HDAC1/2/3-mediated downregulation of neurogranin is involved in cognitive impairment in offspring exposed to maternal subclinical hypothyroidism.

Subclinical hypothyroidism (SCH) in pregnancy is the most common form of thyroid dysfunction in pregnancy, which can affect fetal nervous system development and increase the risk of neurodevelopmental disorders after birth. However, the mechanism of the effect of maternal subclinical hypothyroidism on fetal brain development and behavioral phenotypes is still unclear and requires further study. In this study, we constructed a mouse model of maternal subclinical hypothyroidism by exposing dams to drinking water containing 50 ppm propylthiouracil (PTU) during pregnancy and found that its offspring were accompanied by severe cognitive deficits by behavioral testing. Mechanistically, gestational SCH resulted in the upregulation of protein expression and activity of HDAC1/2/3 in the hippocampus of the offspring. ChIP analysis revealed that H3K9ac on the neurogranin (Ng) promoter was reduced in the hippocampus of the offspring of SCH, with a significant reduction in Ng protein, leading to reduced expression levels of synaptic plasticity markers PSD95 (a membrane-associated protein in the postsynaptic density) and SYN (synaptophysin, a specific marker for presynaptic terminals), and impaired synaptic plasticity. In addition, administration of MS-275 (an HDAC1/2/3-specific inhibitor) to SCH offspring alleviated impaired synaptic plasticity and cognitive dysfunction in offspring. Thus, our study suggests that maternal subclinical hypothyroidism may mediate offspring cognitive dysfunction through the HDAC1/2/3-H3K9ac-Ng pathway. Our study contributes to the understanding of the signaling mechanisms underlying maternal subclinical hypothyroidism-mediated cognitive impairment in the offspring.

Manganese Overexposure Alters Neurogranin Expression and Causes Behavioral Deficits in Larval Zebrafish.

Manganese (Mn), a cofactor for various enzyme classes, is an essential trace metal for all organisms. However, overexposure to Mn causes neurotoxicity. Here, we evaluated the effects of exposure to Mn chloride (MnCl2) on viability, morphology, synapse function (based on neurogranin expression) and behavior of zebrafish larvae. MnCl2 exposure from 2.5 h post fertilization led to reduced survival (60%) at 5 days post fertilization. Phenotypical changes affected body length, eye and olfactory organ size, and visual background adaptation. This was accompanied by a decrease in both the fluorescence intensity of neurogranin immunostaining and expression levels of the neurogranin-encoding genes nrgna and nrgnb, suggesting the presence of synaptic alterations. Furthermore, overexposure to MnCl2 resulted in larvae exhibiting postural defects, reduction in motor activity and impaired preference for light environments. Following the removal of MnCl2 from the fish water, zebrafish larvae recovered their pigmentation pattern and normalized their locomotor behavior, indicating that some aspects of Mn neurotoxicity are reversible. In summary, our results demonstrate that Mn overexposure leads to pronounced morphological alterations, changes in neurogranin expression and behavioral impairments in zebrafish larvae.

Evaluation of neurogranin levels in a rat model of diffuse axonal injury.

Diffuse axonal injury (DAI), one of the most common and devastating type of traumatic brain injury, is the result of the shear force on axons due to severe rotational acceleration and deceleration. Neurogranin (NRGN) is a postsynaptic protein secreted by excitatory neurons, and synaptic dysfunction can alter extracellular NRGN levels. In this study, we examined NRGN levels in serum and cerebrospinal fluid (CSF) after experimental DAI in terms of their diagnostic value. Experimental DAI was induced using the Marmarou technique in male Wistar albino rats. Serum and CSF NRGN levels of the sham group, one­hour, six­hour, 24­hour, and 72­hour post­DAI groups were measured by ELISA method. DAI was verified by staining with hematoxylin­eosin and ß­amyloid precursor protein in the rat brain samples. While no histopathological and immunohistochemical changes were observed in the early hours of the post­DAI groups, the staining of the ß­APP visibly increased over time, with positivity being most frequent and intense in the 72­hour group. It was found that serum NRGN levels were significantly lower in the 6­hour group than in the sham group. The serum NRGN levels in the 24­hour group were significantly higher than those in the sham group. This study showed a dichotomy of post­DAI serum NRGN levels in consecutive time periods. NRGN levels in CSF were higher in the one­hour group than in the sham group and returned to baseline by 72 hours, although not significantly. Our study provides an impression of serum and CSF NRGN levels in a rat DAI model in consecutive time periods. Further studies are needed to understand the diagnostic value of NRGN.

Neuroinflammation driven by human immunodeficiency virus-1 (HIV-1) directs the expression of long noncoding RNA RP11-677M14.2 resulting in dysregulation of neurogranin in vivo and in vitro.

Neuroinflammation and synaptodendritic damage represent the pathological hallmarks of HIV-1 associated cognitive disorders (HAND). The post-synaptic protein neurogranin (Nrgn) is significantly reduced in the frontal cortex of postmortem brains from people with HIV (PWH) and it is associated with inflammatory factors released by infected microglia/macrophages. However, the mechanism involved in synaptic loss have yet to be elucidated. In this study, we characterized a newly identified long non-coding RNA (lncRNA) transcript (RP11-677M14.2), which is antisense to the NRGN locus and is highly expressed in the frontal cortex of HIV-1 individuals. Further analysis indicates an inverse correlation between the expression of RP11-677M14.2 RNA and Nrgn mRNA. Additionally, the Nrgn-lncRNA axis is dysregulated in neurons exposed to HIV-1 infected microglia conditioned medium enriched with IL-1ß. Moreover, in vitro overexpression of this lncRNA impacts Nrgn expression at both mRNA and protein levels. Finally, we modeled the Nrgn-lncRNA dysregulation within an HIV-1-induced inflammatory environment using brain organoids, thereby corroborating our in vivo and in vitro findings. Together, our study implicates a plausible role for lncRNA RP11-677M14.2 in modulating Nrgn expression that might serve as the mechanistic link between Nrgn loss and cognitive dysfunction in HAND, thus shedding new light on the mechanisms underlying synaptodendritic damage.

Delivering synaptic protein mRNAs via extracellular vesicles ameliorates cognitive impairment in a mouse model of Alzheimer's disease.

BACKGROUND: Synaptic dysfunction with reduced synaptic protein levels is a core feature of Alzheimer's disease (AD). Synaptic proteins play a central role in memory processing, learning, and AD pathogenesis. Evidence suggests that synaptic proteins in plasma neuronal-derived extracellular vesicles (EVs) are reduced in patients with AD. However, it remains unclear whether levels of synaptic proteins in EVs are associated with hippocampal atrophy of AD and whether upregulating the expression of these synaptic proteins has a beneficial effect on AD. METHODS: In this study, we included 57 patients with AD and 56 healthy controls. We evaluated their brain atrophy through magnetic resonance imaging using the medial temporal lobe atrophy score. We measured the levels of four synaptic proteins, including synaptosome-associated protein 25 (SNAP25), growth-associated protein 43 (GAP43), neurogranin, and synaptotagmin 1 in both plasma neuronal-derived EVs and cerebrospinal fluid (CSF). We further examined the association of synaptic protein levels with brain atrophy. We also evaluated the levels of these synaptic proteins in the brains of 5×FAD mice. Then, we loaded rabies virus glycoprotein-engineered EVs with messenger RNAs (mRNAs) encoding GAP43 and SNAP25 and administered these EVs to 5×FAD mice. After treatment, synaptic proteins, dendritic density, and cognitive function were evaluated. RESULTS: The results showed that GAP43, SNAP25, neurogranin, and synaptotagmin 1 were decreased in neuronal-derived EVs but increased in CSF in patients with AD, and the changes corresponded to the severity of brain atrophy. GAP43 and SNAP25 were decreased in the brains of 5×FAD mice. The engineered EVs efficiently and stably delivered these synaptic proteins to the brain, where synaptic protein levels were markedly upregulated. Upregulation of synaptic protein expression could ameliorate cognitive impairment in AD by promoting dendritic density. This marks the first successful delivery of synaptic protein mRNAs via EVs in AD mice, yielding remarkable therapeutic effects. CONCLUSIONS: Synaptic proteins are closely related to AD processes. Delivery of synaptic protein mRNAs via EVs stands as a promising effective precision treatment strategy for AD, which significantly advances the current understanding of therapeutic approaches for the disease.

Neurogranin expression regulates mitochondrial function and redox balance in endothelial cells.

Endothelial dysfunction and endothelial activation are common early events in vascular diseases and can arise from mitochondrial dysfunction. Neurogranin (Ng) is a 17kD protein well known to regulate intracellular Ca2+-calmodulin (CaM) complex signaling, and its dysfunction is significantly implicated in brain aging and neurodegenerative diseases. We found that Ng is also expressed in human aortic endothelial cells (HAECs), and depleting Ng promotes Ca2+-CaM complex-dependent endothelial activation and redox imbalances. Endothelial-specific Ng knockout (Cre-CDH5-Ngf/f) mice demonstrate a significant delay in the flow-mediated dilation (FMD) response. Therefore, it is critical to characterize how endothelial Ng expression regulates reactive oxygen species (ROS) generation and affects cardiovascular disease. Label-free quantification proteomics identified that mitochondrial dysfunction and the oxidative phosphorylation pathway are significantly changed in the aorta of Cre-CDH5-Ngf/f mice. We found that a significant amount of Ng is expressed in the mitochondrial fraction of HAECs using western blotting and colocalized with the mitochondrial marker, COX IV, using immunofluorescence staining. Seahorse assay demonstrated that a lack of Ng decreases mitochondrial respiration. Treatment with MitoEbselen significantly restores the oxygen consumption rate in Ng knockdown cells. With the RoGFP-Orp1 approach, we identified that Ng knockdown increases mitochondrial-specific hydrogen peroxide (H2O2) production, and MitoEbselen treatment significantly reduced mitochondrial ROS (mtROS) levels in Ng knockdown cells. These results suggest that Ng plays a significant role in mtROS production. We discovered that MitoEbselen treatment also rescues decreased eNOS expression and nitric oxide (NO) levels in Ng knockdown cells, which implicates the critical role of Ng in mtROS-NO balance in the endothelial cells.

Monitoring synaptic pathology in Alzheimer's disease through fluid and PET imaging biomarkers: a comprehensive review and future perspectives.

Alzheimer's disease (AD) is currently constrained by limited clinical treatment options. The initial pathophysiological event, which can be traced back to decades before the clinical symptoms become apparent, involves the excessive accumulation of amyloid-beta (Aß), a peptide comprised of 40-42 amino acids, in extraneuronal plaques within the brain. Biochemical and histological studies have shown that overaccumulation of Aß instigates an aberrant escalation in the phosphorylation and secretion of tau, a microtubule-binding axonal protein. The accumulation of hyperphosphorylated tau into intraneuronal neurofibrillary tangles is in turn correlated with microglial dysfunction and reactive astrocytosis, culminating in synaptic dysfunction and neurodegeneration. As neurodegeneration progresses, it gives rise to mild clinical symptoms of AD, which may eventually evolve into overt dementia. Synaptic loss in AD may develop even before tau alteration and in response to possible elevations in soluble oligomeric forms of Aß associated with early AD. These findings largely rely on post-mortem autopsy examinations, which typically involve a limited number of patients. Over the past decade, a range of fluid biomarkers such as neurogranin, α-synuclein, visinin-like protein 1 (VILIP-1), neuronal pentraxin 2, and ß-synuclein, along with positron emission tomography (PET) markers like synaptic vesicle glycoprotein 2A, have been developed. These advancements have facilitated the exploration of how synaptic markers in AD patients correlate with cognitive impairment. However, fluid biomarkers indicating synaptic loss have only been validated in cerebrospinal fluid (CSF), not in plasma, with the exception of VILIP-1. The most promising PET radiotracer, [11C]UCB-J, currently faces significant challenges hindering its widespread clinical use, primarily due to the necessity of a cyclotron. As such, additional research geared toward the exploration of synaptic pathology biomarkers is crucial. This will not only enable their extensive clinical application, but also refine the optimization process of AD pharmacological trials.

Cellular Iron Deficiency Disrupts Thyroid Hormone Regulated Gene Expression in Developing Hippocampal Neurons.

BACKGROUND: Developing neurons have high thyroid hormone and iron requirements to support their metabolically demanding growth. Early-life iron and thyroid-hormone deficiencies are prevalent and often coexist, and each independently increases risk of permanently impaired neurobehavioral function in children. Early-life dietary iron deficiency reduces thyroid-hormone concentrations and impairs thyroid hormone-responsive gene expression in the neonatal rat brain, but it is unclear whether the effect is cell-intrinsic. OBJECTIVES: This study determined whether neuronal-specific iron deficiency alters thyroid hormone-regulated gene expression in developing neurons. METHODS: Iron deficiency was induced in primary mouse embryonic hippocampal neuron cultures with the iron chelator deferoxamine (DFO) beginning at 3 d in vitro (DIV). At 11DIV and 18DIV, thyroid hormone-regulated gene messenger ribonucleic acid (mRNA)concentrations indexing thyroid hormone homeostasis (Hairless, mu-crystallin, Type II deiodinase, solute carrier family member 1c1, and solute carrier family member 16a2) and neurodevelopment (neurogranin, Parvalbumin, and Krüppel-like factor 9) were quantified. To assess the effect of iron repletion, DFO was removed at 14DIV from a subset of DFO-treated cultures, and gene expression and adenosine 5'-triphosphate (ATP) concentrations were quantified at 21DIV. RESULTS: At 11DIV and 18DIV, neuronal iron deficiency decreased neurogranin, Parvalbumin, and mu-crystallin, and by 18DIV, solute carrier family member 16a2, solute carrier family member 1c1, Type II deiodinase, and Hairless were increased, suggesting cellular sensing of a functionally abnormal thyroid hormone state. Dimensionality reduction with Principal component analysis reveals that thyroid hormone homeostatic genes strongly correlate with and predict iron status. Iron repletion from 14-21DIV did not restore ATP concentration, and Principal component analysis suggests that, after iron repletion, cultures maintain a gene expression signature indicative of previous iron deficiency. CONCLUSIONS: These novel findings suggest there is an intracellular mechanism coordinating cellular iron/thyroid hormone activities. We speculate this is a part of the homeostatic response to acutely match neuronal energy production and growth signaling. However, the adaptation to iron deficiency may cause permanent deficits in thyroid hormone-dependent neurodevelopmental processes even after recovery from iron deficiency.

Suppression of neurogranin expression by disruption of epigenetic DNA methylation in hippocampal mature granule cells after developmental exposure to neurotoxicants in rats.

We previously performed comprehensive analyses of genes hypermethylated promoter regions and downregulated transcripts in the hippocampal dentate gyrus (DG) of rats upon weaning at postnatal day (PND) 21 after developmental exposure to 6-propyl-2-thiouracil (PTU), valproic acid, and glycidol (GLY), all of which are known to show irreversible effects on hippocampal neurogenesis in adulthood on PND 77. Here, we selected neurotransmitter and neurogenesis-related genes for validation analysis of methylation and expression. As a result, Nrgn by GLY and Shisa7, Agtpbp1, and Cyp46a1 by PTU underwent DNA hypermethylation and sustained downregulation. Immunohistochemical analysis of candidate gene products revealed that the number of neurogranin (NRGN)+ granule cells was decreased in the ventral DG by GLY on PND 21 and 77 and by PTU on PND 21. Among the samples of developmental or 28-day young adult-age exposure to known developmental neurotoxicants in humans, i.e., lead acetate, ethanol, and aluminum chloride, a decrease of NRGN+ cells by ethanol was also observed on PND 77 after developmental exposure. Double immunohistochemistry analysis revealed that NRGN was expressed in mature granule cells, and a similar immunoreactive cell distribution was found for phosphorylated calcium/calmodulin-activated protein kinase, a NRGN downstream molecule. After developmental PTU exposure, the number of activity-regulated cytoskeleton-associated protein+ granule cells was also profoundly decreased in the ventral DG in parallel with the decrease in NRGN+ cells on PND 21. These results suggest that NRGN is a potential marker for suppression of synaptic plasticity in mature granule cells in the ventral DG.

Association between ventricular CSF biomarkers and outcome after shunt surgery in idiopathic normal pressure hydrocephalus.

INTRODUCTION: The relationship between neurochemical changes and outcome after shunt surgery in idiopathic normal pressure hydrocephalus (iNPH), a treatable dementia and gait disorder, is unclear. We used baseline ventricular CSF to explore associations to outcome, after shunting, of biomarkers selected to reflect a range of pathophysiological processes. METHODS: In 119 consecutive patients with iNPH, the iNPH scale was used before and after shunt surgery to quantify outcome. Ventricular CSF was collected perioperatively and analyzed for biomarkers of astrogliosis, axonal, amyloid and tau pathology, and synaptic dysfunction: glial fibrillary acidic protein (GFAP), chitinase-3-like protein 1 (YKL40/CHI3L1), monocyte chemoattractant protein-1 (MCP-1) neurofilament light (NfL), amyloid beta 38 (Aß38), Aß40, Aß42, amyloid beta 42/40 ratio (Aß42/40), soluble amyloid precursor protein alfa (sAPPα), sAPPß, total tau (T-tau), phosphorylated tau (P-tau), growth-associated protein 43 (GAP43), and neurogranin. RESULTS: The neurogranin concentration was higher in improved (68%) compared to unimproved patients (median 365 ng/L (IQR 186-544) vs 330 (205-456); p = 0.046). A linear regression model controlled for age, sex and vascular risk factors including neurogranin, T-tau, and GFAP, resulted in adjusted R2 = 0.06, p = 0.047. The Aß42/40 ratio was bimodally distributed across all samples, as well as in the subgroups of improved and unimproved patients but did not contribute to outcome prediction. The preoperative MMSE score was lower within the low Aß ratio group (median 25, IQR 23-28) compared to the high subgroup (26, 24-29) (p = 0.028). The T-Tau x Aß40/42 ratio and P-tau x Aß40/42 ratio did not contribute to shunt response prediction. The prevalence of vascular risk factors did not affect shunt response. DISCUSSION: A higher preoperative ventricular CSF level of neurogranin, which is a postsynaptic marker, may signal a favorable postoperative outcome. Concentrations of a panel of ventricular CSF biomarkers explained only 6% of the variability in outcome. Evidence of amyloid or tau pathology did not affect the outcome.

Evaluation of cerebrospinal fluid levels of synaptic vesicle protein, VAMP-2, across the sporadic Alzheimer's disease continuum.

BACKGROUND: Synapse loss is an early event that precedes neuronal death and symptom onset and is considered the best neuropathological correlate of cognitive decline in Alzheimer's disease (AD). Vesicle-associated membrane protein 2 (VAMP-2) has emerged as a promising biomarker of AD-related synapse degeneration in cerebrospinal fluid (CSF). The aim of this study was to explore the CSF profile of VAMP-2 across the AD continuum in relation to core AD biomarkers, other synaptic proteins, neurogranin (Ng) and synaptosomal-associated Protein-25 kDa (SNAP-25) and cognitive performance. METHODS: We developed a digital immunoassay on the Single Molecule Array platform to quantify VAMP-2 in CSF and used existing immunoassays to quantify Ng, SNAP-25 and core CSF AD biomarkers. The clinical study included 62 cognitively unimpaired AD biomarker-negative subjects and 152 participants across the AD continuum from the SPIN cohort (Sant Pau Initiative on Neurodegeneration). Cognitive measures of episodic, semantic, executive and visuospatial domains and global cognition were included. Statistical methods included χ2 tests, spearman correlation, and ANCOVA analyses. RESULTS: The VAMP-2 assay had a good analytical performance (repeatability 8.9%, intermediate precision 10.3%). Assay antibodies detected native VAMP-2 protein in human brain homogenates. CSF concentrations of VAMP-2, neurogranin and SNAP-25 were lower in preclinical AD stage 1 compared to controls and higher at later AD stages compared to AD stage 1 and were associated with core AD biomarkers, particularly total tau (adj. r2 = 0.62 to 0.78, p < 0.001). All three synaptic proteins were associated with all cognitive domains in individuals on the AD continuum (adj. r2 = 0.04 to 0.19, p < 0.05). CONCLUSIONS: Our novel digital immunoassay accurately measures VAMP-2 changes in CSF, which reflect AD biomarkers and cognitive performance across multiple domains.

Diagnostic and prognostic value of cerebrospinal fluid SNAP-25 and neurogranin in Creutzfeldt-Jakob disease in a clinical setting cohort of rapidly progressive dementias.

BACKGROUND: The levels of synaptic markers synaptosomal-associated protein 25 (SNAP-25) and neurogranin (Ng) have been shown to increase early in the cerebrospinal fluid (CSF) of patients with Creutzfeldt-Jakob disease (CJD) and to have prognostic potential. However, no validation studies assessed these biomarkers' diagnostic and prognostic value in a large clinical setting cohort of rapidly progressive dementia. METHODS: In this retrospective study, using commercially available immunoassays, we measured the levels of SNAP-25, Ng, 14-3-3, total-tau (t-tau), neurofilament light chain (NfL), and phospho-tau181 (p-tau) in CSF samples from consecutive patients with CJD (n = 220) or non-prion rapidly progressive dementia (np-RPD) (n = 213). We evaluated and compared the diagnostic accuracy of each CSF biomarker and biomarker combination by receiver operating characteristics curve (ROC) analyses, studied SNAP-25 and Ng CSF concentrations distribution across CJD subtypes, and estimated their association with survival using multivariable Cox regression analyses. RESULTS: CSF SNAP-25 and Ng levels were higher in CJD than in np-RPD (SNAP-25: 582, 95% CI 240-1250 vs. 115, 95% CI 78-157 pg/ml, p < 0.0001; Ng: 841, 95% CI 411-1473 vs. 390, 95% CI 260-766 pg/ml, p < 0.001). SNAP-25 diagnostic accuracy (AUC 0.902, 95% CI 0.873-0.931) exceeded that of 14-3-3 (AUC 0.853, 95% CI 0.816-0.889), t-tau (AUC 0.878, 95% CI 0.845-0.901), and the t-tau/p-tau ratio (AUC 0.884, 95% CI 0.851-0.916). In contrast, Ng performed worse (AUC 0.697, 95% CI 0.626-0.767) than all other surrogate biomarkers, except for NfL (AUC 0.649, 95% CI 0.593-0.705). SNAP-25 maintained a relatively high diagnostic value even for atypical CJD subtypes (AUC 0.792, 95% CI 0.729-0.854). In Cox regression analyses, SNAP-25 levels were significantly associated with survival in CJD (hazard ratio [HR] 1.71 95% CI 1.40-2.09). Conversely, Ng was associated with survival only in the most rapidly progressive CJD subtypes (sCJD MM(V)1 and gCJD M1) (HR 1.81 95% CI 1.21-2.93). CONCLUSIONS: In the clinical setting, CSF SNAP-25 is a viable alternative to t-tau, 14-3-3, and the t-tau/p-tau ratio in discriminating the CJD subtypes from other RPDs. Additionally, SNAP-25 and, to a lesser extent, Ng predict survival in CJD, showing prognostic power in the range of CSF t-tau/14-3-3 and NfL, respectively.

The Relationships between Cerebrospinal Fluid Glial (CXCL12, CX3CL, YKL-40) and Synaptic Biomarkers (Ng, NPTXR) in Early Alzheimer's Disease.

In addition to amyloid and tau pathology in the central nervous system (CNS), inflammatory processes and synaptic dysfunction are highly important mechanisms involved in the development and progression of dementia diseases. In the present study, we conducted a comparative analysis of selected pro-inflammatory proteins in the CNS with proteins reflecting synaptic damage and core biomarkers in mild cognitive impairment (MCI) and early Alzheimer's disease (AD). To our knowledge, no studies have yet compared CXCL12 and CX3CL1 with markers of synaptic disturbance in cerebrospinal fluid (CSF) in the early stages of dementia. The quantitative assessment of selected proteins in the CSF of patients with MCI, AD, and non-demented controls (CTRL) was performed using immunoassays (single- and multiplex techniques). In this study, increased CSF concentration of CX3CL1 in MCI and AD patients correlated positively with neurogranin (r = 0.74; p < 0.001, and r = 0.40; p = 0.020, respectively), ptau181 (r = 0.49; p = 0.040), and YKL-40 (r = 0.47; p = 0.050) in MCI subjects. In addition, elevated CSF levels of CXCL12 in the AD group were significantly associated with mini-mental state examination score (r = -0.32; p = 0.040). We found significant evidence to support an association between CX3CL1 and neurogranin, already in the early stages of cognitive decline. Furthermore, our findings indicate that CXCL12 might be a useful marker for tract severity of cognitive impairment.

Serum and cerebrospinal fluid brain damage markers neurofilament light and glial fibrillary acidic protein correlate with tick-borne encephalitis disease severity-a multicentre study on Lithuanian and Swedish patients.

BACKGROUND AND PURPOSE: Our aim was to examine the correlation between biomarkers of neuronal and glial cell damage and severity of disease in patients with tick-borne encephalitis. METHODS: One hundred and fifteen patients with tick-borne encephalitis diagnosed in Lithuania and Sweden were prospectively included, and cerebrospinal fluid (CSF) and serum samples were obtained shortly after hospitalization. Using pre-defined criteria, cases were classified as mild, moderate or severe tick-borne encephalitis. Additionally, the presence of spinal nerve paralysis (myelitis) and/or cranial nerve affection were noted. Concentrations of the brain cell biomarkers glial fibrillary acidic protein (GFAP), YKL-40, S100B, neurogranin, neurofilament light (NfL) and tau were analysed in CSF and, in addition, NfL, GFAP and S100B levels were measured in serum. The Jonckheere-Terpstra test was used for group comparisons of continuous variables and Spearman's partial correlation test was used to adjust for age. RESULTS: Cerebrospinal fluid and serum concentrations of GFAP and NfL correlated with disease severity, independent of age, and with the presence of nerve paralysis. The markers neurogranin, YKL-40, tau and S100B in CSF and S100B in serum were detected, but their concentrations did not correlate with disease severity. CONCLUSIONS: Neuronal cell damage and astroglial cell activation with increased NfL and GFAP in CSF and serum were associated with a more severe disease, independent of age. Increased GFAP and NfL concentrations in CSF and NfL in serum were also indicative of spinal and/or cranial nerve damage. NfL and GFAP are promising prognostic biomarkers in tick-borne encephalitis, and future studies should focus on determining the association between these biomarkers and long-term sequelae.

Cerebrospinal fluid biomarkers in the Longitudinal Early-onset Alzheimer's Disease Study.

INTRODUCTION: One goal of the Longitudinal Early Onset Alzheimer's Disease Study (LEADS) is to define the fluid biomarker characteristics of early-onset Alzheimer's disease (EOAD). METHODS: Cerebrospinal fluid (CSF) concentrations of Aß1-40, Aß1-42, total tau (tTau), pTau181, VILIP-1, SNAP-25, neurogranin (Ng), neurofilament light chain (NfL), and YKL-40 were measured by immunoassay in 165 LEADS participants. The associations of biomarker concentrations with diagnostic group and standard cognitive tests were evaluated. RESULTS: Biomarkers were correlated with one another. Levels of CSF Aß42/40, pTau181, tTau, SNAP-25, and Ng in EOAD differed significantly from cognitively normal and early-onset non-AD dementia; NfL, YKL-40, and VILIP-1 did not. Across groups, all biomarkers except SNAP-25 were correlated with cognition. Within the EOAD group, Aß42/40, NfL, Ng, and SNAP-25 were correlated with at least one cognitive measure. DISCUSSION: This study provides a comprehensive analysis of CSF biomarkers in sporadic EOAD that can inform EOAD clinical trial design.

CSF Synaptic Biomarkers in AT(N)-Based Subgroups of Lewy Body Disease.

BACKGROUND AND OBJECTIVES: Patients with Lewy body disease (LBD) often show a co-occurring Alzheimer disease (AD) pathology. CSF biomarkers allow the detection in vivo of AD-related pathologic hallmarks included in the amyloid-tau-neurodegeneration (AT(N)) classification system. Here, we aimed to investigate whether CSF biomarkers of synaptic and neuroaxonal damage are correlated with the presence of AD copathology in LBD and can be useful to differentiate patients with LBD with different AT(N) profiles. METHODS: We retrospectively measured CSF levels of AD core biomarkers (Aß42/40 ratio, phosphorylated tau protein, and total tau protein) and of synaptic (ß-synuclein, α-synuclein, synaptosomal-associated protein 25 [SNAP-25], and neurogranin) and neuroaxonal proteins (neurofilament light chain [NfL]) in 28 cognitively unimpaired participants with nondegenerative neurologic conditions and 161 participants with a diagnosis of either LBD or AD (at both mild cognitive impairment, AD-MCI, and dementia stages, AD-dem). We compared CSF biomarker levels in clinical and AT(N)-based subgroups. RESULTS: CSF ß-synuclein, α-synuclein, SNAP-25, neurogranin, and NfL levels did not differ between LBD (n = 101, age 67.2 ± 7.8 years, 27.7% females) and controls (age 64.8 ± 8.6 years, 39.3% females) and were increased in AD (AD-MCI: n = 30, AD-dem: n = 30, age 72.3 ± 6.0 years, 63.3% females) compared with both groups (p < 0.001 for all comparisons). In LBD, we found increased levels of synaptic and neuroaxonal degeneration biomarkers in patients with A+T+ (LBD/A+T+) than with A-T- profiles (LBD/A-T-) (p < 0.01 for all), and ß-synuclein showed the highest discriminative accuracy between the 2 groups (area under the curve 0.938, 95% CI 0.884-0.991). CSF ß-synuclein (p = 0.0021), α-synuclein (p = 0.0099), and SNAP-25 concentrations (p = 0.013) were also higher in LBD/A+T+ than in LBD/A+T- cases, which had synaptic biomarker levels within the normal range. CSF α-synuclein was significantly decreased only in patients with LBD with T- profiles compared with controls (p = 0.0448). Moreover, LBD/A+T+ and AD cases did not differ in any biomarker level. DISCUSSION: LBD/A+T+ and AD cases showed significantly increased CSF levels of synaptic and neuroaxonal biomarkers compared with LBD/A-T- and control subjects. Patients with LBD and AT(N)-based AD copathology showed, thus, a distinct signature of synaptic dysfunction from other LBD cases. CLASSIFICATION OF EVIDENCE: This study provides Class II evidence that CSF levels of ß-synuclein, α-synuclein, SNAP-25, neurogranin, and NfL are higher in patients with AD than in patients with LBD.

Cerebrospinal fluid biomarkers of axonal and synaptic degeneration in a population-based sample.

BACKGROUND: Neurofilament light (NfL) and neurogranin (Ng) are promising candidate AD biomarkers, reflecting axonal and synaptic damage, respectively. Since there is a need to understand the synaptic and axonal damage in preclinical Alzheimer's disease (AD), we aimed to determine the cerebrospinal fluid (CSF) levels of NfL and Ng in cognitively unimpaired elderly from the Gothenburg H70 Birth Cohort Studies classified according to the amyloid/tau/neurodegeneration (A/T/N) system. METHODS: The sample consisted of 258 cognitively unimpaired older adults (age 70, 129 women and 129 men) from the Gothenburg Birth Cohort Studies. We compared CSF NfL and Ng concentrations in A/T/N groups using Student's T-test and ANCOVA. RESULTS: CSF NfL concentration was higher in the A-T-N+ group (p=0.001) and the A-T+N+ group (p=0.006) compared with A-T-N-. CSF Ng concentration was higher in the A-T-N+, A-T+N+, A+T-N+, and A+T+N+ groups (p<0.0001) compared with A-T-N-. We found no difference in NfL or Ng concentration in A+ compared with A- (disregarding T- and N- status), whereas those with N+ had higher concentrations of NfL and Ng compared with N- (p<0.0001) (disregarding A- and T- status). CONCLUSIONS: CSF NfL and Ng concentrations are increased in cognitively normal older adults with biomarker evidence of tau pathology and neurodegeneration.

Synaptic biomarkers in the cerebrospinal fluid associate differentially with classical neuronal biomarkers in patients with Alzheimer's disease and frontotemporal dementia.

BACKGROUND: Loss of synaptic functionality has been recently identified as an early-stage indicator of neurological diseases. Consequently, monitoring changes in synaptic protein levels may be relevant for observing disease evolution or treatment responses in patients. Here, we have studied the relationship between fluid biomarkers of neurodegeneration and synaptic dysfunction in patients with Alzheimer's disease (AD), frontotemporal dementia (FTD), and subjective cognitive decline (SCD). METHODS: The exploratory cohort consisted of cerebrospinal fluid (CSF) samples (n = 60) from patients diagnosed with AD (n = 20), FTD (n = 20), and SCD (n = 20) from the Amsterdam Dementia Cohort. We developed two novel immunoassays for the synaptic proteins synaptosomal-associated protein-25 (SNAP25) and vesicle-associated membrane protein-2 (VAMP2). We measured the levels of these biomarkers in CSF, in addition to neuronal pentraxin-2 (NPTX2), glutamate ionotropic receptor-4 (GluR4), and neurogranin (Ng) for this cohort. All in-house immunoassays were validated and analytically qualified prior to clinical application. CSF neurogranin (Ng) was measured using a commercially available ELISA. RESULTS: This pilot study indicated that SNAP25, VAMP2, and Ng may not be specific biomarkers for AD as their levels were significantly elevated in patients with both AD and FTD compared to SCD. Moreover, the strength of the correlations between synaptic proteins was lower in the AD and FTD clinical groups compared to SCD. SNAP25, VAMP2, and Ng correlated strongly with each other as well as with total Tau (Tau) and phosphorylated Tau (PTau) in all three clinical groups. However, this correlation was weakened or absent with NPTX2 and GluR4. None of the synaptic proteins correlated to neurofilament light (NfL) in any clinical group. CONCLUSION: The correlation of the synaptic biomarkers with CSF Tau and PTau but the lack thereof with NfL implies that distinct pathological pathways may be involved in synaptic versus axonal degeneration. Our results reflect the diversity of synaptic pathology in neurodegenerative dementias.

Subtle Differences in Cognition in 70-Year-Olds with Elevated Cerebrospinal Fluid Neurofilament Light and Neurogranin: A H70 Cross-Sectional Study.

BACKGROUND: Most research on cerebrospinal fluid (CSF) neurofilament light protein (NfL) as a marker for neurodegeneration and neurogranin (Ng) for synaptic dysfunction has largely focused on clinical cohorts rather than population-based samples. OBJECTIVE: We hypothesized that increased CSF levels of NfL and Ng are associated with subtle cognitive deficits in cognitively unimpaired (CU) older adults. METHODS: The sample was derived from the Gothenburg H70 Birth Cohort Studies and comprised 258 CU 70-year-olds, with a Clinical Dementia Rating score of zero. All participants underwent extensive cognitive testing. CSF levels of NfL and Ng, as well as amyloid ß1 - 42, total tau, and phosphorylated tau, were measured. RESULTS: Participants with high CSF NfL performed worse in one memory-based test (Immediate recall, p = 0.013) and a language test (FAS, p = 0.016). Individuals with high CSF Ng performed worse on the memory-based test Supra Span (p = 0.035). When stratified according to CSF tau and Aß42 concentrations, participants with high NfL and increased tau performed worse on a memory test than participants normal tau concentrations (Delayed recall, p = 0.003). In participants with high NfL, those with pathologic Aß42 concentrations performed worse on the Delayed recall memory (p = 0.044). In the high Ng group, participants with pathological Aß42 concentrations had lower MMSE scores (p = 0.027). However, in regression analysis we found no linear correlations between CSF NfL or CSF Ng in relation to cognitive tests when controlled for important co-variates. CONCLUSION: Markers of neurodegeneration and synaptic pathology might be associated with subtle signs of cognitive decline in a population-based sample of 70-year-olds.

Neurogranin in Alzheimer's disease and ageing: A human post-mortem study.

Neurogranin (Ng), a post-synaptic protein involved in memory formation, has been investigated as a biomarker in the cerebrospinal fluid (CSF) in Alzheimer's disease (AD) and ageing. CSF Ng levels are elevated in AD relative to healthy controls and correlate with cognition; however, few studies have focused on Ng abundance in the brain. Synapse loss in the brain correlates closely with cognitive decline in AD making synaptic biomarkers potentially important for tracking disease progression, but the links between synaptic protein changes in CSF and brain remain incompletely understood. In the current study, Ng abundance was examined in post-mortem human brain tissue across AD, healthy ageing (HA), and mid-life (ML) cohorts. Ng levels were quantified in three brain regions associated with cognitive change found during ageing and neurodegenerative diseases, namely the middle temporal gyrus, primary visual cortex and the posterior hippocampus using immunohistochemistry. To support immunohistochemical analysis, total homogenate and biochemically enriched synaptic fractions from available temporal gyrus tissues were examined by immunoblot. Finally, we examined whether Ng is associated with lifetime cognitive ageing. Ng levels were significantly reduced in AD relative to HA and ML cases across all regions. Additionally Ng was significantly reduced in HA in comparison to ML in the primary visual cortex. Immunoblotting confirms reduced Ng levels in AD cases supporting immunohistochemical results. Interestingly, there was also a significant reduction of synapse-associated Ng in our group who had lifetime cognitive decline in comparison to the group with lifetime cognitive resilience indicating loss of neurogranin in remaining synapses during ageing is associated with cognitive decline. Our findings indicate that increases in CSF Ng reflect loss of brain neurogranin and support the use of CSF Ng as a biomarker of AD and potentially of cognitive decline in healthy ageing.