mice. Extracts were labeled with stable isotopic tags and fractionated by HPLC, after which relative peptide levels were determined using tandem mass spectrometry. In total, 92 peptides were found, of which 35 were known neuropeptides or related peptides derived from 15 distinct secretory pathway proteins: 7B2, chromogranin A and B, cocaine- and amphetamine-regulated transcript, procholecystokinin, proenkephalin, promelanin concentrating hormone, proneurotensin, propituitary adenylate cyclase-activating peptide, proSAAS, prosomatosatin, provasoactive intestinal peptide, provasopressin, secretogranin III, and VGF. Among the peptides derived from these proteins, approximately 1/3 were decreased in the PC1/3 null mice relative to wild-type mice, approximately 1/3 showed no change, and approximately 1/3 increased in PC1/3 null. Cleavage sites were analyzed in peptides that showed no change or that decreased in PC1/3 mice, and these results were compared with peptides that showed no change or decreased in previous peptidomic studies with PC2 null mice. Analysis of these sites showed that while PC1/3 and PC2 have overlapping substrate preferences, there are particular cleavage site residues that distinguish peptides preferred by each PC.