DISTINCT PHENOTYPIC CHANGES BY PACAP AND VIP IN LIPOPOLYSACCHARIDE STIMULATED BV2 MICROGLIAL CELLS

Background: Aberrant microglial activation plays a key role in the progressive neuronal loss seen in many neurodegenerative diseases. PACAP and VIP are two neuropeptides that elicit robust immunosuppressive functions within the CNS. However, the underlying mechanisms through which these peptides regulate microglia activities are not clear. Aim & Objectives: Using lipopolysaccharide (LPS) to induce BV2 microglial cell polarisation, we aimed at testing whether and how administration of either PACAP or VIP could differentially affect microglial pro-inflammatory profile, polarisation state and morphological appearance to elicit immunosuppressive effects. Methods: Quantitative real-time PCR, Western blot, Griess reactions, immunofluorescence and morphological analyses were conducted in order to determine the effects of PACAP and VIP in BV2 microglial cells exposed or not to 1µg/ml LPS. Results: Our data demonstrated that both PACAP and VIP reduce the expression of pro-inflammatory mediators in LPS-stimulated BV2 cells. We also found that exogenous administration of PACAP and VIP rescued the dysregulations of the endogenous PACAP/VIP levels and attenuated the expression of microglial activation markers caused by LPS. Interestingly, despite the similar anti-inflammatory activities of PACAP and VIP, PACAP mainly reduced the number of M1 polarised cells, whereas VIP acted by increasing the subpopulation of cells exhibiting an ‘intermediate’ phenotype/bipolar-shaped (p<0.001 vs. control), at the expenses of resting/rounded cells. Conclusion: PACAP and VIP both possess immunosuppressive effects in activated BV2 microglial cells, but these effects seem to involve the differential shift of certain cell subpopulations towards distinctive phenotypes

Metformin Treatment Attenuates Brain Inflammation and Rescues PACAP/VIP Neuropeptide Alterations in Mice Fed a High-Fat Diet.

High-fat diet (HFD)-induced comorbid cognitive and behavioural impairments are thought to be the result of persistent low-grade neuroinflammation. Metformin, a first-line medication for the treatment of type-2 diabetes, seems to ameliorate these comorbidities, but the underlying mechanism(s) are not clear. Pituitary adenylate cyclase-activating peptide (PACAP) and vasoactive intestinal peptide (VIP) are neuroprotective peptides endowed with anti-inflammatory properties. Alterations to the PACAP/VIP system could be pivotal during the development of HFD-induced neuroinflammation. To unveil the pathogenic mechanisms underlying HFD-induced neuroinflammation and assess metformin's therapeutic activities, (1) we determined if HFD-induced proinflammatory activity was present in vulnerable brain regions associated with the development of comorbid behaviors, (2) investigated if the PACAP/VIP system is altered by HFD, and (3) assessed if metformin rescues such diet-induced neurochemical alterations. C57BL/6J male mice were divided into two groups to receive either standard chow (SC) or HFD for 16 weeks. A further HFD group received metformin (HFD + M) (300 mg/kg BW daily for 5 weeks) via oral gavage. Body weight, fasting glucose, and insulin levels were measured. After 16 weeks, the proinflammatory profile, glial activation markers, and changes within the PI3K/AKT intracellular pathway and the PACAP/VIP system were evaluated by real-time qPCR and/or Western blot in the hypothalamus, hippocampus, prefrontal cortex, and amygdala. Our data showed that HFD causes widespread low-grade neuroinflammation and gliosis, with regional-specific differences across brain regions. HFD also diminished phospho-AKT(Ser473) expression and caused significant disruptions to the PACAP/VIP system. Treatment with metformin attenuated these neuroinflammatory signatures and reversed PI3K/AKT and PACAP/VIP alterations caused by HFD. Altogether, our findings demonstrate that metformin treatment rescues HFD-induced neuroinflammation in vulnerable brain regions, most likely by a mechanism involving the reinstatement of PACAP/VIP system homeostasis. Data also suggests that the PI3K/AKT pathway, at least in part, mediates some of metformin's beneficial effects

Rapid GFAP and Iba1 Expression Changes in the Female Rat Brain following Spinal Cord Injury

Spinal cord injury (SCI) is a devastating condition often associated with sleep disorders, mood change and depression. Evidence suggests that rapid changes to supporting glia may predispose individuals with SCI to such comorbidities. Here, we interrogated the expression of astrocyte- and microglial-specific markers glial fibrillary acidic protein (GFAP) and ionized calcium binding adaptor molecule 1 (Iba1) in the rat brain in the first 24 hours following spinal cord injury (SCI). Female Sprague Dawley rats underwent thoracic laminectomy; half of the rats received a mild contusion injury at the level of the T10 vertebral body (SCI group), the other half did not (Sham group). Twenty-four hours post-surgery the rats were sacrificed, and the amygdala, periaqueductal grey, prefrontal cortex, hypothalamus, lateral thalamus, hippocampus (dorsal and ventral) were collected. GFAP and Iba1 mRNA and protein levels were measured by real-time qPCR and Western blot. In SCI rats, GFAP mRNA and protein expression increased in the amygdala and hypothalamus (*p<0.05). In contrast, gene and protein expression decreased in the thalamus (**p<0.01) and dorsal hippocampus (*p<0.05 and **p<0.01, respectively). Interestingly, Iba1 transcripts and proteins were significantly diminished only in the dorsal (*p<0.05 and **p<0.01, respectively) and ventral hippocampus, where gene expression diminished (*p<0.05 for both mRNA and protein). Considered together, these findings demonstrate that as early as 24 hours post-SCI there are region-specific disruptions of GFAP and Iba1 transcript and protein levels in higher brain regions

PACAP and VIP Modulate LPS-Induced Microglial Activation and Trigger Distinct Phenotypic Changes in Murine BV2 Microglial Cells.

Pituitary adenylate cyclase-activating polypeptide (PACAP) and vasoactive intestinal peptide (VIP) are two structurally related immunosuppressive peptides. However, the underlying mechanisms through which these peptides regulate microglial activity are not fully understood. Using lipopolysaccharide (LPS) to induce an inflammatory challenge, we tested whether PACAP or VIP differentially affected microglial activation, morphology and cell migration. We found that both peptides attenuated LPS-induced expression of the microglial activation markers Iba1 and iNOS (### p IL-1β, IL-6, Itgam and CD68 (### p < 0.001). In contrast, treatment with PACAP or VIP exerted distinct effects on microglial morphology and migration. PACAP reversed LPS-induced soma enlargement and increased the percentage of small-sized, rounded cells (54.09% vs. 12.05% in LPS-treated cells), whereas VIP promoted a phenotypic shift towards cell subpopulations with mid-sized, spindle-shaped somata (48.41% vs. 31.36% in LPS-treated cells). Additionally, PACAP was more efficient than VIP in restoring LPS-induced impairment of cell migration and the expression of urokinase plasminogen activator (uPA) in BV2 cells compared with VIP. These results suggest that whilst both PACAP and VIP exert similar immunosuppressive effects in activated BV2 microglia, each peptide triggers distinctive shifts towards phenotypes of differing morphologies and with differing migration capacities

Robust Dopaminergic Differentiation and Enhanced LPS-Induced Neuroinflammatory Response in Serum-Deprived Human SH-SY5Y Cells: Implication for Parkinson's Disease.

Parkinson's disease (PD) is a chronic neurodegenerative condition characterized by motor symptoms such as bradykinesia, resting tremor, and rigidity. PD diagnosis is based on medical history, review of signs, symptoms, neurological and physical examinations. Unfortunately, by the time the disease is diagnosed, dopamine (DA) neuronal loss is often extended, thereby resulting in ineffective therapies. Recent evidence suggests that neuroinflammation may be pivotal during PD onset and progression. However, suitable cellular models and biomarkers to detect early signs of neuroinflammation are still missing. In this study, we developed a well-differentiated DAergic neuronal cell line where we triggered a neuroinflammatory response to assess the temporal expression of the tissue- and urokinase plasminogen activators (tPA and uPA) and their endogenous inhibitor (PAI-1) along with that of pro-inflammatory mediators and the neuronal marker nNOS. Human neuroblastoma cells SH-SY5Y were differentiated into DAergic neuronal-like cells using a combination of 12-O-tetradecanoylphorbol-13-acetate (TPA) and serum depletion. Terminally-differentiated neurons were then exposed to lipopolysaccharide (LPS) for short (up to 24 h) or long term (up to 10 days) to mimic acute or chronic inflammation. Results demonstrated that uPA protein expression was stably upregulated during chronic inflammation, whereas the expression of nNOS protein better reflected the cellular response to acute inflammation. Additional studies revealed that the temporal induction of uPA was associated with increased AKT phosphorylation, but did not seem to involve cAMP-responsive element-binding protein (CREB) activation, nor the mitogen-activated protein kinase (MAPK) pathway. In conclusion, our in vitro data suggests that nNOS and uPA may serve as viable candidate biomarkers of acute and chronic neuroinflammation

Adeno-associated virus vector gene delivery elevates Factor I levels and down-regulates the complement alternative pathway in vivo.

The complement system is a key component of innate immunity but impaired regulation influences disease susceptibility, including age-related macular degeneration (AMD) and some kidney diseases. Whilst complete complement inhibition has been used successfully to treat acute kidney disease, key unresolved challenges include strategies to modulate rather than completely inhibit the system and to deliver therapy potentially over decades. Elevating concentrations of complement regulator factor I (CFI) restricts complement activation in vitro and this approach was extended in the current study to modulate complement activation in vivo. Sustained increases in CFI levels were achieved using an adeno-associated virus (AAV) vector to target the liver, inducing a 4- to 5-fold increase in circulating CFI levels. This led to decreased activity of the alternative pathway as demonstrated by a reduction in the rate of iC3b deposition and more rapid formation of C3 degradation products. In addition, vector application in a mouse model of systemic lupus erythematosus (NZBWF1), where tissue injury is in part complement dependent, resulted in reduced complement C3 and IgG renal deposition. Collectively, these data demonstrate that sustained elevation of CFI reduces complement activation in vivo providing proof-of-principle support for the therapeutic application of AAV gene delivery to modulate complement activation

Gain-of-function factor H–related 5 protein impairs glomerular complement regulation resulting in kidney damage

Genetic variation within the factor H–related (FHR) genes is associated with the complement-mediated kidney disease, C3 glomerulopathy (C3G). There is no definitive treatment for C3G, and a significant proportion of patients develop end-stage renal disease. The prototypical example is CFHR5 nephropathy, through which an internal duplication within a single CFHR5 gene generates a mutant FHR5 protein (FHR5mut) that leads to accumulation of complement C3 within glomeruli. To elucidate how abnormal FHR proteins cause C3G, we modeled CFHR5 nephropathy in mice. Animals lacking the murine factor H (FH) and FHR proteins, but coexpressing human FH and FHR5mut (hFH-FHR5mut), developed glomerular C3 deposition, whereas mice coexpressing human FH with the normal FHR5 protein (hFH-FHR5) did not. Like in patients, the FHR5mut had a dominant gain-of-function effect, and when administered in hFH-FHR5 mice, it triggered C3 deposition. Importantly, adeno-associated virus vector-delivered homodimeric mini-FH, a molecule with superior surface C3 binding compared to FH, reduced glomerular C3 deposition in the presence of the FHR5mut. Our data demonstrate that FHR5mut causes C3G by disrupting the homeostatic regulation of complement within the kidney and is directly pathogenic in C3G. These results support the use of FH-derived molecules with enhanced C3 binding for treating C3G associated with abnormal FHR proteins. They also suggest that targeting FHR5 represents a way to treat complement-mediated kidney injury