Journal of the Neurological Sciences 369 (2016) 242249

Contents lists available at ScienceDirect

Journal of the Neurological Sciences

journal homepage: www.elsevier.com/locate/jns

Chitinase expression in Alzheimer's disease and non-demented

brains regions
C. Sanlippo a, L. Malaguarnera b, M. Di Rosa b,
a
b

Section of Neurosciences, Department G.F. Ingrassia, University of Catania, Via Santa Soa, 78, 95123 Catania, Italy
Department of Biomedical and Biotechnological Sciences, University of Catania, Italy

a r t i c l e

i n f o

Article history:
Received 21 June 2016
Received in revised form 3 August 2016
Accepted 12 August 2016
Available online 16 August 2016
Keywords:
LOAD
Chitinase
Visual cortex
CHI3L1
CHIT1

a b s t r a c t
Background: Alzheimer disease is the most typical form of dementia. The causes of AD are not yet completely understood, but they include a combination of genetic, environmental and lifestyle factors that inuence ja person's
risk for developing the disease. New biomarkers related to these processes could be important for the diagnosis
and follow-up of AD patients.
Objective: The intent of this study was to weigh the expression levels of chitinases genes in brain regions of lateonset AD (LOAD) patients.
Materials and methods: We analysed three microarray datasets obtained from the NCBI in order to verify the expression levels of chitinase genes family in brain biopsies (CR, DLPFC and VC) of LOAD patients compared to
healthy subjects. We also divided the sample in function of sex difference and ages.
Results: The analysis showed that all chitinases genes were modulated in LOAD brain regions compared to
healthy subjects. Furthermore positively correlation was identied between chitinases gene expression and
healthy age's subjects. Moreover, it has been shown that CHI3L1 and CHI3L2 were regulated differently in healthy
and LOAD brain depending on the sex.
Conclusion: It is possible to conclude that all chitinases could be considered new potential markers for LOAD
disease.
2016 Elsevier B.V. All rights reserved.

1. Introduction
Albeit many efforts to understand the pathogenesis of Alzheimer's
disease (AD) were done, there is no univocal vision about it so far. To
date, after several years of research, Amyloid beta (A) and tau pathology still play a central role in the brain degeneration that leads to AD.
Furthermore, the extracellular A deposits and intracellular tau aggregates are essential to make the denite neuropathological diagnosis of
Alzheimer disease (AD) [1]. Many other proteins and molecular mechanism were taken in consideration to explain the pathogenesis of this
complex and multifactorial disease. Recently immune system and its activation seem to have an interesting role [2]. Accumulating evidence indicates that there is a close link between AD pathogenesis and neuroinammatory cascades in the brain. It is now evident that CNS infection

Abbreviations: CR, cerebellum; VC, visual cortex; DLPFC, dorsolateral prefrontal cortex;
EC, Brodmann's areas; BA, entorhinal cortex; SFG, superior frontal gyrus; HIP,
hippocampus; MTG, middle temporal gyrus; PCG, posterior cingulate cortex; MeV,
MultiExperiment Viewer; CHIT1, chitotriosidase; AMcase or CHIA, acidic mammalian
chitinase; CHI3L1, chitinase 3-like-1; CHI3L2, chitinase 3-like-2; CHID1, chitinase
domaincontaining 1; AD, Alzheimer disease; LOAD, late-onset AD; A, amyloid beta;
A42, -amyloid 42; CLP or chilectins ChiLs, chitinase-like proteins; CTBS, chitobiase;
OVGP1, oviductin.
Corresponding author.
E-mail address: mdirosa@unict.it (M. Di Rosa).

http://dx.doi.org/10.1016/j.jns.2016.08.029
0022-510X/ 2016 Elsevier B.V. All rights reserved.

and neurological diseases generate local inammation and consequently activation of the immune response. In particular, the response to injury is driven by the resident immune cells, the microglia distributed
throughout the normal adult brain [3,4].
Specically, AD is characterized by an inammatory response to A,
inducing the activation of microglia and the enrolment of astrocytes to
the sites where A deposits occur [5]. The role of these activated glial
cells is a eld of great scientic interest as, on the one hand, glial activation has been considered as an endogenous defensive apparatus against
plaque deposition, while on the other hand, the persistent activation
and associated inammation may also contribute to the progression of
AD [6]. Besides the classical CSF biomarker analytics that reect the
core neuro-pathologies in Alzheimer disease, -amyloid (42) (A42 is
the primary constituent of amyloid plaques), total tau (a marker of neuronal damage and/or death), and hyper-phosphorylated tau (P-tau;
forms intra-neuronal neurobrillary tangles), demonstrate outstanding
diagnostic and prognostic utility in research cohorts [7,8]. Other recently identied biomarkers, including chitotriosidase and chitinase-3-like
protein 1 (YKL-40), encompassed to the chitinases family (markers of
macrophage activation and neuroinammation) have also demonstrated clinical utility in AD, especially when combined in an algorithm with
CSF A42 [9,10]. Chitinases represent a class of evolutionarily ancient
enzymes which catalyze the chitin hydrolysis to simple sugars and are
found in bacteria, archaea, protists, fungi, plants and animals [11]. The

C. Sanlippo et al. / Journal of the Neurological Sciences 369 (2016) 242249

human's chitinases have three catalytically active sites and have been
classied on the basis of their function: two endo-chitinases cleaving
the chitin randomly at internal sites, described chitinase1 or
chitotriosidase (CHIT1) and acidic mammalian chitinase (CHIA or
AMCase), and an exochitinases termed chitobiase (CTBS) [1214]. A
family of enzymatically inactive chitinases completely or partially lacking of the catalytic motif have been identied [15]. These enzymes are
collectively designated chitinase-like proteins (CLP) or chilectins
(ChiLs) because they have maintained active-site carbohydrate binding
[16]. The active chitinases are highly conserved across mammals, while
the CLPs identify more novel gene duplication events with subsequent
loss-of-function mutations [17,18]. This emerges a charming situation
in which all mammals express the conserved active enzymes CHIT1
and CHIA but additionally express a broad range of diverse CLPs without
well-dened function [19]. So far, in humans have been identied
four CLPs, termed chitinase-3-like 1 (CHI3L1), oviductin (OVGP1),
chitinase-3-like 2 (CHI3L2) and stabilin-1 interacting chitinase-likeprotein (SI-CLP) or CHID1, which is only distantly related to the others
[20]. Recently, the human chitinase family has come under increasing
investigation due to their excess secretion into the serum or in
tissues that are chronically inamed [21,22]. CHIT1, that is produced
by activated monocyte/macrophages, and patients with Gaucher's disease (lysosomal storage disease) have increased levels in plasma [23].
CSF levels of the enzyme have been assessed in AD patients and nondemented controls, with increased levels found in the rst group,
although with an overlap between the groups [24,25]. Another inammatory biomarker is CHI3L1, a glycoprotein that structurally resembles
chitotriosidase, [26] and is mainly expressed in astrocytes in the brain
[9,27]. Some [9,28], but not all [21], studies have found increased levels
of CHI3L1 in AD patients. Our purpose is to analyze the RNA expression
levels of these proteins in the different brain regions, involved in the degeneration during AD, to better understand their possible role. Overall
how their levels change in the different brain region, comparing AD to
control.

243

neurologically healthy (10 males and 4 females) with a mean age of

79.8 9.1 years and clinically and neuro-pathologically classied
LOAD subjects (15 males and 18 females) with a mean age of 79.9
6.9 years. These brain regions selected are either histo-pathologically
or metabolically pertinent to AD and aging; the entorhinal cortex, superior frontal gyrus, hippocampus, primary visual cortex, middle temporal
gyrus, and the posterior cingulate cortex. Complete experimental details
can be retrieved in the relative publication [32,33].
The MultiExperiment Viewer (MeV) software was used to determine differentially expressed genes and to generate heatmaps of expression genes (Supplementary Fig. 1C). In cases where multiple
probes belonging to the same NCBI GeneID, we selected those with
the highest variance. The expression of gene was indicated as the
mean-log ratio of individual microarray intensities relative to average
intensities of all samples. In order to identify chitinases commonly modulated in different microarray datasets, Venn diagrams' analysis were
performed using the web-based utility Venn Diagram Generator
(http://www.bioinformatics.lu) (Supplementary Fig. 1A).
2.2. Statistical analysis
For statistical analysis, Prism 7 software (GraphPad Software, La
Jolla, CA, USA) was used. Based on Shapiro-Wilk test, almost all data
were skewed, so nonparametric tests were used. Differences between
groups were evaluated using the MannWhitney U test and KruskalWallis test was performed to compare data between all groups followed
by Dunn's post hoc test. Correlations were determined using
Spearman's correlation. All tests were two-sided and signicance
was assigned at p b 0.05. To analyze a 2 2 contingency table, a Chisquare with Yates correction was performed by GraphPad Prism
software (http://graphpad.com/quickcalcs/contingency1.cfm). The association between rows (groups) and columns (outcomes) with
p b 0.0001 was considered to be extremely statistically signicant.
3. Results

2. Materials and methods

2.1. Bioinformatics analysis

3.1. Different expression levels of chitinase-like proteins in CR, PFG and VC

of LOAD patients

In this paper, we wanted to analyze the expression levels of

chitinases genes in different brain regions of late-onset Alzheimer's disease (LOAD) patients. For this reason we have selected the microarray
datasets from the NCBI GEO databank under accession number
GSE44772 (SuperSeries) including the regional/specic Subseries
GSE44768 (CR), GSE44770 (DLPFC), GSE44771(VC). The GSE44772
SuperSeries was composed of 630 tissue samples from cerebellum
(CR), visual cortex (VC) and dorsolateral prefrontal cortex (DLPFC).
These brain regions were chosen because DLPFC is frequently affected
in LOAD while VC and CR remain less affected throughout most of the
disease progression [29]. All autopsied brains were selected from subjects with LOAD diagnosis or from normal non-demented subjects
(healthy). These samples were composed by 129 LOAD patients (67 female and 62 male) and 101 non-demented (19 female and 82 male)
healthy controls. The middle ages for LOAD and healthy men and
woman were very similar (Supplementary Table 1). Complete experimental details can be retrieved in the relative publication [30].
In order to verify the results, we analysed two more microarray
datasets, GSE15222 and GSE5281. From GSE15222 we selected the
data of post mortem prefrontal cortex from LOAD (n = 176 cases) and
healthy donor (n = 187). Patients' samples that had a clinical history
of cerebrovascular disease, stroke, Lewy bodies, or comorbidity with
any other neurological disease were excluded. LOAD or control neuropathology was conrmed by plaque and tangle evaluation. Complete
experimental details can be retrieved in the relative publication [31].
As regard to the microarray dataset GSE5281, this included the
data of six brain regions from individuals clinically identied as

The GSE44772 SuperSeries was used in order to determine the expression levels of the chitinase genes family in LOAD brain regions patients in comparison to individuals who had died from a nonneurological disease (healthy). The results obtained were conrmed
by the Venn diagram analysis of other two microarray dataset
(GSE15222 and GSE5281) (p b 0.0001 by Chi Square with Yates' correction) (Supplementary Fig. 1A). In comparison with healthy controls,
LOAD cases had signicantly high expression levels of all chitinases except for CHID1. We observed signicant high levels of CHI3L1 in all
LOAD brain regions comparing to healthy individuals (p b 0.0001)
(Fig. 1A). The analysis of single brain regions conrmed that CHI3L1
was signicant upregulated in VC (p b 0.0001), CR (p b 0.0001) and
DLPFC (p b 0.0001) of LOAD patients compared to healthy subjects
(Fig. 1A). Furthermore, we showed that in healthy subjects the CHI3L1
levels were signicantly upregulated in DLPFC, VC compared to CR
(p b 0.00001) and in VC compared to DLPFC (p b 0.001) (Fig. 2A). Moreover, in healthy subjects the CHI3L1 expression levels were lower compared to the expression levels of the others chitinases (Fig. 2A). As
regard to the CHI3L1 expression in LOAD brain regions, we showed a
signicant upregulation in VC compared to CR (p b 0.0001) and DLPFC
(p b 0.001) (Fig. 2B). No signicant modulation was observed when
we compared CR to DLPFC.
Similar results were obtained when we compared the CHI3L2 expression levels in brain regions of LOAD patients and healthy subject.
In fact, we showed a signicant upregulation in CR (p b 0.001), DLPFC
(p b 0.0001) and VC (p b 0.0001) of LOAD patients compared to healthy
subjects (Fig. 2B). Furthermore, the healthy brain regions expression

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Fig. 1. Chitinases family expression in brain regions of LOAD patients and healthy subjects. The microarray analysis showed a strongly signicant upregulation of (A) CHI3L1, (B) CHI3L2,
(D) CHIT1, (D) CHIA and (C) CHID1 downregulation in the different brain section (CR, DLPFC and VC) of LOAD patients compared to healthy subject (GSE44772). Data are expressed as log2
intensity expression levels and presented as dot plot. p-Values b0.01 were considered to be statistically signicant (*p b 0.01; **p b 0.001; ***p b 0.0001; ****p b 0.0001).

levels analysis showed that CHI3L2 was signicantly downregulated in

DLPFC (p b 0.00001) and VC (p b 0.0001) compared to CR (p b 0.0001)
(Fig. 2A). This result was conrmed in LOAD brain regions (Fig. 2B).
No signicant modulation was observed when we compared DLPFC vs
VC.
As regard to CHID1 expression levels, we observed opposite results. CHID1 was signicantly downregulated in LOAD patients
(p b 0.0001) compared to healthy subjects (Fig. 1C). The analysis of
single brain regions conrmed that CHID1 levels were signicant
downregulated in VC (p b 0.0001), CR (p b 0.0001) and DLPFC
(p b 0.0001) in LOAD patients compared to healthy subjects
(Fig. 1C). No signicant modulation was observed when we compared CHID1 expression levels in the different brain regions of
healthy subject and LOAD patients (Fig. 2A/B).
3.2. Chitinases with chitinolytic activity are signicantly expressed in VC of
LOAD patients
We also decided to perform an expression levels analysis of
chitinases with enzymatic activity. The CHIT1 and CHIA were signicantly upregulated in LOAD brain regions compared to healthy subjects
(p b 0.01 and p b 0.00001 respectively) (Fig. 1DE). The analysis of single brain regions showed that CHIT1 and CHIA were signicantly
expressed only in VC of LOAD patients (Fig. 1DE) compared to VC of
healthy subjects. When we compared the CHIT1 expression levels in
healthy brain regions, we showed an upregulation in VC compared to

DLPFC (p b 0.01) (Fig. 2A). As regards to CHIA, we also observed a significantly upregulation in DLPFC (p b 0.001) compared to CR (Fig. 2A). As
regard to CHIT1 no signicant modulation was observed when we compared CR vs DLPFC and vs VC (Fig. 2A). As regard to CHIA, no signicant
modulation was observed when we compared CR vs VC and DLPFC vs
VC (Fig. 2A). The LOAD brain regions analysis showed a signicant upregulation of CHIT1 when we compared the expression levels in CR/
DLPFC vs VC (p b 0.00001) (Fig. 2B). Similar results were obtained
when we compared CHIA expression levels in LOAD brain regions (CR
vs DLPFC with p b 0.001; CR vs VC with p b 0.00001; DLPFC vs VC with
p b 0.001) (Fig. 2B).
3.3. Chitinase-like proteins are age-related
We performed a correlation analysis between the chitinases
expression levels and the ages of LOAD and healthy patients.
LOAD and healthy ages were available in the microarray dataset
GSE44772. CHI3L1 and CHI3L2 expression levels were positively
correlated with the ages of healthy subject (r s = 0.4, p b 0.0001
and rs = 0.19, p b 0.0008 respectively) but not with LOAD ages patients (Fig. 3A/B) (Table 1). At the contrary, the CHID1 expression
levels were negatively correlated to healthy ages (and rs = 0.1304,
p = 0.0023) but no signicant correlation with LOAD ages was
found (Fig. 3C; Table 1). When we considered all samples' ages, we
found that the chitinases genes correlated were CHI3L1, CHI3L2,
CHID1 and CHIA (Table 1). Furthermore, we analysed the correlation

C. Sanlippo et al. / Journal of the Neurological Sciences 369 (2016) 242249

245

Fig. 2. Chitinases expression comparison in CR, DLPFC and VC of LOAD patients and healthy subjects. Chitinases expression levels in healthy (A) and LOAD (B) brain regions. Data are
expressed as log2 intensity expression levels and presented as dot plot. p-Values b0.01 were considered to be statistically signicant (*p b 0.01; **p b 0.001; ***p b 0.0001; ****p b 0.0001).

between the chitinases levels in the brain regions of all samples

(Table 2). Positive and negative correlation between the chitinaselike proteins and chitinases with chitinolytic activity was found.

Among the noteworthy results, we observed the strongly positively

correlation between CHI3L1 vs CHI3L2 (p b 0.000001) and CHIT1 vs
CHIA (p = 0) (Table 2). As regard to CHID1, negative correlation

Fig. 3. Chitinase-like proteins correlation with ages of healthy subjects. The LOAD and healthy ages were available in the microarray dataset GSE44772. The CHI3L1 and CHI3L2 expression
levels were positively correlated (CHI3L1, p b 0.0001 and rs = 0.400; CHI3L2 p b 0.0008 and rs = 0.191) with the ages of healthy subject but not with LOAD ages patients. The CHID1
expression levels were negatively correlated to the ages of healthy (p = 0.0023 and rs = 0.1304) but not with LOAD ages patients.

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Table 1
Chitinase expression vs patients' ages.
Gene

CHI3L1

CHI3L2

CHID1

CHIT1

CHIA

Healthy patients' ages

p = 4.23e13
rs = 0.400
p = 0.064
rs = 0.094
p=0
rs = 0.572

p = 0.00083
rs = 0.191
p = 0.067
rs = 0.092
p = 3.36e16
rs = 0.303

p = 0.023
rs = 0.130
p = 0.791
rs = 0.0134
p = 9.37e24
rs = 0.369

p = 0.69
rs = 0.022
p = 0.81
rs = 0.0120
p = 0.055
rs = 0.072

p = 0.52
rs = 0.0368
p = 0.16
rs = 0.070
p = 0.0002
rs = 0.138

AD patients' ages
All patients' ages

with CHI3L1 (p b 0.0001) and positive correlation with CHIT1 and

CHIA (p = 0.0094 and p = 0.0002) were found (Table 2).
3.4. CHI3L1 and CHI3L2 different expression in male and female
CHI3L1 was signicant higher in healthy (p b 0.01) and LOAD
(p b 0.00001) female patients compared to male (Fig. 4A). Similar results were obtained when we analysed the CHI3L2 expression levels
in healthy female brain compared to male (Fig. 4B). The results obtained
were compatible with the signicant increase of CHI3L2 levels in LOAD
brain male compared to the healthy. Surprisingly, no signicantly correlation was observed when we compared the CHI3L2 expression levels
between female healthy vs female LOAD and female vs male in LOAD
group (Fig. 4B).
4. Discussion
In this paper we examine the chitinase family genes expression
levels in autopsied tissues from cerebellum (CR), dorsolateral prefrontal
cortex (DLPFC) and visual cortex (VC) in 230 brains of 129 LOAD

patients and 101 non-demented healthy controls available in

GSE44772 microarray platform. The analysis demonstrates that all
chitinases genes were modulated in LOAD brain regions compared to
healthy subjects (Fig. 5). Furthermore, we have found, positively correlation between chitinase genes expression and healthy age's subjects.
Moreover, we discovered that CHI3L1 and CHI3L2 were regulated differently in healthy and LOAD brain depending on the sex. All these ndings
seem to suggest that the chitinases are related to the AD disease.
In the last years, the chitinases expression analysis was associated to
several neurological diseases [3437]. The CHI3L1, CHI3L2 and CHIT1
are considered novel candidate biomarkers to diagnose, predict and
monitor Alzheimer's disease [24,38,39]. As regard to CHID1 and CHIA
there are no correlation study with Alzheimer's disease.
It has been shown that CHI3L1 and CHI3L2 are selective molecule associated to inammation and in particular to neuro-inammatory damage [37,40,41]. It is known that inammation has an essential role in the
progression of Alzheimer's disease (AD), since amyloid- (A) is able to
recruit microglia, initiating an inammatory response, determining different consequences for neuronal survival. In this contest, CHI3L1 and
CHI3L2 could play an important role in local immuno activation. In the

Table 2
Correlations of chitinase expression.
Gene

CHI3L1

CHI3L2

CHID1

CHIT1

CHIA

CHI3L1

CHI3L2

p = 1.43e22
rs = 0.360
p = 7.35e16
rs = 0.300
p = 0.30
rs = 0.0394
p = 3.14e05
rs = 0.157

p = 1.43e22
rs = 0.360
1

p = 7.35e16
rs = 0.300
p = 0.13
rs = 0.057
1

p = 0.30
rs = 0.039
p = 0.055
rs = 0.0730
p = 0.009
rs = 0.0988
1

p = 3.14e05
rs = 0.157
p = 0.10
rs = 0.061
p = 0.00027
rs = 0.1381
p=0
rs = 0.498
1

CHID1
CHIT1
CHIA

p = 0.13
rs = 0.0573
p = 0.055
rs = 0.0730
p = 0.10
rs = 0.061

p = 0.0094
rs = 0.0988
p = 0.0002
rs = 0.138

p=0
rs = 0.498

Fig. 4. CHI3L1 and CHI3L2 different expression in male and female. (A)The CHI3L1 was signicant higher in healthy (p b 0.01) and LOAD (p b 0.00001) female patients compared to male.
(B) Similar results were obtained when we analysed the CHI3L2 expression levels in healthy female brain compared to male. Dataset accession number GSE44772. Data are expressed as
log2 intensity expression levels and presented as dot plot. p-Values b0.01 were considered to be statistically signicant (*p b 0.01; **p b 0.001; ***p b 0.0001; ****p b 0.0001).

C. Sanlippo et al. / Journal of the Neurological Sciences 369 (2016) 242249

247

Fig. 5. Chitinases expression representation in healthy and LOAD brain regions. Chitinase expression comparison in CR, DLPFC and VC of LOAD patients and healthy subjects. Gene
expression values are color coded from bright red (most upregulated) to dark blue (most downregulated).

current study we have showed that CHI3L1 and CHI3L2 were closely related. The signicant chitinases up-regulation in all LOAD brain regions
exanimated, suggested the important role played in the brain and overall in AD. On the other hand, the analysis of brain regions showed that in
the healthy subject and in LOAD patients, CHI3L1 and CHI3L2 had different expression. In healthy subject and LOAD patients, CHI3L1 was highly
expressed in VC compared to CR and DLPFC. Visual decits are often reported as one of the rst symptoms of AD [42]. Furthermore, it has been
shown that in late-stage Alzheimer's disease, pro-inammatory and
pro-apoptotic genes are expressed into the primary visual sensory cortex [43]. During the normal aging and the AD progression, the increase
of neuroinammation in VC could justify the CHI3L1 upregulation, a
typical pro-inammatory molecule [44]. As regard to CHI3L2 expression
levels, we showed that was upregulated in CR compared to VC and
DLPFC in healthy subjects and LOAD patients. It is likely to note that
CR is the most vulnerable regions to age-dependent loss of volume
[45] and CHI3L2 is associated to cells' proliferation in glioma cells [46].
The CHI3L2 upregulation in healthy CR compared to the other brain regions could play a compensatory reply to age-dependent loss of volume.
Additional analyses that have displayed statistically signicant were
the CHI3L1, CHI3L2 and CHID1 expression levels correlated to the
healthy ages. The positive correlation found for CHI3L1 and CHI3L2 expression and the healthy ages could be explained by the increase of neuroinammation related to the aging. Surprisingly, we found no
correlation in LOAD patients. This discrepancy could be due to the increasing of chitinases levels related to AD inammation. The chitinase
expression high levels in AD patients did not allow discriminating in
function of ages variation.
Regarding the negative correlation found between CHID1 and ages
in healthy subjects, it is very likely to be related to the physiological neuronal death in the elderly [47].
Additionally, during microarray analysis we obtained another interesting result. We found that CHI3L1 was signicant up-regulated in
healthy and LOAD female brain compared to the male. As regard to
CHI3L2 expression, we found signicant upregulation only in healthy
female brain compared to male. It has been shown that the main risk
factors for developing Alzheimer's disease (AD) are age and gender
[48]. The proportion of individuals suffering AD is always higher in
women than in men. An explanation for the high incidence of dementia
in women is that they live longer than men, suffer of obesity, diabetes
and other pathologies, which increase the likelihood of developing AD.
All these pathological condition are linked to the CHI3L1 activation
[4951]. Furthermore, in a large autopsy study it has been shown that
amyloid plaque and neurobrillary tangle pathology were greatest
among women [52,53]. The activated microglia by amyloid plaque,
could increase the CHI3L1 and CHI3L2 levels in LOAD brain and the
subsequent chitinase production could exacerbate the pathological
condition.
In our analysis we have found that CHID1, another chitinase-like
protein, was signicant reduced in LOAD brain regions compared to

healthy brain. No difference in the expression was observed in the comparison of healthy and LOAD brain regions. The CHID1 expression is associated to monocyte/macrophages immuno-activation [20]. There are
no studies that attest the CHID1 modulation in degenerative neurological diseases. It has been demonstrated that high levels of CHID1 were
measured in pediatric brain tumors [54]. The CHID1 results were in contrast to the other chitinases measurements. We showed that CHID1 is
the most upregulated chitinase in the healthy brain. In the past, we
have demonstrated that CHID1 is highly expressed in healthy cerebral
cortex not only in glia cells but also in neuronal cells [54]. It was largely
demonstrated that during the AD progression there is a volume loss of
whole brain [55]. The neuron death in AD patients could justify the
CHID1 reduction.
Additional factors that have displayed statistically signicant were
the CHIT1 and CHIA expression levels in LOAD brain regions. In comparison with healthy subjects, LOAD cases had signicantly higher expression levels of CHIT1 and CHIA in VC compared to CR and DLPFC. AD
patients can present variable range of visual symptoms, from lower
level deciency such as changes in visual eld coverage, color discrimination, contrast sensitivity, visuospatial perception, and visual processing speed [56] to higher level decits such as problems in visual
attention and in recognition of complex objects such as faces [57]. All
these decits could be attributed to a neurodegeneration of visual cortex [58]. In addition, in several publications have found that AD patients
have an axonal degeneration of the optic nerves, a reduction of retinal
ganglion cells [59] and increasing of neurobrillary tangles and neurotic
senile plaques at level of visual cortex [60]. Moreover, it has been hypothesized that chitin-like polysaccharides provide a scaffolding for amyloid-beta deposition [61]. In this context, the CHIT1 and CHIA could
interact with amyloid-beta via chitin-like polysaccharides. Furthermore,
these two chitinases are the only with enzymatic activity. There are several papers regarding the hypothetical role played by CHIT1 as biomarker in Alzheimer's disease and its activity determination in CSF. It seems
reasonable to think that the chitinases' levels in CSF of AD patients could
be determined by the VC microglia immuno secretion during the
neuroinammation.
5. Conclusions
From the outcome of our investigation it is possible to conclude that
chitinases are produced in brain regions of LOAD patients. The CHI3L1,
CHI3L2, CHIT1 and CHIA expression levels could be connected to the
immuno-activation of microglia and as regard to CHID1, could be connected to the neuron death. The expression of CHI3L1 and CHI3L2
could be linked to the increase of neuroinammation during aging.
The CHI3L1 high expression levels discovered in healthy female and in
LOAD brain regions could explain the increase susceptibility to develop
AD in women rather than men.
In light of the complexity of AD pathogenesis, new strategies are
needed to boost the probability of identifying new target genes.

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Supplementary data to this article can be found online at http://dx.

doi.org/10.1016/j.jns.2016.08.029.
Conict of interests
The authors declare that they have no conict of interests.
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