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A disturbed balance between excitation and inhibition (E/I balance) is increasingly recognized as a key driver of neurodegeneration in multiple sclerosis (MS), a chronic inflammatory disease of the central nervous system. To understand how chronic hyperexcitability contributes to neuronal loss in MS, we transcriptionally profiled neurons from mice lacking inhibitory metabotropic glutamate signaling with shifted E/I balance and increased vulnerability to inflammation-induced neurodegeneration. This revealed a prominent induction of the nuclear receptor NR4A2 in neurons. Mechanistically, NR4A2 increased susceptibility to excitotoxicity by stimulating continuous VGF secretion leading to glycolysis-dependent neuronal cell death. Extending these findings to people with MS (pwMS), we observed increased VGF levels in serum and brain biopsies. Notably, neuron-specific deletion of Vgf in a mouse model of MS ameliorated neurodegeneration. These findings underscore the detrimental effect of a persistent metabolic shift driven by excitatory activity as a fundamental mechanism in inflammation-induced neurodegeneration.

The pathogenesis of neuropsychiatric systemic lupus erythematosus (NPSLE) is widely unknown, and the role of autoantibodies is still undetermined. To identify brain-reactive autoantibodies possibly related to NPSLE, immunofluorescence (IF) and transmission electron microscopy (TEM) on rat and human brains were performed. ELISA was used to reveal the presence of known circulating autoantibodies, while western blot (WB) was applied to characterize potential unknown autoantigen(s). We enrolled 209 subjects, including patients affected by SLE (n=69), NPSLE (n=36), Multiple Sclerosis (MS, n=22), and 82 age- and gender-matched healthy donors (HD). Autoantibody reactivity by IF was observed in almost the entire rat brain (cortex, hippocampus, and cerebellum) using sera from NPSLE and SLE patients and was virtually negative in MS and HD. NPSLE showed higher prevalence (OR 2.4; p = 0.047), intensity, and titer of brain-reactive autoantibodies than SLE patients. Most of the patient sera with brain-reactive autoantibodies (75%) also stained human brains. Double staining experiments on rat brains mixing patients' sera with antibodies directed against neuronal (NeuN) or glial markers showed autoantibody reactivity restricted to NeuN-containing neurons. Using TEM, the targets of brain-reactive autoantibodies were located in the nuclei and, to a lesser extent, in the cytoplasm and mitochondria. Given the high degree of colocalization between NeuN and brain-reactive autoantibodies, we assumed NeuN was a possible autoantigen. However, WB analysis with HEK293T cell lysates expressing or not expressing the gene encoding for NeuN protein (RIBFOX3) showed that patients' sera carrying brain-reactive autoantibodies did not recognize the NeuN corresponding band size. Among the panel of NPSLE-associated autoantibodies (e.g., anti-NR2, anti-P-ribosomal protein, antiphospholipid) investigated by ELISA assay, only the anti-β2-glycoprotein-I (aβ2GPI) IgG was exclusively found in those sera containing brain-reactive autoantibodies. In conclusion, SLE and NPSLE patients possess brain-reactive autoantibodies but with higher frequency and titers found in NPSLE patients. Although many target antigens of brain-reactive autoantibodies are still undetermined, they likely include β2GPI.

This review summarized different studies reporting the presence of autoantibodies reacting against cells of the pituitary (APAs) and/or hypothalamus (AHAs). Both APAs and AHAs have been revealed through immunofluorescence using different kinds of substrates. Autoantibodies against gonadotropic cells were mainly found in patients affected by cryptorchidism and hypogonadotropic hypogonadism while those against prolactin cells were found in different kinds of patients, the majority without pituitary abnormalities. APAs to growth hormone (GH) cells have been associated with GH deficiency while those against the adrenocorticotropic cells have distinguished central Cushing’s disease patients at risk of incomplete cure after surgical adenoma removal. AHAs to vasopressin cells have identified patients at risk of developing diabetes insipidus. APAs have been also found together with AHAs in patients affected by idiopathic hypopituitarism, but both were also present in different kinds of patients without abnormalities of the hypothalamic–pituitary axis. Despite some data being promising, the clinical use of pituitary and hypothalamus autoantibodies is still limited by the low diagnostic sensitivity, irreproducibility of the results, and the absence of autoantigen/s able to discriminate the autoimmune reaction involving the pituitary or the hypothalamus from the other autoimmune states.

Background Diagnosis of dementia with Lewy bodies (DLB) is challenging, largely due to a lack of diagnostic tools. Cerebrospinal fluid (CSF) biomarkers have been proven useful in Alzheimer’s disease (AD) diagnosis. Here, we aimed to identify novel CSF biomarkers for DLB using a high-throughput proteomic approach. Methods We applied liquid chromatography/tandem mass spectrometry with label-free quantification to identify biomarker candidates to individual CSF samples from a well-characterized cohort comprising patients with DLB ( n  = 20) and controls ( n  = 20). Validation was performed using (1) the identical proteomic workflow in an independent cohort ( n  = 30), (2) proteomic data from patients with related neurodegenerative diseases ( n  = 149) and (3) orthogonal techniques in an extended cohort consisting of DLB patients and controls ( n  = 76). Additionally, we utilized random forest analysis to identify the subset of candidate markers that best distinguished DLB from all other groups. Results In total, we identified 1995 proteins. In the discovery cohort, 69 proteins were differentially expressed in DLB compared to controls ( p  < 0.05). Independent cohort replication confirmed VGF, SCG2, NPTX2, NPTXR, PDYN and PCSK1N as candidate biomarkers for DLB. The downregulation of the candidate biomarkers was somewhat more pronounced in DLB in comparison with related neurodegenerative diseases. Using random forest analysis, we identified a panel of VGF, SCG2 and PDYN to best differentiate between DLB and other clinical groups (accuracy: 0.82 (95%CI: 0.75–0.89)). Moreover, we confirmed the decrease of VGF and NPTX2 in DLB by ELISA and SRM methods. Low CSF levels of all biomarker candidates, except PCSK1N, were associated with more pronounced cognitive decline (0.37 < r < 0.56, all p  < 0.01). Conclusion We identified and validated six novel CSF biomarkers for DLB. These biomarkers, particularly when used as a panel, show promise to improve diagnostic accuracy and strengthen the importance of synaptic dysfunction in the pathophysiology of DLB.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by a progressive degeneration in the neurons of the frontal cortex, spinal cord, and brainstem, altering the correct release of neurotransmitters. The disease affects every muscle in the body and could cause death three to five years after symptoms first occur. There is currently no efficient treatment to stop the disease’s progression. The lack of identification of potential therapeutic strategies is a consequence of the delayed diagnosis due to the absence of accurate ALS early biomarkers. Indeed, neurotransmitters altered in ALS are not measurable in body fluids at quantities that allow for testing, making their use as diagnostic tools a challenge. Contrarily, neuroproteins and neuropeptides are chemical messengers produced and released by neurons, and most of them have the potential to enter bodily fluids. To find out new possible ALS biomarkers, the research of neuropeptides and proteins is intensified using mass spectrometry and biochemical-based assays. Neuropeptides derived from the proVGF precursor protein act as signaling molecules within neurons. ProVGF and its derived peptides are expressed in the nervous and endocrine systems but are also widely distributed in body fluids such as blood, urine, and cerebrospinal fluid, making them viable options as disease biomarkers. To highlight the proVGF and its derived peptides’ major roles as ALS diagnostic biomarkers, this review provides an overview of the VGF peptide alterations in spinal cord and body fluids and outlines the limitations of the reported investigations.

TLQP-21, a peptide encoded by the highly conserved vgf gene, is expressed in neuroendocrine cells and has been the most prominent VGF-derived peptide studied in relation to control of energy balance. The recent discovery that TLQP-21 is the natural agonist for the complement 3a receptor 1 (C3aR1) has revived interest in this peptide as a potential drug target for obesity. We have investigated its function in Siberian hamsters (Phodopus sungorus), a rodent that displays natural seasonal changes in body weight and adiposity as an adaptation to survive winter. We have previously shown that intracerebroventricular administration of TLQP-21 reduced food intake and body weight in hamsters in their long-day fat state. The aim of our current study was to determine the systemic actions of TLQP-21 on food intake, energy expenditure and body weight, and to establish whether adiposity affected these responses. Peripheral infusion of TLQP-21 (1mg/kg/day for 7 days) in lean hamsters exposed to short photoperiods (SP) reduced cumulative food intake in the home cage (p<0.05), and intake when measured in metabolic cages (P<0.01). Energy expenditure was significantly increased (p<0.001) by TLQP-21 infusion, this was associated with a significant increase in uncoupling protein 1 mRNA in brown adipose tissue (BAT) (p<0.05), and body weight was significantly reduced (p<0.05). These effects of systemic TLQP-21 treatment were not observed in hamsters exposed to long photoperiod (LP) with a fat phenotype. C3aR1 mRNA and protein were abundantly expressed in the hypothalamus, brown and white adipose tissue in hamsters, but changes in expression cannot explain the differential response to TLQP-21 in lean and fat hamsters.

VGF mRNA is widely expressed in areas of the nervous system known to degenerate in Amyotrophic Lateral Sclerosis (ALS), including cerebral cortex, brainstem and spinal cord. Despite certain VGF alterations are reported in animal models, little information is available with respect to the ALS patients. We addressed VGF peptide changes in fibroblast cell cultures and in plasma obtained from ALS patients, in parallel with spinal cord and plasma samples from the G93A-SOD1 mouse model. Antisera specific for the C-terminal end of the human and mouse VGF proteins, respectively, were used in immunohistochemistry and enzyme-linked immunosorbent assay (ELISA), while gel chromatography and HPLC/ESI-MS/MS were used to identify the VGF peptides present. Immunoreactive VGF C-terminus peptides were reduced in both fibroblast and plasma samples from ALS patients in an advanced stage of the disease. In the G93A-SOD1 mice, the same VGF peptides were also decreased in plasma in the late-symptomatic stage, while showing an earlier down-regulation in the spinal cord. In immunohistochemistry, a large number of gray matter structures were VGF C-terminus immunoreactive in control mice (including nerve terminals, axons and a few perikarya identified as motoneurons), with a striking reduction already in the pre-symptomatic stage. Through gel chromatography and spectrometry analysis, we identified one form likely to be the VGF precursor as well as peptides containing the NAPP- sequence in all tissues studied, while in the mice and fibroblasts, we revealed also AQEE- and TLQP- peptides. Taken together, selective VGF fragment depletion may participate in disease onset and/or progression of ALS.

To address the possible involvement of VGF peptides in obesity and diabetes, we studied type 2 diabetes (T2D) and obese patients, and high-fat diet induced obese mice. Two VGF peptides (NAPP-19 and QQET-30) were identified in human plasma by HPLC-ESI-MS. The VGF C-terminus, the above two cleaved peptides, and the TLQP-21 related peptide/s were studied using ELISA and immunohistochemistry. In euglycemic patients, plasma NAPPE and TLQP like peptides were significantly reduced with obesity (74±10 vs. 167±28, and 92±10 vs. 191±19 pmol/ml, mean+SEM, n = 10 and 6, obese vs. normal BMI, respectively, p<0.03). Upon a standard glucose load, a distinct response was shown for VGF C-terminus, TLQP and QQET-like (ERVW immunoreactive) peptides in euglycemic normal BMI patients, but was virtually abolished in euglycemic obese, and in T2D patients independently of BMI. High-fat diet induced obese mice showed reduced plasma VGF C-terminus, NAPPE and QQET-like (ERVW) peptide/s (3±0.2 vs. 4.6±0.3, 22±3.5 vs. 34±1.3, and 48±7 vs. 100±7 pmol/ml, mean+SEM, n = 8/group, obese vs. slim, respectively, p<0.03), with a loss of the response to glucose for all VGF peptides studied. In immunohistochemistry, TLQP and/or VGF C-terminus antibodies labelled VGF containing perikarya in mouse celiac ganglia, pancreatic islet cells and thin beaded nerve fibres in brown adipose tissues, with fewer in white adipose tissue. Upon the glucose load, tyrosine hydroxylase and VGF C-terminus immunoreactive axons became apparent in pancreatic islets of slim animals, but not in obese animals. Alltogether, a significant loss of VGF peptide immunoreactivity and/or their response to glucose was demonstrated in obese patients, with or without T2D, in parallel with a similar loss in high-fat diet induced obese mice. An involvement of VGF in metabolic regulations, including those of brown and/or white adipose tissues is underlined, and may point out specific VGF peptides as potential targets for diagnosis and/or treatment.

VGF (non-acronymic) was first highlighted to have a role in energy homeostasis through experiments involving dietary manipulation in mice. Fasting increased VGF mRNA in the Arc and levels were subsequently reduced upon refeeding. This anabolic role for VGF was supported by observations in a VGF null (VGF-/-) mouse and in the diet-induced and gold-thioglucose obese mice. However, this anabolic role for VGF has not been supported by a number of subsequent studies investigating the physiological effects of VGF-derived peptides. Intracerebroventricular (ICV) infusion of TLQP-21 increased resting energy expenditure and rectal temperature in mice and protected against diet-induced obesity. Similarly, ICV infusion of TLQP-21 into Siberian hamsters significantly reduced body weight, but this was due to a decrease in food intake, with no effect on energy expenditure. Subsequently NERP-2 was shown to increase food intake in rats via the orexin system, suggesting opposing roles for these VGF-derived peptides. Thus to further elucidate the role of hypothalamic VGF in the regulation of energy homeostasis we utilised a recombinant adeno-associated viral vector to over-express VGF in adult male Siberian hamsters, thus avoiding any developmental effects or associated functional compensation. Initially, hypothalamic over-expression of VGF in adult Siberian hamsters produced no effect on metabolic parameters, but by 12 weeks post-infusion hamsters had increased oxygen consumption and a tendency to increased carbon dioxide production; this attenuated body weight gain, reduced interscapular white adipose tissue and resulted in a compensatory increase in food intake. These observed changes in energy expenditure and food intake were associated with an increase in the hypothalamic contents of the VGF-derived peptides AQEE, TLQP and NERP-2. The complex phenotype of the VGF-/- mice is a likely consequence of global ablation of the gene and its derived peptides during development, as well as in the adult.

Background: Dementia with Lewy bodies (DLB) is a neurodegenerative disease where synaptic loss and reduced synaptic integrity are important neuropathological substrates. Neuronal Pentraxin 2(NPTX2) is a synaptic protein that drives the GABAergic inhibitory circuit. Our aim was to examine if NPTX2 cerebral spinal fluid (CSF) levels in DLB patients were altered and how these levels related to other synaptic protein levels and to cognitive function and decline. Methods: NPTX2, VGF, and α-synuclein levels were determined in CSF of cognitive healthy (n = 27), DLB (n = 48), and AD (n = 20) subjects. Multiple cognitive domains were tested, and data were compared using linear models. Results: Decreased NPTX2 levels were observed in DLB (median = 474) and AD (median = 453) compared to cognitive healthy subjects (median = 773). Strong correlations between NPTX2, VGF, and α-synuclein were observed dependent on diagnosis. Combined, these markers had a high differentiating power between DLB and cognitive healthy subjects (AUC = 0.944). Clinically, NPTX2 levels related to global cognitive function and cognitive decline in the visual spatial domain. Conclusion: NPTX2 CSF levels were reduced in DLB and closely correlated to decreased VGF and α-synuclein CSF levels. CSF NPTX2 levels in DLB related to decreased functioning in the visual spatial domain.