Association of APOE genotype and cerebrospinal fluid Aß and tau biomarkers with cognitive and motor phenotype in amyotrophic lateral sclerosis.

OBJECTIVE: Little is known about amyotrophic lateral sclerosis (ALS)-nonspecific cognitive deficits - most notably memory disturbance - and their biological underpinnings. We investigated the associations of the Alzheimer's disease (AD) genetic risk factor APOE and cerebrospinal fluid (CSF) biomarkers Aß and tau proteins with cognitive and motor phenotype in ALS. METHODS: APOE haplotype was determined in 281 ALS patients; for 105 of these, CSF levels of Aß42, Aß40, total tau (T-tau), and phosphorylated tau (P-tau181) were quantified by chemiluminescence enzyme immunoassay (CLEIA). The Edinburgh Cognitive and Behavioural ALS Screen (ECAS) was employed to evaluate the neuropsychological phenotype. RESULTS: APOE-E4 allele was associated with worse ECAS memory score (median, 14.0 in carriers vs. 16.0 in non-carriers) and lower CSF Aß42 (-0.8 vs. 0.1, log-transformed values) and Aß42/40 ratio (-0.1 vs. 0.3). Some 37.1% of ALS patients showed low Aß42 levels, possibly reflecting cerebral Aß deposition. While lower Aß42/40 correlated with lower memory score (ß = 0.20), Aß42 positively correlated with both ALS-specific (ß = 0.24) and ALS-nonspecific (ß = 0.24) scores. Although Aß42/40 negatively correlated with T-tau (ß = -0.29) and P-tau181 (ß = -0.33), we found an unexpected positive association of Aß42 and Aß40 with both tau proteins. Regarding motor phenotype, lower levels of Aß species were associated with lower motor neuron (LMN) signs (Aß40: ß = 0.34; Aß42: ß = 0.22). CONCLUSIONS: APOE haplotype and CSF Aß biomarkers are associated with cognitive deficits in ALS and particularly with memory impairment. This might partly reflect AD-like pathophysiological processes, but additional ALS-specific mechanisms could be involved.

Relationship between cerebrospinal fluid/serum albumin quotient and phenotype in amyotrophic lateral sclerosis: a retrospective study on 328 patients.

BACKGROUND: We analysed the relationship between cerebrospinal fluid (CSF)/serum albumin quotient (Q-Alb) and phenotype in a large cohort of patients with amyotrophic lateral sclerosis (ALS). METHODS: Three hundred twenty-eight single-centre consecutive patients with ALS were evaluated for Q-Alb, basic epidemiological and clinical data, motor phenotype, cognitive/behavioural impairment, clinical staging, clinical and neurophysiological indexes of upper (UMN) and lower motor neuron (LMN) dysfunction, and presence of ALS gene mutations. RESULTS: Q-Alb did not correlate with age but was independently associated with sex, with male patients having higher levels than female ones; the site of onset was not independently associated with Q-Alb. Q-Alb was not associated with motor phenotype, cognitive/behavioural impairment, disease stage, progression rate, survival, or genetic mutations. Among measures of UMN and LMN dysfunction, Q-Alb only had a weak positive correlation with an electromyography-based index of active limb denervation. CONCLUSION: Previous work has documented increased Q-Alb in ALS compared to unaffected individuals. This, together with the absence of associations with nearly all ALS phenotypic features in our cohort, suggests dysfunction of the blood-CSF barrier as a shared, phenotype-independent element in ALS pathophysiology. However, correlation with the active denervation index could point to barrier dysfunction as a local driver of LMN degeneration.

Lower semantic fluency scores and a phonemic-over-semantic advantage predict abnormal CSF P-tau181 levels in Aß + patients within the Alzheimer's disease clinical spectrum.

BACKGROUND: The present study aimed to determine whether patients with mild cognitive impairment (MCI) and dementia due to Alzheimer's disease (AD), semantic verbal fluency (SVF), and the semantic-phonemic discrepancy (SPD) could predict abnormal cerebrospinal fluid (CSF) phosphorylated tau (P-tau181) and total tau (T-tau) levels. METHODS: Phonemic verbal fluency (PVF) and SVF scores of N = 116 Aß-positive patients with either MCI due to AD (N = 39) or probable AD dementia (ADD; N = 77) were retrospectively collected. The SPD was computed by subtracting PVF scores from SVF ones (positive and negative values corresponding to a semantic and phonemic advantage, respectively). Patients were cognitively phenotyped via a thorough test battery and profiled according to the amyloidosis/tauopathy/neurodegeneration (ATN) framework via CSF analyses. Two separate sets of logistic regressions were run to predict normal vs. abnormal P-tau181 and T-tau levels by encompassing as predictors SVF + PVF and SPD and covarying for demographic, disease-related features, and cognitive profile. RESULTS: Lower SVF, but not PVF, scores, as well as a greater phonemic advantage (i.e., negative SPD values), predicted abnormal CSF P-tau181 levels (p ≤ .01). Moreover, lower SVF scores were selectively predictive of abnormal CSF T-tau levels too (p = .016), while the SPD was not. DISCUSSION: SVF and the SPD are able to predict tauopathy across the AD spectrum, thus supporting their status of valid, and sufficiently specific, cognitive markers of AD.

Cerebrospinal fluid/serum albumin quotient (Q-Alb) is not increased in Alzheimer's disease compared to neurological disease controls: a retrospective study on 276 patients.

BACKGROUND: The cerebrospinal fluid (CSF)/serum albumin quotient (Q-Alb) is a marker of the blood-CSF barrier (BCSFB) and possibly of the blood-brain barrier (BBB). The latter is known to be altered in Alzheimer's disease (AD) based on neuropathological and neuroimaging studies. Following investigations performed on clinically diagnosed cohorts, we aimed at comparing Q-Alb in cognitively impaired patients with neurochemical demonstration of AD pathophysiology and neurological disease controls (NDCs). METHODS: We evaluated N = 144 AD patients (MCI, N = 43; AD dementia - ADD, N = 101) and N = 132 NDCs. AD patients were all A + according to the A/T/N framework and were neurochemically classified based on T and N parameters. RESULTS: Q-Alb did not significantly differ between AD patients and NDCs. Moreover, it was not associated with disease stage (MCI vs. ADD), MMSE score, or CSF AD biomarkers. DISCUSSION: Our study indicates that BCSFB dysfunction is not a specific feature of AD. When interpreting Q-Alb as a marker of the BBB, the lack of difference from NDCs might be due to BBB dysfunction widely occurring in other neurological, non-degenerative, conditions or - more probably - to low sensitivity of this biochemical parameter towards subtle BBB alterations causing leakage of molecules smaller than albumin. Furthermore, Q-Alb is not associated with the degree of global cognitive deterioration in AD, nor with CSF AD neurochemical biomarkers.

Influence of kidney function and CSF/serum albumin ratio on plasma Aß42 and Aß40 levels measured on a fully automated platform in patients with Alzheimer's disease.

INTRODUCTION: Alzheimer's disease  (AD) is characterized by decreased cerebrospinal fluid (CSF) Aß42 and Aß42/Aß40 ratio. Aß peptides can now be measured also in plasma and are promising peripheral biomarkers for AD. We evaluated the relationships of plasma Aß species with their CSF counterparts, kidney function, and serum/CSF albumin ratio (Q-Alb) in AD patients. MATERIALS AND METHODS: We measured plasma Aß42 and Aß40, as well as CSF AD biomarkers, with the fully automated Lumipulse platform in a cohort of N = 30 patients with clinical and neurochemical diagnosis of AD. RESULTS: The two plasma Aß peptides correlated strongly with each other (r = 0.7449), as did the corresponding CSF biomarkers (r = 0.7670). On the contrary, the positive correlations of plasma Aß42, Aß40, and Aß42/Aß40 ratio with their CSF counterparts and the negative correlation of plasma Aß42/Aß40 ratio with CSF P-tau181 were not statistically significant. Plasma levels of both Aß species negatively correlated with estimated glomerular filtration rate (eGFR) (Aß42: r = -0.4138; Aß40: r = -0.6015), but plasma Aß42/Aß40 ratio did not. Q-Alb did not correlate with any plasma Aß parameter. DISCUSSION: Plasma Aß42 and Aß40 are critically influenced by kidney function; however, their ratio is advantageously spared from this effect. The lack of significant correlations between plasma Aß species and their CSF counterparts is probably mainly due to small sample size and inclusion of only Aß + individuals. Q-Alb is not a major determinant of plasma Aß concentrations, highlighting the uncertainties about mechanisms of Aß transfer between CNS and periphery.

Phosphorylated tau in plasma could be a biomarker of lower motor neuron impairment in amyotrophic lateral sclerosis.

INTRODUCTION: Plasma levels of phosphorylated tau (P-tau181) have been recently reported to be increased in amyotrophic lateral sclerosis (ALS) and associated with lower motor neuron (LMN) impairment. PATIENTS AND METHODS: We quantified plasma P-tau181 (pP-tau181) in a cohort of 29 deeply phenotyped ALS patients using the new fully automated Lumipulse assay and analysed phenotype-biomarker correlations. RESULTS: pP-tau181 levels correlated positively with a clinical LMN score (r = 0.3803) and negatively, albeit not significantly, with a composite index of muscle strength (r = - 0.3416; p = 0.0811), but not with Penn Upper Motor Neuron (UMN) Score. Accordingly, pP-tau181 correlated with electromyographic indices of spinal active and chronic denervation (r = 0.4507 and r = 0.3864, respectively) but not with transcranial magnetic stimulation parameters of UMN dysfunction. pP-tau181 levels did not correlate with those in the cerebrospinal fluid (CSF), serum NFL, serum GFAP, CSF/serum albumin ratio, or estimated glomerular filtration rate, but correlated with plasma creatine kinase levels (r = 0.4661). Finally, while not being associated with neuropsychological phenotype, pP-tau181 correlated negatively with pH (r = - 0.5632) and positively with partial pressure of carbon dioxide (PaCO2; r = 0.7092), bicarbonate (sHCO3-; r = 0.6667) and base excess (r = 0.6611) on arterial blood gas analysis. DISCUSSION: pP-tau181 has potential as ALS biomarker and could be associated with LMN impairment. Its raised levels might reflect pathophysiological processes (tau hyperphosphorylation and/or release) occurring in the axons of LMNs distantly from the CNS and the CSF. pP-tau181 could also be associated with respiratory dysfunction.

A + T ± status across MCI and dementia due to AD: a clinic-based, retrospective study.

BACKGROUND: This study aimed at comparing, within the 2018 NIA-AA amyloidosis/tauopathy/neurodegeneration (ATN) framework, the distribution of T ± profiles across A + patients with MCI and dementia in a retrospective, single-center, clinic-based cohort. METHODS: We retrospectively collected data on N = 168 A + patients with either MCI due to AD (N = 50) or probable AD dementia (ADD; N = 118). ATN status was assigned, according to the 2018 NIA-AA framework, based on cerebrospinal fluid (CSF) biomarker concentrations. A χ2-test for independent samples was run to compare the distribution of A + T + vs. A + T- profiles, regardless of N status, across MCI and dementia patients. RESULTS: The most represented ATN profile in both groups was A + T + N + (MCI: 54%; dementia: 70.3%); 3.4% of dementia patients and none within the MCI cohort presented with an A + T-N + profile. When grouping ATN profiles solely based on A and T dimensions, the prevalence of A + T + was of 76.3% and 66% in dementia and MCI patients, respectively. No association between clinical diagnoses (i.e., MCI vs. dementia status) and AT profiles (i.e., A + T + vs. A + T-) was detected. DISCUSSION: The distribution of A + T + vs. A + T- does not differ between MCI and ADD, with A + T + profiles being predominant in both clinical categories. This does not support the common notion of A + T- profiles being relatively more prevalent in MCI patients, as indexing an earlier and/or less severe disease. Hence, caution should be exerted in attributing a case of MCI to prodromal AD solely based on A-positivity in the presence of a T-negative profile.

CSF Aß40 and P-Tau181 Might Differentiate Atypical from Typical AD Phenotypes: Preliminary Evidence.

OBJECTIVES: This study aimed at testing whether CSF levels of amyloid ß42 (Aß42), Aß40, total tau, and phosphorylated tau (P-tau181) individually contribute to the identification of atypical phenotypes among a retrospective cohort of probable Alzheimer's disease (AD) patients diagnosed by means of the ratio between Aß42 and Aß40 (Aß42/40). METHODS: The retrospective study cohort comprised 50 probable AD patients diagnosed by means of the ratio between Aß42 and Aß40 (Aß42/40) and for whom total tau and P-tau181 values were also available. Patients were clinically classified as typical, amnestic-predominant AD (N = 39; 16 males; mean age: 73.4 ± 7.6 years; mean disease duration: 27.4 ± 24.7 months), or atypical phenotypes (N = 11; 6 males; mean age: 70.2 ± 6.5 years; mean disease duration: 35.5 ± 24.9 months) - i.e., posterior cortical atrophy (N = 4), logopenic-variant primary progressive aphasia (N = 4), and behavioural variant AD (N = 3). A logistic regression allowed predicting the occurrence of atypical versus typical phenotypes based on age, sex, and Aß42, Aß40, total tau, and P-tau181 levels. RESULTS: Atypical and typical AD patients were comparable for Aß42/40 values. Only Aß40 and P-tau181 levels positively (p = 0.015) and negatively (p = 0.019) predicted the occurrence of atypical AD phenotypes, respectively. Classification precision was of 86%, yielding excellent specificity (94.9%) but poor sensitivity (54.5%). CONCLUSIONS: The present study delivers promising, albeit preliminary, evidence on the utility of Aß40 and P-tau181 CSF biomarkers in differentiating atypical from typical Aß42/40-confirmed AD phenotypes, prompting further research and confirmation on larger cohorts.

Quantification of serum TDP-43 and neurofilament light chain in patients with amyotrophic lateral sclerosis stratified by UNC13A genotype.

Amyotrophic Lateral Sclerosis (ALS) is a fatal neurodegenerative condition affecting upper and/or lower motor neurons and characterized neuropathologically by TDP-43 proteinopathy. Given its role in ALS pathobiology, it is currently under debate whether TDP-43 might represent a suitable ALS biomarker to be measured in patients' biofluids. The rs12608932 A > C single nucleotide polymorphism in the UNC13A gene is a risk factor for ALS and patients homozygous for the high-risk C allele display a higher burden of TDP-43 neuropathology than homozygotes for the low-risk A allele, although the association with TDP-43 levels in biofluids has never been evaluated. In this study, we measured serum levels of TDP-43 and neurofilament light chain (NFL) by Simoa technology in a cohort of 69 ALS patients stratified according to the UNC13A rs12608932 genotype compared to 43 neurologically healthy controls. By multiple linear regression analysis, serum TDP-43 was significantly elevated in ALS patients compared to controls, with UNC13A AA and AC, but not CC, ALS patients showing higher serum TDP-43 levels than controls. We also confirmed that serum NFL concentration was increased in ALS patients, without any correlation with the UNC13A genotype. Our results indicate that serum TDP-43 is higher in ALS patients compared to controls and that, in contrast to NFL, this increase is specifically associated with the UNC13A rs12608932 AA and AC genotypes, but not with the high-risk CC genotype. Studies in larger cohorts will be needed to confirm these findings and to elucidate the biological link between serum TDP-43 levels and UNC13A genotype.

Exploiting the role of CSF NfL, CHIT1, and miR-181b as potential diagnostic and prognostic biomarkers for ALS.

Amyotrophic lateral sclerosis (ALS) is a rare neurodegenerative disorder characterized by relentless and progressive loss of motor neurons. A molecular diagnosis, supported by the identification of specific biomarkers, might promote the definition of multiple biological subtypes of ALS, improving patient stratification and providing prognostic information. Here, we investigated the levels of neurofilament light chain (NfL), chitotriosidase (CHIT1) and microRNA-181b (miR-181b) in the cerebrospinal fluid (CSF) of ALS subjects (N = 210) as well as neurologically healthy and neurological disease controls (N = 218, including N = 74 with other neurodegenerative diseases) from a large European multicentric cohort, evaluating their specific or combined utility as diagnostic and prognostic biomarkers. NfL, CHIT1 and miR-181b all showed significantly higher levels in ALS subjects compared to controls, with NfL showing the most effective diagnostic performance. Importantly, all three biomarkers were increased compared to neurodegenerative disease controls and, specifically, to patients with Alzheimer's disease (AD; N = 44), with NfL and CHIT1 being also higher in ALS than in alpha-synucleinopathies (N = 22). Notably, ALS patients displayed increased CHIT1 levels despite having, compared to controls, a higher prevalence of a polymorphism lowering CHIT1 expression. While no relationship was found between CSF miR-181b and clinical measures in ALS (disease duration, functional disability, and disease progression rate), CSF NfL was the best independent predictor of disease progression and survival. This study deepens our knowledge of ALS biomarkers, highlighting the relative specificity of CHIT1 for ALS among neurodegenerative diseases and appraising the potential diagnostic utility of CSF miR-181b.

Serum levels of glial fibrillary acidic protein in patients with amyotrophic lateral sclerosis.

OBJECTIVE: To compare serum levels of the astrocyte biomarker glial fibrillary acidic protein (GFAP) in patients with amyotrophic lateral sclerosis (ALS) and neurologically healthy controls and to analyze the relations between serum GFAP (sGFAP) and phenotype in ALS. METHODS: We studied 114 ALS patients and 38 controls. sGFAP was quantified with single molecule array (Simoa) technology. RESULTS: In both ALS patients and controls, sGFAP moderately correlated with age. ALS patients had higher sGFAP levels compared to controls, but this yielded a weak discriminative performance (AUC = 0.6198). In ALS, sGFAP was not associated with most of the motor phenotypic features, including site of onset, functional status, disease progression rate, disease stage, and indices of upper (UMN) and lower motor neuron (LMN) impairment. However, sGFAP negatively correlated with cognitive scores regarding ALS-nonspecific functions, particularly memory (r = -0.2082) and tended to be higher in ALS patients with eye movement abnormalities (p = 0.0628). sGFAP also correlated with polysomnographic indices of oxygen desaturation (ODI; r = 0.2639) and apnea-hypopnea (AHI; r = 0.2858). In a multivariate analysis, sGFAP was negatively associated with survival (HR = 1.005). Relevantly, we found a negative correlation between sGFAP and estimated glomerular filtration rate (eGFR; r = -0.3500). INTERPRETATION: Our work provides neurochemical evidence of astrocyte involvement in ALS pathophysiology and particularly in the development of extra-motor manifestations (namely, cognitive - memory - impairment) and respiratory dysfunction. The negative correlation between sGFAP and eGFR has practical relevance and should not be disregarded in future investigations.

Phenotypic correlates of serum neurofilament light chain levels in amyotrophic lateral sclerosis.

Objective: To investigate the relationship between serum levels of the neuroaxonal degeneration biomarker neurofilament light chain (NFL) and phenotype in ALS. Materials and methods: Serum NFL (sNFL) concentration was quantified in 209 ALS patients and 46 neurologically healthy controls (NHCs). Results: sNFL was clearly increased in ALS patients and discriminated them from NHCs with AUC = 0.9694. Among ALS patients, females had higher sNFL levels, especially in case of bulbar onset. sNFL was more increased in phenotypes with both upper (UMN) and lower motor neuron (LMN) signs, and particularly in those with UMN predominance, compared to LMN forms. At the same time, primary lateral sclerosis (PLS) had significantly lower levels compared to UMN-predominant ALS (AUC = 0.7667). sNFL correlated negatively with disease duration at sampling and ALSFRS-R score, positively with disease progression rate, differed among King's stages, and was negatively associated with survival. It also correlated with clinical/neurophysiological indices of UMN and LMN dysfunction (Penn UMN Score, LMN score, MRC composite score, active spinal denervation score). On the contrary, sNFL was not associated with cognitive deficits nor with respiratory parameters. Notably, we found a negative correlation between sNFL and estimated glomerular filtration rate (eGFR). Interpretation: We confirm that ALS is characterized by increased sNFL levels, whose main determinant is the rate of degeneration of both UMNs and LMNs. sNFL is a biomarker of only motor, not of extra-motor, disease. The negative correlation with kidney function might reflect varying renal clearance of the molecule and deserves further investigation before introducing sNFL measurement as routine test in clinical care of ALS patients.

Serum neurofilament light chain levels in Covid-19 patients without major neurological manifestations.

BACKGROUND: Increased serum levels of neurofilament light chain (sNFL), a biomarker of neuroaxonal damage, have been reported in patients with Covid-19. We aimed at investigating whether sNFL is increased in Covid-19 patients without major neurological manifestations, is associated with disease severity, respiratory and routine blood parameters, and changes longitudinally in the short term. METHODS: sNFL levels were measured with single molecule array (Simoa) technology in 57 hospitalized Covid-19 patients without major neurological manifestations and in 30 neurologically healthy controls. Patients were evaluated for PaO2/FiO2 ratio on arterial blood gas, Brescia Respiratory Covid Severity Scale (BRCSS), white blood cell counts, serum C-reactive protein (CRP), plasma D-dimer, plasma fibrinogen, and serum creatinine at admission. In 20 patients, NFL was also measured on serum samples obtained at a later timepoint during the hospital stay. RESULTS: Covid-19 patients had higher baseline sNFL levels compared to controls, regardless of disease severity. Baseline sNFL correlated with serum CRP and plasma D-dimer in patients with mild disease, but was not associated with measures of respiratory impairment. Longitudinal sNFL levels tended to be higher than baseline ones, albeit not significantly, and correlated with serum CRP and plasma D-dimer. The PaO2/FiO2 ratio was not associated with longitudinal sNFL, whereas BRCSS only correlated with longitudinal sNFL variation. CONCLUSIONS: We provide neurochemical evidence of subclinical axonal damage in Covid-19 also in the absence of major neurological manifestations. This is apparently not fully explained by hypoxic injury; rather, systemic inflammation might promote this damage. However, a direct neurotoxic effect of SARS-CoV-2 cannot be excluded.

The Frontal Assessment Battery (FAB) effectively discriminates between MCI and dementia within the clinical spectrum of neurochemically confirmed Alzheimer's disease.

Background: This study aimed at testing the ability of the frontal assessment battery (FAB) to differentiate between patients with mild cognitive impairment (MCI) and dementia due to Alzheimer's disease (AD), as well as comparing its discriminative power to that of the Mini-Mental State Examination (MMSE). Methods: The present retrospective cohort included N = 107 Aß-positive patients diagnosed with either MCI due to AD (N = 40) or probable AD dementia (ADD; N = 67). A two-step multiple logistic regression (MLR) was run to predict an MCI vs. ADD diagnosis based on FAB scores. Within the baseline step, demographics, disease duration, MMSE scores, and information on cognitive phenotypes were entered, with the FAB being added within the second step. Receiver-operating characteristics analyses were also run to derive intrinsic and post-test diagnostics. Results: Within the baseline MLR step, only lower MMSE scores predicted the occurrence of ADD; by adding the FAB, which likewise was able to discriminate between MCI and ADD (p = 0.016), a significant increase in model fit was detected (p = 0.007). The diagnostic efficiency of the FAB (AUC = 0.85) was comparable (p = 0.583) to that of the MMSE (AUC = 0.82), also yielding good intrinsic and post-test diagnostics, which were comparable to those of the MMSE. Discussion: The FAB is a diagnostically sound screener to discriminate between MCI and ADD, independently of patients' overall cognitive profile. In doing so, the FAB is comparable to the MMSE, and the complementation of the latter with the former is advisable in order to increase the accuracy in differentiating between MCI and ADD within screening sessions.