ARDD 2020: from aging mechanisms to interventions

Aging is emerging as a druggable target with growing interest from academia, industry and investors. New technologies such as artificial intelligence and advanced screening techniques, as well as a strong influence from the industry sector may lead to novel discoveries to treat age-related diseases. The present review summarizes presentations from the 7th Annual Aging Research and Drug Discovery (ARDD) meeting, held online on the 1st to 4th of September 2020. The meeting covered topics related to new methodologies to study aging, knowledge about basic mechanisms of longevity, latest interventional strategies to target the aging process as well as discussions about the impact of aging research on society and economy. More than 2000 participants and 65 speakers joined the meeting and we already look forward to an even larger meeting next year. Please mark your calendars for the 8th ARDD meeting that is scheduled for the 31st of August to 3rd of September, 2021, at Columbia University, USA

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. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s12987-022-00335-x

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.</p

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. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s12987-022-00348-6

Posthemorrhagic hydrocephalus associates with elevated inflammation and CSF hypersecretion via activation of choroidal transporters

Abstract Introduction Posthemorrhagic hydrocephalus (PHH) often develops following hemorrhagic events such as intraventricular hemorrhage (IVH) and subarachnoid hemorrhage (SAH). Treatment is limited to surgical diversion of the cerebrospinal fluid (CSF) since no efficient pharmacological therapies are available. This limitation follows from our incomplete knowledge of the molecular mechanisms underlying the ventriculomegaly characteristic of PHH. Here, we aimed to elucidate the molecular coupling between a hemorrhagic event and the subsequent PHH development, and reveal the inflammatory profile of the PHH pathogenesis. Methods CSF obtained from patients with SAH was analyzed for inflammatory markers using the proximity extension assay (PEA) technique. We employed an in vivo rat model of IVH to determine ventricular size, brain water content, intracranial pressure, and CSF secretion rate, as well as for transcriptomic analysis. Ex vivo radio-isotope assays of choroid plexus transport were employed to determine the direct effect of choroidal exposure to blood and inflammatory markers, both with acutely isolated choroid plexus and after prolonged exposure obtained with viable choroid plexus kept in tissue culture conditions. Results The rat model of IVH demonstrated PHH and associated CSF hypersecretion. The Na+/K+-ATPase activity was enhanced in choroid plexus isolated from IVH rats, but not directly stimulated by blood components. Inflammatory markers that were elevated in SAH patient CSF acted on immune receptors upregulated in IVH rat choroid plexus and caused Na+/K+/2Cl- cotransporter 1 (NKCC1) hyperactivity in ex vivo experimental conditions. Conclusions CSF hypersecretion may contribute to PHH development, likely due to hyperactivity of choroid plexus transporters. The hemorrhage-induced inflammation detected in CSF and in the choroid plexus tissue may represent the underlying pathology. Therapeutic targeting of such pathways may be employed in future treatment strategies towards PHH patients.http://deepblue.lib.umich.edu/bitstream/2027.42/173172/1/12987_2022_Article_360.pd

Membrane transporters control cerebrospinal fluid formation independently of conventional osmosis to modulate intracranial pressure

Background: Disturbances in the brain fluid balance can lead to life-threatening elevation in the intracranial pressure (ICP), which represents a vast clinical challenge. Nevertheless, the details underlying the molecular mechanisms governing cerebrospinal fluid (CSF) secretion are largely unresolved, thus preventing targeted and efficient pharmaceutical therapy of cerebral pathologies involving elevated ICP. Methods: Experimental rats were employed for in vivo determinations of CSF secretion rates, ICP, blood pressure and ex vivo excised choroid plexus for morphological analysis and quantification of expression and activity of various transport proteins. CSF and blood extractions from rats, pigs, and humans were employed for osmolality determinations and a mathematical model employed to determine a contribution from potential local gradients at the surface of choroid plexus. Results: We demonstrate that CSF secretion can occur independently of conventional osmosis and that local osmotic gradients do not suffice to support CSF secretion. Instead, the CSF secretion across the luminal membrane of choroid plexus relies approximately equally on the Na$^{+}$/K$^{+}$/2Cl$^{−}$ cotransporter NKCC1, the Na$^{+}$/HCO$_{3}$$^{−}$ cotransporter NBCe2, and the Na$^{+}$/K$^{+}$-ATPase, but not on the Na$^{+}$/H$^{+}$ exchanger NHE1. We demonstrate that pharmacological modulation of CSF secretion directly affects the ICP. Conclusions: CSF secretion appears to not rely on conventional osmosis, but rather occur by a concerted effort of different choroidal transporters, possibly via a molecular mode of water transport inherent in the proteins themselves. Therapeutic modulation of the rate of CSF secretion may be employed as a strategy to modulate ICP. These insights identify new promising therapeutic targets against brain pathologies associated with elevated ICP

Targeted deletion of the aquaglyceroporin AQP9 is protective in a mouse model of Parkinson’s disease

<div><p>More than 90% of the cases of Parkinson’s disease have unknown etiology. Gradual loss of dopaminergic neurons of substantia nigra is the main cause of morbidity in this disease. External factors such as environmental toxins are believed to play a role in the cell loss, although the cause of the selective vulnerability of dopaminergic neurons remains unknown. We have previously shown that aquaglyceroporin AQP9 is expressed in dopaminergic neurons and astrocytes of rodent brain. AQP9 is permeable to a broad spectrum of substrates including purines, pyrimidines, and lactate, in addition to water and glycerol. Here we test our hypothesis that AQP9 serves as an influx route for exogenous toxins and, hence, may contribute to the selective vulnerability of nigral dopaminergic (tyrosine hydroxylase-positive) neurons. Using <i>Xenopus</i> oocytes injected with <i>Aqp9</i> cRNA, we show that AQP9 is permeable to the parkinsonogenic toxin 1-methyl-4-phenylpyridinium (MPP⁺). Stable expression of AQP9 in HEK cells increases their vulnerability to MPP+ and to arsenite—another parkinsonogenic toxin. Conversely, targeted deletion of <i>Aqp9</i> in mice protects nigral dopaminergic neurons against MPP⁺ toxicity. A protective effect of <i>Aqp9</i> deletion was demonstrated in organotypic slice cultures of mouse midbrain exposed to MPP⁺ <i>in vitro</i> and in mice subjected to intrastriatal injections of MPP⁺ <i>in vivo</i>. Seven days after intrastriatal MPP⁺ injections, the population of tyrosine hydroxylase-positive cells in substantia nigra is reduced by 48% in <i>Aqp9</i> knockout mice compared with 67% in WT littermates. Our results show that AQP9 –selectively expressed in catecholaminergic neurons—is permeable to MPP⁺ and suggest that this aquaglyceroporin contributes to the selective vulnerability of nigral dopaminergic neurons by providing an entry route for parkinsonogenic toxins. To our knowledge this is the first evidence implicating a toxin permeable membrane channel in the pathophysiology of Parkinson’s disease.</p></div

HEK293 cells expressing EGFP-<i>h</i>AQP9 are more sensitive to arsenite than HEK293 cells expressing YFP-<i>h</i>DAT.

<p>A-C) Native HEK293 cells and HEK293 cells expressing EGFP-<i>h</i>AQP9 or YFP-<i>h</i>DAT were grown in 96-well plates and exposed to different concentrations of arsenite (eight wells for each concentration). Cell viability was assessed after 24 hours using the MTT assay. Data were collected from independent plates (n = 3 for each construct) and normalized to respective untreated cells. Both EGFP-<i>h</i>AQP9 and YFP-<i>h</i>DAT expressing cells showed higher sensitivity to arsenite, than native HEK293 cells, with EGFP-<i>h</i>AQP9 cells being the most sensitive. At the arsenite concentration of 10 μM, stably transfected EGFP-<i>h</i>AQP9 were the only cells showing toxin sensitivity (A). The curve showing IC50 values for arsenite calculated by nonlinear regression, log(inhibitor) vs response (three parameters) is shown (B). For log transformed data, the concentration 0 was set to 1 nM. Comparison of the IC50 values shows a significantly lower IC50 value for the HEK293 cells expressing EGFP-<i>h</i>AQP9 compared to the native HEK293 cells or HEK293 cells expressing YFP-<i>h</i>DAT (C). Bars are mean ± SEM. Asterisks: significantly different from untreated controls; *p<0.05, **p<0.01, ***p<0.001; crosses: significantly different from previous data point: ++ p<0.01.</p