Cerebrospinal fluid substance P concentrations are elevated in patients with Alzheimer's disease

Neuroscience Letters 609 (2015) 58–62

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Research paper

Cerebrospinal fluid substance P concentrations are elevated in patients with Alzheimer’s disease Per Johansson a,b , Erik G. Almqvist c , Anders Wallin d , Jan-Ove Johansson b , Ulf Andreasson d , Kaj Blennow d , Henrik Zetterberg d,e , Johan Svensson b,c,∗ a

Department of Neuropsychiatry, Skaraborg Central Hospital, SE-521 85 Falköping, Sweden Institute of Medicine, Department of Internal Medicine, Sahlgrenska Academy, University of Gothenburg, SE-413 45 Gothenburg, Sweden c Department of Endocrinology, Skaraborg Central Hospital, SE-541 85 Skövde, Sweden d Institute of Neuroscience and Physiology, Department of Psychiatry and Neurochemistry, The Sahlgrenska Academy at University of Gothenburg, SE-431 80 Mölndal, Sweden e UCL Institute of Neurology, Queen Square, London WC1N 3BG, UK b

h i g h l i g h t s • • • • •

Cross-sectional study of 60 patients and 20 matched controls. Neuropeptides were measured in cerebrospinal fluid (CSF). CSF substance P level was increased in Alzheimer’s disease (AD). Substance P correlated with ␤-amyloid1–42 (A␤1–42 ) in CSF of AD patients. CSF orexin A level was marginally reduced in other dementias.

a r t i c l e

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Article history: Received 25 June 2015 Received in revised form 22 September 2015 Accepted 2 October 2015 Available online 8 October 2015 Keywords: Alzheimer’s disease Cerebrospinal fluid Dementia Substance P Orexin A Neurotensin

a b s t r a c t The neuropeptides substance P, orexin A (hypocretin-1) and neurotensin are signaling molecules that influence brain activity. We examined their cerebrospinal fluid (CSF) levels in a study population consisting of Alzheimer’s disease (AD) dementia or mild cognitive impairment (MCI) diagnosed with AD dementia upon follow-up (n = 32), stable MCI (SMCI, n = 13), other dementias (n = 15), and healthy controls (n = 20). CSF substance P level was increased in AD patients compared to patients with other dementias and healthy controls (P < 0.05 and P < 0.01, respectively). Patients with other dementia or SMCI had lower CSF orexin A level than AD patients (both P < 0.05) and marginally lower level than healthy controls (both P = 0.05). CSF neurotensin level was similar in all groups. In the total study population (n = 80), CSF substance P level correlated positively with CSF levels of T-tau and P-tau, and in AD patients (n = 32), CSF substance P level correlated positively with CSF A␤1–42 level. In conclusion, CSF substance P level was elevated in AD patients and correlated with CSF A␤1–42 level, a well established marker of senile plaque pathology. The role of low CSF orexin A level in other dementias or SMCI needs to be explored in further studies. © 2015 Elsevier Ireland Ltd. All rights reserved.

1. Introduction The 11-amino acid neuropeptide substance P is involved in the regulation of anxiety and emotionality by actions on its main receptor neurokinin 1 (NK-1) [1]. In animal studies, substance P improved

∗ Corresponding author at: Department of Internal Medicine, Gröna Stråket 8, Sahlgrenska University Hospital, SE−413 45 Gothenburg, Sweden. Fax: +46 31 821524. E-mail address: [email protected] (J. Svensson). http://dx.doi.org/10.1016/j.neulet.2015.10.006 0304-3940/© 2015 Elsevier Ireland Ltd. All rights reserved.

memory functions [2,3] and provided protection from age-related memory decline [4]. Co-administration of substance P prevented the cognitive impairment induced by infusion of amyloid-␤ (A␤) [5]. In most studies of postmortem Alzheimer’s disease (AD) brains, substance P immunoreactivity was decreased [6,7]. Both decreased and increased CSF substance P levels have been observed in manifest AD [8–10]. Orexin A (hypocretin-1) participates in the regulation of arousal, wakefulness, sleep and appetite [11]. In mice, intracerebral administration of orexin A improved memory functions [12,13]. A study of postmortem AD brains found decreased number of orexin A

P. Johansson et al. / Neuroscience Letters 609 (2015) 58–62

immunoreactive neurons and lower orexin A level in postmortem CSF [14]. However, in vivo CSF samples have shown unaltered orexin level in AD patients [15,16]. Neurotensin induces a variety of effects, among them improved learning and memory [17]. In postmortem AD brains, neurotensin immunoreactivity was decreased in amygdaloid regions [18], but little is known of the CSF level of neurotensin in AD. Finally, hyperactivity of the hypothalamic-pituitary-adrenal axis is a recognized feature of clinically manifest AD [19]. However, in early AD, the CSF level of the adrenal-derived hormone cortisol could either be normal [20] or elevated [21]. In a mono-center study, we determined CSF levels of the neuropeptides substance P, orexin A, and neurotensin as well as cortisol using a multiplex panel. We also studied whether there were associations with CSF levels of AD biomarkers. 2. Materials and methods 2.1. Study participants Sixty consecutive Caucasian patients (30 men and 30 women) admitted to a memory clinic in Falköping, Sweden were recruited 2000–2008 [22]. Inclusion criteria were age 65–80 years, body mass index (BMI) 20–26 kg/m2 , and waist:hip ratio 0.65–0.90 in women and 0.70–0.95 in men. Exclusion criteria included serum creatinine > 175 mmol/L, diabetes mellitus, and medication with glucocorticoids or acetylcholine esterase inhibitors. Twenty control subjects (10 men and 10 women) were recruited contemporaneously from the same geographical area among spouses of the included patients (n = 5) and by advertisements in local newspapers (n = 15). The controls had no subjective symptoms of cognitive dysfunction but otherwise, inclusion and exclusion criteria were similar as those in the patients [evaluated at a visit to a specialized physician (P.J.)]. The diagnosis of dementia was performed by an independent specialized physician according to the DSM-IV criteria. Patients with dementia were classified as suffering of AD [23], vascular dementia (VaD) according to NINDS–AIREN [24] or the guidelines for subcortical VaD [25]. Frontotemporal dementia and dementia with Lewy bodies (LBD) were diagnosed as described previously [22]. Mild cognitive impairment (MCI) was diagnosed in patients not fulfilling the criteria for dementia [26]. During follow-up visits for a median of 3 (range 1–7) years, 13 MCI patients remained in stable cognitive function (SMCI). Others progressed and were diagnosed with AD (n = 7), VaD (n = 3), or frontotemporal lobe dementia (n = 1). In total, the study population consisted of AD dementia or MCI diagnosed with AD dementia upon follow-up (n = 32), SMCI (n = 13), other dementias (n = 15), and healthy controls (n = 20). The distribution of diagnoses in the other dementia group were VAD or MCI diagnosed with VAD upon follow-up (n = 10), DLB (n = 4), and MCI that later converted to frontotemporal lobe dementia (n = 1). 2.2. Ethical considerations The study was approved by the ethical committee of Göteborg University, and informed consent was obtained from all participants. 2.3. Cognitive and physical examination Before the test day, a mini-mental state examination (MMSE) was performed. On the test day, in the fasted state, body weight and height were measured as well as waist circumference and hip girth.

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2.4. Biochemical procedures CSF samples were collected by lumbar puncture at the standardized time point 8.30–9.00 am. and stored at − 80 ◦ C, without being thawed and re-frozen. All analyses were performed with the analyst blinded to the clinical diagnoses. CSF concentrations of substance P, orexin A, cortisol, neurotensin, ␣-MSH, ␤-endorfin, melatonin, and oxytocin were analyzed using a multiplex panel (Millipore Human Neuropeptide Magnetic Bead Panel© , Merck-Millipore, Stockholm, Sweden, Cat#: HNPMAG-35K) following the instructions from the manufacturer. Data was acquired with a MAGPIX (Luminex ’s-Hertogenbosch, The Netherlands) instrument and concentrations calculated against calibration curves using the xPONENT software (Luminex). CSF levels of ␣-MSH, ␤-endorfin, melatonin, and oxytocin were not measurable in the majority of the participants and are therefore not reported. Lower limits of quantification for substance P, orexin A, neurotensin, and cortisol were 12, 301, 119, and 509 pg/mL, respectively. The corresponding intra/inter-assay coefficients of variation (CVs) were <10%/<20%, <10%/<20%, <10%/<10%, and <10%/<20%, respectively. The CSF biomarkers A␤1–42, total-tau (T-tau), and tau phosphorylated at threonine 181 (P-tau181) were measured using INNOTEST® ELISA assays [22]. Lower limits of quantification for A␤1–42 , Ttau, and P-tau were 125, 75, and 15.6 pg/mL, respectively. The corresponding intra/inter-assay CVs were 6.3%/11%, 10%/11%, and 3.6%/18%, respectively. 2.5. Statistical analyses Values are given as the median (25th–75th percentile). Between-group differences were assessed using the nonparametric Kruskal–Wallis test for multiple variables, followed by the Mann–Whitney U test for pair-wise comparisons. Correlations were sought using the Spearman rank order correlation test. A two-tailed P-value ≤ 0.05 was considered statistically significant. 3. Results Patients and controls were comparable in terms of age, gender, BMI, and waist:hip ratio (Table 1). AD biomarkers in CSF have been reported previously [22]. None of the CSF biomarkers correlated with age or CSF/serum albumin ratio (P–> 0.05). 3.1. CSF levels of neuropeptides (Table 2) CSF substance P level was increased in AD patients compared to both patients with other dementias and healthy controls (P–< 0.05 and P–< 0.01, respectively). Patients with other dementia or SMCI had lower CSF orexin A level than AD patients (both P–< 0.05) and marginally lower level compared to healthy controls (both P = 0.05). CSF levels of neurotensin and cortisol were similar in all groups . 3.2. Correlation analysis (Fig. 1) We evaluated whether variables that differed between groups (substance P and orexin A) correlated with CSF AD biomarkers. In the total study population (n = 80), CSF substance P level correlated positively with CSF levels of T-tau (r–= 0.36, P < 0.01) and P-tau (r = 0.43, P < 0.001) but not with CSF A␤1–42 level (r = 0.05). In AD patients (n = 32), CSF substance P level correlated positively with CSF A␤1–42 level (r = 0.41, P < 0.05) but not with CSF levels of T-tau or P-tau (both r = 0.15) . In the total study population (n = 80), CSF orexin A level correlated positively with CSF levels of T-tau (r = 0.47, P < 0.001) and

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P-tau (r = 0.51, P–< 0.001). However, in AD patients (n = 32), CSF orexin A level was not correlated with CSF AD biomarkers. 4. Discussion In the present study, AD patients had elevated CSF level of substance P, which correlated positively with the CSF AD biomarker A␤1–42 . CSF orexin A level was unchanged in AD patients whereas in patients with other dementia and SMCI, CSF orexin A level was lower than that in AD patients and marginally lower compared to healthy controls. CSF levels of neurotensin as well as cortisol were similar in all groups. CSF substance P level may be affected by body composition and also by age [8]. However, our patients and controls were matched in terms of age, gender, BMI, and waist:hip ratio, and none of the participants had diabetes mellitus or received treatment with acetylcholine esterase inhibitors or glucocorticoids. One study limitation is the cross-sectional design, and changes over time could therefore not be studied. Another limitation is that no measurements were performed in serum, which could have given additional information. We observed elevated CSF substance P level in relatively early AD (median MMSE score 23, median age 75 years). One previous study showed that CSF substance P level was decreased in late AD (mean age 87 years; MMSE was not performed) but not in younger AD patients [8]. In another study, AD patients with a mean age of 68 years and MMSE score of 17.7 had decreased CSF substance P level compared to healthy controls [9]. In one additional study, CSF substance P level was unchanged in the total AD group whereas late onset AD (mean age 76 years, MMSE = 14.5) showed increased CSF substance P level compared to both early onset AD (mean age 58 years, MMSE = 14.0) and healthy controls [10]. In summary, previous studies in more advanced AD than that in the present study

have shown conflicting results and the impact of age on CSF substance P level in AD is relatively unclear in these studies. In the total study population (n = 80), CSF substance P level correlated positively with the AD biomarkers T-tau and P-tau. Furthermore, in AD patients (n = 32), CSF substance P level correlated positively with CSF A␤1–42 level. CSF A␤1–42 level is typically decreased in AD [22], likely secondary to peptide sequestration within plaques. Therefore, the results of the correlation analysis might suggest that there is an association between CSF substance P level and AD disease status. In rats, co-administration of substance P provided protection from the cognitive impairment induced by A␤ infusion [5]. Substance P administrated into the cerebral cortex prevented A␤ induced neuronal loss [27]. Furthermore, substance P administration improved memory functions [2,3] and prevented aging-related memory decline [4]. Substance P also stimulates nonamyloidogenic APP processing, which might reduce the generation of toxic A␤ peptides [28]. In postmortem AD brains, decreased substance P immunoreactivity has been observed in most studies [6,7]. It could therefore be hypothesized that the increased CSF substance P level in early AD observed in the present study represents a compensatory mechanism to maintain substance P activity in the CNS and to protect the AD brain from pathological A␤ exposure. It is less likely that administration of substance P will be used as a treatment of AD. Substance P is present also in organs like immune cells and liver and induces multiple effects including risk of emesis and nausea [1]. However, substance P could be involved in the effects induced by memantine, a NMDA receptor blocker used for the treatment of AD [29]. In the rat AD brain, memantine treatment normalized the reduction of substance P caused by infusion of ibotenic acid [29], and memantine attenuated the A␤-induced changes in substance P level in the rat hippocampus [30]. Finally,

Table 1 Characterization of the study population in terms of age, anthropometric measures, MMSE score, and CSF biomarkers, which has been published previously [22]. AD Men/women Age (years) BMI (kg/m2 ) Waist/hip ratio MMSE score A␤1–42 (pg/mL) T-tau (pg/mL) P-tau (pg/mL)

(n = 32)

15/17 75 (71–77) 23.6 (21.4–25.0) 0.86 (0.76–0.92) 23 (19–25)*,** 420 (336–493)*,†† 584 (434–747)*,**,## 98 (78–113)*,**,##

Other dementias(n = 15)

SMCI (n = 13)

Controls (n = 20)

P-value

10/5 74 (72–77) 24.9 (21.4–25.8) 0.90 (0.85–0.93) 24 (20–26)***,† 404 (346–823)# 311 (254–381) 47 (34–63)

5/8 72 (69–74) 24.1 (23.0–25.9) 0.82 (0.78–0.89) 29 (27–29) 671 (528–851) 270 (224–393) 60 (38–76)

10/10 75 (70–78) 23.7 (22.8–25.3) 0.88 (0.82–0.91) 29(27–29) 990 (786–1038) 327 (223–398) 65 (50–79)

0.25 0.66 0.12 <0.0001 < 0.0001 < 0.0001 < 0.0001

Values are given as the median (25th–75th percentile). P-values in the right column refer to differences between all four groups using the Kruskal–Wallis test. Comparisons between two separate groups were performed using the Mann–Whitney U test. * P < 0.0001 vs. controls. ** P < 0.0001 vs. SMCI. *** P = 0.0002 vs. controls. † P = 0.0003 vs. SMCI. †† P = 0.0002 vs. SMCI. # P = 0.005 vs. controls. ## P < 0.0001 vs. other dementias. Table 2 CSF levels of substance P, orexin A, neurotensin, and cortisol in the study population of 60 patients with cognitive impairment and 20 healthy matched controls. AD Substance P (pg/mL) Orexin A (pg/mL) Neurotensin (pg/mL) Cortisol (pg/mL)

(n–= 32)

38.0 (34.8-45.0)*,** 1597 (1335–1866) 148 (130–163) 6989 (6289–7970)

Other dementias(n = 15)

SMCI

34.0 (25.8–36.8) 1318 (1176–1442)†,†† 141 (119–154) 6933 (6166–8440)

35.0 (30.0–39.0) 1323 (1107–1405)†,†† 167 (161–178) 6001 (4664–6473)

(n = 13)

Controls (n = 20)

P-value

31.5 (27.0–40.0) 1585 (1351–1828) 147 (131–186) 6665 (5705–7450)

0.02 0.02 0.34 0.11

Values are given as the median (25th–75th percentile). P-values in the right column refer to differences between all four groups using the Kruskal–Wallis test. Comparisons between two separate groups were performed using the Mann–Whitney U test. * P < 0.01 vs. controls. ** P < 0.05 vs. other dementias. † P = 0.05 vs. controls. †† P < 0.05 vs. AD group.

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Fig. 1. Correlations between CSF substance P level and CSF levels of the AD biomarkers A␤1–42 and P-tau. (A) CSF substance P level did not correlate with CSF A␤1–42 level in the total study population (n = 80; r = 0.05), whereas (B) CSF substance P level correlated positively with CSF A␤1–42 level in the AD group (n = 32; r = 0.41, P < 0.05). (C) CSF substance P level correlated positively with CSF P-tau level in the total study population (n = 80; r = 0.43, P < 0.001), whereas (D) there was no significant correlation in the AD group (n = 32; r = 0.15). Note the logarithmic scale on the y-axis. Correlations were sought using the Spearman rank order correlation test.

substance P can evoke release of acetylcholine [31], but little is known whether cholinesterase inhibitors affect substance P levels. In the present study, CSF orexin A (hypocretin-1) level was unchanged in AD patients. Patients with other dementia or SMCI had lower CSF orexin A level than AD patients, and marginally lower level compared to healthy controls. In postmortem brains with advanced AD, decreased number of orexin A immunoreactive neurons and lower orexin A level in postmortem CSF were observed [14]. However, in vivo CSF orexin A level was similar in AD patients compared to healthy controls [15,16] whereas reduced CSF orexin A level in vivo was found in Lewy body dementia (LBD) [16]. Furthermore, reduced numbers of orexin A-positive neurons in lateral hypothalamus were found in LBD patients compared to AD patients and healthy controls [32]. Therefore, our results concur with previous in vivo studies and suggest unchanged CSF orexin A level in AD whereas low CSF orexin A level could be of importance for cognitive dysfunction in other dementias or SMCI. However, this needs to be explored in more detail in further studies as the number of SMCI patients was relatively low in our study and the number of each specific diagnosis was small in the other dementia group. One previous study of postmortem AD brains showed reduced neurotensin immunoreactivity in amygdaloid regions [18], but little is known of the CSF level of neurotensin in AD. In our study,

CSF neurotensin level was similar in all groups. Furthermore, CSF cortisol level was unaltered. Studies in the early phases of AD have shown either normal [20] or increased [21] CSF cortisol level whereas CSF cortisol level has been increased in established AD [19]. 5. Conclusions Patients with early AD had elevated CSF substance P level, which correlated with biomarkers of AD disease status. Possibly, increased CSF substance P level could represent a compensatory mechanism to protect the AD brain from pathological A␤ exposure. CSF orexin A level was lower in patients with other dementias or SMCI compared to AD patients and marginally lower than in healthy controls. However, the number of patients in the other dementia and SMCI groups was relatively small and further studies are needed to clarify the role of CSF orexin A level in dementing disorders other than AD. Conflict of interest There is nothing to disclose. None of the authors have any conflict of interest.

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Funding This work was supported by the Swedish Research Council (523-2007-7111), the ALF/LUA grant in Gothenburg (ALFGBG146841), the Lundberg Foundation, the Torsten and Ragnar Söderberg’s Foundation, the Lundbeck Foundation, Sahlgrenska University Hospital, Sahlgrenska Academy, Stiftelsen Psykiatriska Forskningsfonden, Stiftelsen Gamla Tjänarinnor, Uppsala Universitets Medicinska Fakultet stiftelse för psykiatrisk och neurologisk forskning, the Alzheimer Foundation, Sweden and the Dementia Association, Sweden. None of the funding sources had any role in the study design; in the collection, analysis and interpretation of data; in the writing of the report; or in the decision to submit the paper for publication. Contributors All authors contributed to the design of the study, to the collection and analysis of data and to drafting, editing and reviewing the manuscript. All authors have approved the final article. Acknowledgements The authors thank Carina Borén at the Department of Neuropsychiatry, Skaraborg Hospital, Falköping and Eva Bringman at the Department of Psychiatry, Sahlgrenska University Hospital, Mölndal, for excellent technical assistance. References ˜ ˜ [1] M. Munoz, R. Covenas, Involvement of substance P and the NK-1 receptor in human pathology, Amino Acids 46 (2014) 1727–1750. [2] E. Kertes, K. László, B. Berta, L. Lénárd, Effects of substance P microinjections into the globus pallidus and central nucleus of amygdala on passive avoidance learning in rats, Behav. Brain Res. 198 (2009) 397–403. [3] X. Liu, S. Shu, C. Zeng, Y. Cai, K. Zhang, C. Wang, J. Wang, The role of substance P in the marginal division of the neostriatum in learning and memory is mediated through the neurokinin 1 receptor in rats, Neurochem. Res. 36 (2011) 1896–1902. [4] R. Hasenöhrl, C. Frisch, S. Nikolaus, J. Huston, Chronic administration of neurokinin SP improves maze performance in aged Rattus norvegicus, Behav. Neural Biol. 62 (1994) 110–120. [5] P. Campolongo, P. Ratano, M. Ciotti, F. Florenzano, S. Nori, R. Marolda, M. Palmery, A. Rinaldi, C. Zona, R. Possenti, P. Calissano, C. Severini, Systemic administration of substance P recovers beta amyloid-induced cognitive deficits in rat: involvement of Kv potassium channels, PLoS One 8 (2013) e78036. [6] M. Beal, M. Mazurek, Substance P-like immunoreactivity is reduced in Alzheimer’s disease cerebral cortex, Neurology 37 (1987) 1205. [7] B. Quigley Jr, N. Kowall, Substance P-like immunoreactive neurons are depleted in Alzheimer’s disease cerebral cortex, Neuroscience 41 (1991) 41–60. [8] H. Cramer, D. Schaudt, K. Rissler, D. Strubel, J. Warter, F. Kuntzmann, Somatostatin-like immunoreactivity and substance-P-like immunoreactivity in the CSF of patients with senile dementia of Alzheimer type, multi-infarct syndrome and communicating hydrocephalus, J. Neurol. 232 (1985) 346–351. [9] M. Martinez, A. Frank, A. Hernanz, Relationship of interleukin-1 beta and beta 2-microglobulin with neuropeptides in cerebrospinal fluid of patients with dementia of the Alzheimer type, J. Neuroimmunol. 48 (1993) 235–240. [10] N. Rösler, I. Wichart, K. Jellinger, Clinical significance of neurobiochemical profiles in the lumbar cerebrospinal fluid of Alzheimer’s disease patients, J. Neural Transm. 108 (2001) 231–246. [11] D. Salin-Pascual, M. Gerashchenko, C. Blanco-Centurion, P. Shiromani, Hypothalamic regulation of sleep, Neuropsychopharmacology 25 (Suppl. 5) (2001) S21–S27.

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