Junling Yang

Jinghong Kou

Robert Lalonde

MD Ken-Ichiro Fukuchi
email: kfukuchi@uic.edu

Intracranial IL-17A overexpression decreases cerebral amyloid angiopathy by upregulation of ABCA1 in an animal

Keywords: Alzheimer's disease, ATP-binding cassette transporters, blood-brain barrier, cerebral amyloid angiopathy, Aβ, interleukin 17, mouse behavior, ERK signaling, endothelial

de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

cells

Introduction

Alzheimer's disease (AD) is the most prevalent form of dementia (Fratiglioni et al., 2000;[START_REF] Kawas | Age-specific incidence rates of Alzheimer's disease: the Baltimore Longitudinal Study of Aging[END_REF][START_REF] Kukull | Dementia and Alzheimer disease incidence: a prospective cohort study[END_REF], characterized by extracellular amyloid β (Aβ) deposition in the brain parenchyma and cerebral blood vessels. Additionally, neuroinflammation is a consistent feature of AD and thought to influence the disease pathogenesis [START_REF] Mcmanus | Respiratory infection promotes T cell infiltration and amyloid-beta deposition in APP/PS1 mice[END_REF]. Immune responses occurring in brains of AD patients can be driven by resident immune cells and/or brain infiltrating peripheral immunocompetent cells [START_REF] Ghosh | Stress granules modulate SYK to cause microglial cell dysfunction in Alzheimer's disease[END_REF][START_REF] Togo | Occurrence of T cells in the brain of Alzheimer's disease and other neurological diseases[END_REF]. Microglia, the innate immune cells in the central nervous system (CNS), are activated by Aβ deposits and secrete inflammatory cytokines and chemokines, contributing to AD progression [START_REF] Hickman | Microglial dysfunction and defective beta-amyloid clearance pathways in aging Alzheimer's disease mice[END_REF][START_REF] Ridolfi | The role of the innate immune system in Alzheimer's disease and frontotemporal lobar degeneration: an eye on microglia[END_REF][START_REF] Rodriguez | Complex and differential glial responses in Alzheimer's disease and ageing[END_REF].

Central-memory CD4 + T lymphocytes account for more than 80% of the cells in the cerebrospinal fluid (CSF) and routinely penetrate the CNS. Lymphocytes in the CNS are replenished by newly immigrating cells approximately twice per day [START_REF] Engelhardt | The ins and outs of T-lymphocyte trafficking to the CNS: anatomical sites and molecular mechanisms[END_REF][START_REF] Hickey | Basic principles of immunological surveillance of the normal central nervous system[END_REF][START_REF] Smolders | Characteristics of differentiated CD8(+) and CD4 (+) T cells present in the human brain[END_REF]. Numerous lines of evidence indicate the presence of T cells in the brains of AD patients [START_REF] Ferretti | T-cell brain infiltration and memory deficits in a mouse model of Alzheimer's disease[END_REF][START_REF] Lombardi | Characterization of cytokine production, screening of lymphocyte subset patterns and in vitro apoptosis in healthy and Alzheimer's Disease (AD) individuals[END_REF][START_REF] Monsonego | Increased T cell reactivity to amyloid beta protein in older humans and patients with Alzheimer disease[END_REF][START_REF] Shalit | T lymphocyte subpopulations and activation markers correlate with severity of Alzheimer's disease[END_REF][START_REF] Togo | Occurrence of T cells in the brain of Alzheimer's disease and other neurological diseases[END_REF] although their possible roles in AD pathogenesis have been studied less than those of microglia. T cells in the brains of AD patients increased as compared with subjects with non-AD degenerative dementias and age-matched controls [START_REF] Lueg | Clinical relevance of specific T-cell activation in the blood and cerebrospinal fluid of patients with mild Alzheimer's disease[END_REF][START_REF] Togo | Occurrence of T cells in the brain of Alzheimer's disease and other neurological diseases[END_REF]. T cells breaching the brain vasculature and parenchyma can be either detrimental or beneficial for brain homeostasis [START_REF] Kipnis | T cell deficiency leads to cognitive dysfunction: implications for therapeutic vaccination for schizophrenia and other psychiatric conditions[END_REF][START_REF] Monsonego | Abeta-induced meningoencephalitis is IFNgamma-dependent and is associated with T cell-dependent clearance of Abeta in a mouse model of Alzheimer's disease[END_REF][START_REF] Ziv | Immune cells contribute to the maintenance of neurogenesis and spatial learning abilities in adulthood[END_REF]. Depletion of regulatory T (Treg) cells in an AD mouse model accelerated the onset of cognitive deficits without altering brain Aβ load [START_REF] Dansokho | Regulatory T cells delay disease progression in Alzheimer-like pathology[END_REF]. Administration of Aβ-specific type 1 T helper (Th1) cells into the brain lateral ventricle of an AD mouse model enhanced removal of cerebral Aβ deposits and promoted neurogenesis without harmful effects (Fisher et al., 2014). Thus, these T cells play beneficial roles in the AD pathophysiology. In contrast, intravenous administration of Aβ-specific Th1 cells caused brain infiltration, increased microglial activation and Aβ deposition, and impaired cognitive function in an AD mouse model but similarly administered Aβ-specific IL-17-producing helper T (Th17) cells infiltrated into the brain without exacerbating microglia activation, Aβ deposition and cognitive deficits in the same mice [START_REF] Browne | IFN-gamma production by amyloid beta-specific Th1 cells promotes microglial activation and increases plaque burden in a mouse model of Alzheimer's disease[END_REF].

IL-17A or IL-17 is a signature cytokine of Th17 cells and is involved in antimicrobial host defense and inflammation. A plethora of evidence supports the notion that Th17 cells and IL-17A play a pathogenetic role in certain autoimmune diseases such as multiple sclerosis, rheumatoid arthritis, psoriasis, and inflammatory bowel disease. Respiratory infection with Bordetella pertussis in an AD mouse model [START_REF] Mcmanus | Respiratory infection promotes T cell infiltration and amyloid-beta deposition in APP/PS1 mice[END_REF] and intrahippocampal administration of Aβ42 into rats [START_REF] Zhang | Th17 cell-mediated neuroinflammation is involved in neurodegeneration of abeta1-42-induced Alzheimer's disease model rats[END_REF] induced infiltration of Th17 cells and expression of IL-17A in the brain, which were associated with exacerbation of an AD-like phenotype in these experimental animals. It is interesting to note that plasma IL-17A levels have been identified as one of the plasma biomarkers for AD diagnosis and neocortical Aβ load [START_REF] Burnham | A blood-based predictor for neocortical Abeta burden in Alzheimer's disease: results from the AIBL study[END_REF][START_REF] Doecke | Blood-based protein biomarkers for diagnosis of Alzheimer disease[END_REF]. However, the role of IL-17A in AD pathophysiology is unclear. In this study, we investigated the effects of IL-17A overexpression via intracranial delivery of recombinant adeno-associated virus serotype 5 (rAAV5)-mediated IL-17A on Aβ load, microglia activation, glucose metabolism and behavioral function using a transgenic animal model of AD.

Material and Methods

Experimental animals and intracranial injection of rAAV-IL-17A

A rAAV vector, rAAV5-IL-17A, was prepared as described previously [START_REF] Yang | Intracranial delivery of interleukin-17A via adeno-associated virus fails to induce physical and learning disabilities and neuroinflammation in mice but improves glucose metabolism through AKT signaling pathway[END_REF]. A control rAAV vector encoding LacZ, rAAV5-LacZ, was similarly prepared.

B6.Cg-Tg (APPswe, PSEN1dE9) 85Dbo/J mice (TgAPPswe/PS1dE9 mice) purchased from Jackson Laboratory, were used as an AD mouse model to study a potential role of IL-17A in the AD pathogenesis. For intracranial injection, 2-month-old TgAPPswe/PS1dE9 mice were anesthetized by Nembutal® (pentobarbital) and placed on a stereotaxic instrument with a motorized stereotaxic injector (Stoelting, Wood Dale, IL). A midline incision was made to expose the bregma. A hole in the skull was made by a drill 0.5 mm posterior to the bregma and 1.0 mm right to the midline. rAAV5-IL-17A

(1.5 x 10 10 vector genomes (vg) in 10 µl PBS/mouse) was injected unilaterally into the right lateral brain ventricle at the depth of 2 mm at a rate of 1 µl/min. Mice subjected to rAAV5-IL-17A injection are referred to as IL-17A-overexpressing mice (n=8). Mice similarly treated with 10 µl PBS or rAAV5-LacZ (1.5 x 10 10 vg/mouse) served as controls and are referred to as control mice (n=9): Four PBS-injected and 5 rAAV5-LacZ-injected mice were combined together because no phenotypic differences between 2 controls were found (more information in Supplementary Figure 1). Due to the difference in brain amyloid content between male and female TgAPP/PS1 mice [START_REF] Akhter | Cyclic ozone exposure induces gender-dependent neuropathology and memory decline in an animal model of Alzheimer's disease[END_REF]Gallagher et al., 2013;[START_REF] Sierksma | Behavioral and neurobiological effects of prenatal stress exposure in male and female APPswe/PS1dE9 mice[END_REF][START_REF] Taniuchi | Decreased proliferation of hippocampal progenitor cells in APPswe/PS1dE9 transgenic mice[END_REF][START_REF] Wang | Gender differences in the amount and deposition of amyloidbeta in APPswe and PS1 double transgenic mice[END_REF], we used only male mice for this study. All animal protocols were prospectively approved by the Institutional Animal Care and Use Committee of the University of Illinois College of Medicine at Peoria.

Behavioral tests

Behavioral schedule and statistics

A battery of behavioral tests was performed on experimental mice at 12 months of age as previously described [START_REF] Lim | MyD88 deficiency ameliorates beta-amyloidosis in an animal model of Alzheimer's disease[END_REF]. After measuring body weight and adapting mice to handling, the tests were conducted over a 16-day period as follows:

spontaneous alternation (days 1-10), open-field (days 6-8), elevated plus-maze (days 9 and 10), and Morris water maze (days 11-16). In each test, whenever possible, the apparatus was wiped clean with a wet cloth and dried before the next mouse was introduced to minimize odor cues. For T-maze, Mann-Whitney U test was used to assess the chance of alternation compared with theory (50%) and unpaired t-test was used for the intergroup differences. For open-field, the intergroup differences were assessed by 2 x 3 analysis of variance (ANOVA) for 2 independent groups and 3 days of testing with repeated measures on the second factor. The unpaired t-test was used for the elevated plus-maze and the probe test of the Morris water maze and a 2 x 5 ANOVA for 2 independent groups and 5 days of testing with repeated measures on the second factor was used for 5-day acquisition of the Morris water maze. Results are expressed as mean ± standard error of the mean (SEM). In all cases, P < 0.05 was considered to be significant.

Exploration and anxiety

Spontaneous alternation was measured in a T-maze, made of white acrylic and consisting of a central stem flanked on each side by 2 arms. The maze width was 9 cm, the wall height 20 cm, and each arm 30 cm in length. On the initial trial, the mice were placed in the stem with the right arm blocked by a plastic barrier (forced choice). After entering the available arm, the mice were kept in it for 1 min by closing the barrier behind them. The mice were then retrieved and after removing the barrier, placed back in the stem for a free-choice trial, either into the same arm or the opposite arm (4-paw criterion). On the following 10 days, the same 2-trial procedure was repeated, except that the blocked arm was switched from right on odd days to left on even days.

The number of alternations and the latencies before responding during the choice trial were measured. In the absence of any decision within 1 min, the mice were briefly prodded from behind, usually not more than once, far from the choice point so that a response could be recorded on every trial.

Motor activity was measured in the open-field chamber that was made of white acrylic with a 50 cm × 50 cm surface area. Each mouse was placed in a corner of the open-field. The activity in central (25 cm × 25 cm surface area) and peripheral zones was recorded in a 5-min session for 3 consecutive days and analyzed by video tracking software (SD Instruments, San Diego, CA). The distance traveled and the time spent resting (<2 cm/s), moving slow (2-5 cm/s), or moving fast (>5 cm/s) in each zone were measured, as well as the time spent in the periphery and center of the apparatus.

The elevated plus-maze apparatus consisted of 4 arms in a cross-shaped form 70 cm in length with a 10 cm × 10 cm central region. Two of the arms were enclosed on 3 sides by walls (10 cm in height) facing each other, while the other two were open, except for a minimal border (0.5 cm in height) used to minimize falls. A mouse was placed in the central region and then the number of entries and the time spent inside enclosed and open arms were measured in a 5-min session on 2 consecutive days with the same video tracking system. The open/total arm entries and duration ratios were also calculated.

Spatial learning and memory

The Morris water maze consisted of a pool of blue opaque plastic, 116 cm in diameter with 75-cm high walls, filled with water (20°C) at a height of 31 cm. Powdered milk was evenly spread over the water surface to camouflage the escape platform (8 x 8 cm) made of white plastic and covered with a wire mesh grid to ensure a firm grip. The watered milk was removed every day after a few hours of training and the pool rinsed with clean water. The pool was contained in a room with external visual cues such as light fixtures and a ladder. The mice were placed next to and facing the wall successively in north (N), east (E), south (S), and west (W) positions, with the escape platform hidden 1 cm below water level in the middle of the NW quadrant. The same video tracking equipment was used to estimate path length and escape latencies in 4trial sessions for 5 days with a 15-min inter-trial interval. The mice remained on the platform for at least 5 s. When the mice failed to reach the escape platform within the 1 min cut-off period, they were retrieved from the pool and placed on the platform for 5 s.

After swimming, the mice were kept dry in a plastic holding cage filled with paper towels.

In the morning after the acquisition phase, a probe trial was conducted by removing the platform and placing the mouse next to and facing the N side. The time spent in the previously correct quadrant was measured for a single 1-min trial. In the afternoon, the visible platform subtask was conducted, with the escape platform lifted 1 cm above water level and shifted to the SE quadrant. A 17-cm high pole was inserted on top of the escape platform as a viewing aid. The same procedure was adopted as with place learning except that the subtest was conducted on a single day.

Intraperitoneal glucose tolerance test (IPGTT) and insulin tolerance test (ITT)

Forty-two and forty-four weeks after rAAV injection, IPGTT and ITT were conducted, respectively, as previously described [START_REF] Ize-Ludlow | Progressive erosion of beta-cell function precedes the onset of hyperglycemia in the NOD mouse model of type 1 diabetes[END_REF]. Animals were fasted for 16 h, weighed and intraperitoneally injected with D-glucose (1.5 g/kg) for IPGTT. Blood was obtained using the tail nick method before and at 30, 60, 90 and 120 min after the D-glucose injection for monitoring glucose levels using Glucometer (ReliOn® Prime, Arkray USA, Minneapolis MN). Plasma insulin levels were determined by an insulin ELISA kit (Crystal Chem Inc., Downers Grove, IL). For ITT, mice were fasted for 4 h, weighed, and intraperitoneally injected with human insulin (0.75 units/kg; Novolin® R, Novo Nordisk, Princeton, NJ). Blood sugar was monitored before and at 15, 30, 60, and 90 min.

Murine CSF isolation

Forty-four weeks after rAAV5-IL-17A intracranial injection, mice were deeply anesthetized with Nembutal® (pentobarbital) and CSF was isolated from the cisterna magna compartment using the method previously described [START_REF] Demattos | Plaque-associated disruption of CSF and plasma amyloidbeta (Abeta) equilibrium in a mouse model of Alzheimer's disease[END_REF].

Mice were anesthetized by Nembutal® (pentobarbital) and fixed face down on a narrow platform. An incision was made from the top of the skull to the dorsal thorax. The musculature from the base of the skull to the first vertebrae was carefully removed to expose the meninges overlying the cisterna magna. The surrounding area was gently cleaned with PBS using cotton swabs to remove any residual blood or other interstitial fluid. The arachnoid membrane covering the cistern was punctured with a 29 gauge insulin syringe. A polypropylene narrow bore pipette was immediately placed in the hole to collect CSF. As the primary CSF exiting the compartment was collected, a second collection was performed after the cistern was refilled within 2 min. About 10 to 15 µl CSF was collected from each mouse.

Immunohistochemical analyses

Forty-four weeks after rAAV5-IL-17A intracranial injection, mice were deeply anesthetized with Nembutal® (pentobarbital) and cardinally perfused with cold PBS and their brains were quickly removed. The neocortices and hippocampi of the left hemispheres were separately dissected and stored at -80°C for further studies. The right hemispheres were fixed in 4% paraformaldehyde for 48 h, stored overnight in 30% sucrose in 0.1 M PBS and frozen in Tissue-Tek optimal cutting temperature compound.

Coronal sections (35 µm) of the brains were immuno-stained with anti-IBA1 antibody (Wako, Osaka, Japan) for detection of activated microglia. Endogenous peroxidase was eliminated by 1% H 2 O 2 in 10% methanol Tris-buffered saline (TBS) treatment. After washing with 0.1 M Tris buffer (pH 7.5) and 0.1M TBS (pH 7.4), sections were blocked with 5% normal serum in 0.1 M TBS with 0.5% triton-X-100 (TBS-T) and then incubated with primary antibodies in 2% serum in TBS-T for 18-48 h at 4ºC. For the negative controls, slides were processed without primary antibody. After rinsing, the sections were incubated with biotinylated secondary antibodies in 2% serum TBS-T for 2 h at room temperature. The avidin-biotin peroxidase method using 3, 3'-diaminobenzidine as a substrate (Vector, Burlingame, CA) was performed. Sections were counterstained with hematoxylin. For the detection of fibrillar Aβ plaque, tissue sections were stained in 1% thioflavin S (Sigma) and rinsed with 70% ethanol. After washing with H 2 O, the sections were mounted in 75% glycerol in H 2 O. Histomorphometry was performed for quantification of amyloid deposits and reactive/activated glial cells using an Olympus BX61 automated microscope, Olympus Fluoview system, and Image Pro Plus v4 image analysis software (Media Cybernetics, Silver Spring, MD, USA) capable of color segmentation and automation via programmable macros. Five coronal brain sections from each mouse were analyzed, each separated by an approximately 400-µm interval, starting at 1.3 mm posterior to the bregma to caudal. Both neocortex and hippocampus were found in all the brain sections and analyzed separately. Stained areas were expressed as a percentage of total neocortex or hippocampus, respectively. For cerebral amyloid angiopathy (CAA), thioflavin S-positive blood vessels in 3 to 4 right hemisphere sections for each mouse were enumerated and expressed as a number of thioflavin S-positive blood vessels per square millimeter. Data were expressed as mean ± SEM [START_REF] Kou | Catalytic immunoglobulin gene delivery in a mouse model of Alzheimer's disease: prophylactic and therapeutic applications[END_REF].

Quantification of plasma and CSF IL-17A by ELISA

Approximately 100 µl of blood/mouse was drawn from the tail vein at 3, 7, and 11 months after injection by using microhematocrit heparin tubes (Fisher Scientific, Pittsburgh, PA) and centrifuged for 20 min at 2,000 xg for plasma isolation. Levels of IL-17A in plasma and CSF were determined by ELISA (eBioscience, Inc. San Diego, CA).

Quantification of brain Aβ and CSF by ELISA, and ABCA1 by western blotting

The neocortex and hippocampus were removed from -80°C, lysed using the Bio-Plex cell lysis kit (Bio-Rad Laboratories, Hercules, CA) and homogenized according to the manufacturer's protocol, and centrifuged at 16,000 ×g for 30 min at 4°C. The supernatants containing buffer soluble Aβ were collected and the protein concentrations in the supernatants were determined by Bio-Rad Protein Assay (Bio-Rad Laboratories, Hercules, CA). The pellets containing insoluble Aβ were further dounce homogenized in guanidine hydrochloride (final concentration, 5 M) and then rock-shaken for 3 to 4 h at room temperature. Levels of buffer soluble and insoluble Aβ in the neocortex and hippocampus, and Aβ in the CSF were quantified by Aβ42 and Aβ40 ELISA kits (Invitrogen, Carlsbad, CA) according to the manufacturer's protocol.

The protein lysates were used to determine ABCA1 levels by western blotting, also. After determining protein concentrations, the proteins were electrophoresed under reducing conditions in 10% SDS-PAGE gels and transferred to PVDF membranes. The membranes were incubated overnight at 4°C anti-ABCA1 antibody (1:1000 dilution) (Abcam, Cambridge, MA), or anti-β-actin antibody (Sigma, St. Louis, MO) and specific bands were visualized by an enhanced chemiluminescence system (Amersham, Arlington Heights, IL). The optical densities of the protein bands were determined by densitometric scanning using an HP Scanjet G3010 Photo Scanner and Image J V1.40 (NIH, MD).

Cell culture and western blot

The bEnd.3 cells (ATCC® CRL2299™) were grown in ATCC-formulated Dulbecco's modified eagle's medium containing 10% fetal bovine serum (Atlanta Biologicals, Flowery Branch, GA) in a humidified 37°C incubator with a 5% CO 2 atmosphere. Cells were plated on culture dishes at 4 x 10 4 /ml cells. Forty-eight hours after seeding, bEnd.3 cells were treated with 0, 1, 10, 100 ng/ml of IL-17A (Cell Signaling Technology, Danvers, MA) for 24 h to determine ABCA1 protein expression.

Other sets of bEnd.3 cells were treated with 100 ng/ml of IL-17A for 0 (no IL-17A), 5, 15 and 30 min to detect the activation of p-ERK1/2. Different sets of bEnd.3 cells were pretreated with U0126 (25 µM) or DMSO (vehicle) for 1 h followed by treatment with or without IL-17A (100 ng/ml) for 30 min or 24 h to determine expression levels of ERK or ABCA1, respectively. The cell lysates were prepared by using radioimmune precipitation assay (RIPA) lysis buffer containing complete mini-protease-inhibitor and phosphatase inhibitor cocktail tablets (Roche Diagnostics Corporation, Indianapolis, IN) and centrifuged at 12,000 xg for 10 min at 4°C for collecting supernatants. After determining protein concentrations, the proteins were electrophoresed under reducing conditions in 10% SDS-PAGE gels and transferred to PVDF membranes. The membranes were incubated overnight at 4°C with anti-phospho-ERK1/2, anti-ERK1/2 antibody (1:1000 dilution) (Cell Signaling Technology, Danvers, MA), anti-ABCA1, or anti-β-actin antibody and specific bands were visualized by an enhanced chemiluminescence system (Amersham, Arlington Heights, IL). The optical densities of the protein bands were determined by densitometric scanning using an HP Scanjet G3010 Photo Scanner and Image J V1.40 (NIH, MD). The optical density of p-ERK1/2 band was divided by that of total ERK1/2 band on the same lane from the same membrane for normalization. β-actin was used to normalize expression levels of ABCA1.

Statistical analysis

Data were expressed as mean ± SEM. The data were analyzed using SPSS version 22 software (IBM, Armonk, NY). One-way ANOVA followed by a leastsignificant-difference test was used for statistical comparisons among multiple groups and t-test for statistical comparisons between two groups. P values less than 0.05 were considered statistically significant.

Results

Overexpression of IL-17A via intracranial delivery of rAAV5 improves glucose metabolism

The bioactivity of IL-17A produced by the AAV-IL-17A vector was confirmed using 3T3 cells described previously [START_REF] Yang | Intracranial delivery of interleukin-17A via adeno-associated virus fails to induce physical and learning disabilities and neuroinflammation in mice but improves glucose metabolism through AKT signaling pathway[END_REF]. To explore the potential role of IL-17A in AD, we intracranially delivered rAAV5-IL-17A into 2-month-old TgAPPswe/PS1dE9 mice. Three, seven, and eleven months after injection, the average plasma IL-17A levels in IL-17A-overexpressing mice were 14.88 ± 1.95, 14.69 ± 1.28, and 9.31 ± 0.95 ng/ml respectively and the average CSF IL-17A level in IL-17Aoverexpressing mice was 270.73 ± 48.83 ng/ml 11 months after injection, while IL-17A was undetectable in control mice by ELISA (Figure 1A). The regional distribution of rAAV5 in brain was found mostly in hippocampal neuronal cells determined by beta-gal immunostaining (supplementary Figure 2A andB). Consistently, the copy number of rAAV5 in hippocampus is 2.5-fold and 12-fold higher than those in cortex and in cerebellum by qPCR method, respectively (supplementary Figure 2C) (more information in supplementary material and methods). And the distribution of rAAV5 in peripheral tissues, heart, lung, liver, spleen, kidney and intestine were undetectable by using the current method (data not shown).

Blood glucose and insulin levels in IL-17A-overexpressing mice were significantly lower than those in control mice throughout IPGTT (Figure 1B andC). There was no significant difference in ITT between groups (data not shown).

Overexpression of IL-17A improves anxiety and spatial learning but not long-term memory

Exploration and anxiety

In the T-maze spontaneous alternation test, mice in each group alternated above chance (P<0.05) (Figure 2A) and choice latencies showed no difference between groups (Figure 2B). In the open-field, IL-17A-overexpressing mice had a higher center/total time ratio than control mice (Figures 3A andB) (P<0.01). Resting time and moving slow time increased over days in both groups, whereas moving fast time decreased over days (Figures 3C andD). There was a significant group x day interaction, because moving slow time increased and moving fast time decreased relatively more in IL-17A-overexpressing mice than controls (Figures 3D and3E). In contrast, distance travelled declined equally in both groups (Figure 3F). In the elevated plus-maze test, open arm duration was higher in IL-17A-overexpressing mice on day 1 but open/total arm entries lower in that group on day 2 (P<0.05) (Table 1).

Spatial learning and memory

In the acquisition phase of the Morris water maze test, there were significant reductions in latencies and path lengths between days in both groups (P<0.05) (Figure 4A andB). Furthermore, escape latencies in IL-17A-overexpressing mice were quicker than control mice (P<0.05) which is an overall group effect irrespective of days (Figure 4A). In the probe trial, the percentages of time spent in the correct quadrant were 27.40 ± 3.09 s and 26.73 ± 3.58 s for IL-17A-overexpressing and control mice, respectively (P>0.05) (Figure 4C). There were no intergroup differences in the visible platform subtask, also (data not shown).

Overexpression of IL-17A in mouse brain does not increase microglia activation but reduces Aβ in CSF and CAA

Fibrillar Aβ deposits are closely associated with activated microglia. Microglia can be activated by various endogenous and exogenous factors through their receptors including toll-like receptors, scavenger receptors, and numerous cytokine and chemokine receptors [START_REF] Kierdorf | Factors regulating microglia activation[END_REF]. IL-17A overexpression may aggravate microglia activation in an AD mouse model. Activation of microglia is characterized by cell retraction and thickening and increased body size. Microglial cell body to cell size ratio has been shown to be positively correlated with microglial activation using IbaI immunostaining (Hovens IB, Nyakas C, Schoemaker RG. A novel method for evaluating microglial activation using ionized calcium-binding adaptor protein-1 staining: cell body to cell size ratio. Neuroimmunol Neuroinflammation 2014;1(2):82-8.).

Therefore, the brain sections were subjected to immunohistochemical analysis with anti-Iba1 antibody for detection of activated microglia. Microglia stained with Iba1 did not increase in IL-17A-overexpressing mice (Figure 5A). Microglial cell body to cell size ratio (Figure 5B) and the total number of microglial cells did not show significant differences between groups (Figure 5C).

To determine the effect of IL-17A overexpression on Aβ load in the brain, Aβ deposits were visualized by thioflavin S fluorescence and quantified by morphometry (Figure 6A andB). The areas positive for thioflavin S fluorescence in the neocortex and hippocampus showed no statistic differences between groups: 0.36 ± 0.03% and 0.27 ± 0.03% for IL-17A-overexpressing mice, 0.37± 0.03% and 0.21 ± 0.22% in control mice, respectively. However, both CSF Aβ40 and Aβ42 were dramatically decreased in IL-17A-overexpressing mice (1.88 ± 0.37 and 0.50 ± 0.09 ng/ml, respectively) compared with that in control mice (7.35 ± 1.58 and 2.05 ± 0.35 ng/ml, respectively) (P<0.01 and P<0.001, respectively) (Figure 6C). Hippocampal soluble Aβ40 levels in IL-17Aoverexpressing mice (0.20 ± 0.05 ng/mg protein) were also significantly decreased compared with that in control mice (0.81 ± 0.39 ng/mg protein) (P<0.05) while no differences were found in hippocampal soluble Aβ42 levels and in neocortical soluble Aβ40 and Aβ42 levels between groups (Figure 6D andE). No differences were found in insoluble Aβ40 and Aβ42 levels in the neocortex and hippocampus between groups (Figure 6F and G) (P>0.05).

Vascular Aβ deposition (CAA) is associated with vascular dysfunction and thought to play an important role in the AD pathogenesis (de la [START_REF] De La Torre | Is Alzheimer's disease a neurodegenerative or a vascular disorder? Data, dogma, and dialectics[END_REF][START_REF] Smith | Beta-amyloid, blood vessels, and brain function[END_REF]. We enumerated the number of blood vessels positive for thioflavin S fluorescence in the right neocortices. The number of thioflavin S stained blood vessels in IL-17A-overexpressing mice (0.05 ± 0.02/mm 2 ) considerably decreased compared with that in control mice (0.24 ± 0.06) (Figure 6H and I) (P<0.01).

IL-17A increases ABCA1 expression by ERK1/2 activation

In order to investigate possible mechanisms by which IL-17A decreases Aβ in CSF and CAA, protein levels of ABCA1 in IL-17A-overexpressing mice were determined by immunoblot analysis. Hippocampal ABCA1 protein increased in IL-17Aoverexpressing mice compared with control mice (P<0.05) (Figure 7 A and B), while there were no changes in cortex (data not shown). To further explore signal pathways of the increase in ABCA1 by IL-17A a mouse brain endothelial cell line, bEnd.3 cell, was used to determine the level of ERK1/2. Immunoblot analysis consistently revealed a dose-dependent increase in ABCA1 expression by IL-17A treatment (control vs 100 ng/ml IL-17A, P<0.05) (Figure 8A). Moreover, phospho-ERK1/2 (p-ERK1/2) levels were significantly increased in bEnd.3 cells (P<0.05) by 100 ng/ml IL-17A (Figure 8B). U0126 (25 µM), an ERK1/2 kinase inhibitor, completely inhibited both the activation of ERK1/2 and the upregulation of ABCA1 expression by IL-17A (Figure 8C and D) (P<0.05).

Discussion

IL-17A is produced by Th17 cells and other immune cells, such as neutrophils, macrophages, dendritic cells, and natural killer, lymphoid tissue inducer and γδ-T cells [START_REF] Korn | IL-17 and Th17 Cells[END_REF][START_REF] Zenaro | Neutrophils promote Alzheimer's disease-like pathology and cognitive decline via LFA-1 integrin[END_REF]. Th17 cells infiltrate into the brain parenchyma in AD patients and mouse models, suggesting that Th17 cells may contribute to neuroinflammation by release of proinflammatory cytokines IL-17A and IL-22 (McManus et al., 2014;[START_REF] Zhang | Th17 cell-mediated neuroinflammation is involved in neurodegeneration of abeta1-42-induced Alzheimer's disease model rats[END_REF]. IL-17A is released by neutrophils surrounding Aβ deposits in two transgenic mouse models of AD (5XFAD and 3xTg-AD mice), which suggests that IL-17A may be involved in AD pathogenesis and cognitive impairment [START_REF] Zenaro | Neutrophils promote Alzheimer's disease-like pathology and cognitive decline via LFA-1 integrin[END_REF]. However, the role of the IL-17A in the AD brain has not been well studied. To the best of our knowledge, this is the first study to demonstrate the effects of IL-17A overexpression on AD-like pathophysiology in an AD mouse model. The distribution of rAAV5 was mainly expressed in the brain not in peripheral tissues (such as heart, lung, liver, spleen, kidney and intestine). However, plasma IL-17A levels in IL-17A overexpressing mice were one twenty-ninth of the CSF IL-17A levels. There exist several possibilities. Despite of undetectable viral in peripheral tissue, we still cannot exclude the possibility that a few AAV5 leaked into brain vessels during intraventricular injection. On the other hand, because the blood-brain barrier plays an important role in regulating the rate of transport in (K in ) and out (K out ) of the brain (PMID: 24009582), the difference in K in and/or K out may be the main cause to distribution of IL-17A in CSF and plasma in IL-17A overexpressing mice.

Insulin signaling impairment/ insulin resistance (IR) is a common and important pathophysiologic alteration between type 2 diabetes mellitus (T2DM) and AD [START_REF] Craft | Insulin and neurodegenerative disease: shared and specific mechanisms[END_REF][START_REF] Sebastiao | Insulin as a bridge between type 2 diabetes and Alzheimer disease -How anti-diabetics could be a solution for dementia[END_REF][START_REF] Verdile | Inflammation and oxidative stress: The molecular connectivity between insulin resistance, obesity, and Alzheimer's disease[END_REF], although the cellular and molecular mechanism that associates T2DM with AD is unknown [START_REF] Rios | Is Alzheimer's disease related to metabolic syndrome? A Wnt signaling conundrum[END_REF]. IR may be one of the causes of AD. Hyperinsulinemia/insulin resistance may lead to alterations of protein expression or posttranslational modification central to AD pathogenesis, including tau, Aβ and the Aβ-degrading protease neprilysin [START_REF] Morales-Corraliza | Brain-wide insulin resistance, tau phosphorylation changes, and hippocampal neprilysin and amyloid-beta alterations in a monkey model of Type 1 diabetes[END_REF]. A longitudinal study found that peripheral IR is associated with a higher risk of AD [START_REF] Schrijvers | Insulin metabolism and the risk of Alzheimer disease: the Rotterdam Study[END_REF]. IR in the CNS is involved in AD progression [START_REF] Biessels | Hippocampal insulin resistance and cognitive dysfunction[END_REF] and intranasal insulin delivery improved cognitive function in a subset of patients with AD or mild cognitive impairment [START_REF] Benedict | Intranasal insulin as a therapeutic option in the treatment of cognitive impairments[END_REF][START_REF] Claxton | Long-acting intranasal insulin detemir improves cognition for adults with mild cognitive impairment or early-stage Alzheimer's disease dementia[END_REF][START_REF] Craft | Intranasal insulin therapy for Alzheimer disease and amnestic mild cognitive impairment: a pilot clinical trial[END_REF]. Inversely, AD may be one of the causes of IR.

Fasting glucose levels and/or insulin sensitivity are altered in 81% of AD patients suggesting that AD may lead to a diabetic phenotype [START_REF] Janson | Increased risk of type 2 diabetes in Alzheimer disease[END_REF]. A recent study demonstrated that intracerebroventricular infusion of Aβ oligomers induces peripheral insulin resistance possibly by triggering neuroinflammation [START_REF] Clarke | Alzheimer-associated Abeta oligomers impact the central nervous system to induce peripheral metabolic deregulation[END_REF]. Therefore, AD appears to be reciprocally associated with IR, suggesting that improving IR may alleviate AD and vice versa. In this study, IL-17A significantly improved glucose metabolism in IL-17A-overexpressing mice compared with that in control mice. Although the IL-17A level in CSF was about 29 times higher than that in plasma, it is unclear whether the effect of IL-17A on glucose metabolism is due to activation of IL-17A signal transduction pathways in CNS, peripheral tissues or both.

Aβ accumulation occurs in the brain parenchyma and CAA and plays a causal role in AD pathogenesis. CAA is the deposition of Aβ in the walls of cerebral blood vessels and identified at autopsy in 75-98% of AD patients [START_REF] Jellinger | Incidence of cerebrovascular lesions in Alzheimer's disease: a postmortem study[END_REF]. Aβ deposition in CAA affects cerebral blood flow, alters BBB permeability, interferes with Aβ clearance or efflux from the brain and triggers deleterious inflammatory responses, contributing to development of vascular fragility, hemorrhages and, ultimately, dementia [START_REF] Cupino | Alzheimer's silent partner: cerebral amyloid angiopathy[END_REF][START_REF] Ghiso | Amyloidosis associated with cerebral amyloid angiopathy: cell signaling pathways elicited in cerebral endothelial cells[END_REF][START_REF] Kalaria | The blood-brain barrier and cerebral microcirculation in Alzheimer disease[END_REF]. According to the two-hit vascular hypothesis of AD, brain vascular damages (hit 1) initiate Aβ accumulation (hit 2) in the brain, leading to neurodegeneration and memory decline in AD (Sagare et al., 2012). Clinical imaging, epidemiological and pharmacotherapy studies support the notion that vascular changes play a central role in early AD pathogenesis (de la [START_REF] De La Torre | Is Alzheimer's disease a neurodegenerative or a vascular disorder? Data, dogma, and dialectics[END_REF][START_REF] Iturria-Medina | Early role of vascular dysregulation on late-onset Alzheimer's disease based on multifactorial data-driven analysis[END_REF]. If not all, a subset of AD patients have disruptions of the BBB [START_REF] Erickson | Blood-brain barrier dysfunction as a cause and consequence of Alzheimer's disease[END_REF], resulting in the reduction of cerebral blood flow [START_REF] Spetsieris | Metabolic resting-state brain networks in health and disease[END_REF] and Aβ clearance in late-onset AD [START_REF] Mawuenyega | Decreased clearance of CNS betaamyloid in Alzheimer's disease[END_REF]. We found that IL-17A-overexpressing mice show an approximately 4-fold decrease in Aβ deposition in CAA compared with control mice. Soluble Aβ levels in the hippocampus and CSF are similarly decreased in IL-17A-overexpressing mice. Contrary to this observation, a decrease in CSF Aβ, especially Aβ42, is accompanied by an increase in brain Aβ deposition in the early stages of AD patients [START_REF] Kuhlmann | CSF Abeta1-42 -an excellent but complicated Alzheimer's biomarker -a route to standardisation[END_REF][START_REF] Maia | Changes in amyloid-beta and Tau in the cerebrospinal fluid of transgenic mice overexpressing amyloid precursor protein[END_REF].

Therefore, CSF Aβ levels do not always reflect Aβ load in the brain. Altogether, IL-17A may protect the brain vasculature from Aβ accumulation and prevent CAA. It is worth noting that IL-17A does not alter microglia activation in our experiments. Microglial cells are known to be important for amyloid plaque formation and clearance. However, eliminating microglia in AD mice failed to change amyloid plaque formation and modulate Aβ pathology [START_REF] Grathwohl | Formation and maintenance of Alzheimer's disease beta-amyloid plaques in the absence of microglia[END_REF][START_REF] Spangenberg | Eliminating microglia in Alzheimer's mice prevents neuronal loss without modulating amyloid-beta pathology[END_REF]. Thus, microglia may not be involved in reduction of Aβ deposits in IL-17A-overexpressing mice.

Cognitive impairment along with anxiety in AD animal models, which resembles that in AD patients, has been reported (Foidl et al., 2016;[START_REF] Pietrzak | Amyloid-beta, anxiety, and cognitive decline in preclinical Alzheimer disease: a multicenter, prospective cohort study[END_REF][START_REF] Sil | Cox-2 plays a vital role in the impaired anxiety like behavior in colchicine induced rat model of Alzheimer disease[END_REF][START_REF] Starkstein | The construct of generalized anxiety disorder in Alzheimer disease[END_REF]. Protofibrillar Aβ 1-42 injection induced anxiety and AD-like pathology in rats [START_REF] Sharma | Alzheimer's disease like pathology induced six weeks after aggregated amyloid-beta injection in rats: increased oxidative stress and impaired long-term memory with anxiety-like behavior[END_REF], suggesting the deleterious effect of elevated Aβ levels on behavior in AD. In our current study, IL-17A-overexpressing mice manifested behavioral alterations relative to controls. IL-17A-overexpressing mice were characterized by more rapid habituation in the open-field test, as indicated by more pronounced increases in moving slowly and more pronounced decreases in moving quickly across days of testing. Moreover, IL-17A-overexpressing mice had a higher center/total time ratio, indicating less anxiety in exploring the open-field. The lesser anxiety displayed by this group was also observed in the elevated plus-maze test, where open arm duration was increased relative to controls on day 1 though not on day 2 of testing when exploration is usually minimal. Therefore, IL-17A overexpression may improve anxiety by reducing soluble Aβ levels in the hippocampus. During acquisition of the Morris water maze task, IL-17A-overexpressing mice reached the escape platform faster than control mice. In contrast, there was no change in probe or visible platform subtasks, indicating cognitive improvement in the acquisition process but not long-term memory, visuomotor control, or swimming speed. Our results are consistent with the previous studies reporting the lack of association between Aβ deposits in brain and behavioral changes (Heikkinen et al. 2004;Holcomb et al. 1999;Howlett et al. 2004;Montarolo et al. 2013;Morgan 2003). It is possible that spatial memory deficits in AD model mice correlate with the selective modulation of the levels of hippocampal Aβ 42 (Faucher et al. 2015;Puolivali et al. 2002), which are unchanged in IL-17Aoverexpressing mice.

ABCA1 is one of the routes of Aβ clearance from the brain across the BBB. The depletion and overexpression of ABCA1 increases and decreases brain Aβ in AD mouse models, respectively [START_REF] Wahrle | Deletion of Abca1 increases Abeta deposition in the PDAPP transgenic mouse model of Alzheimer disease[END_REF][START_REF] Wahrle | Overexpression of ABCA1 reduces amyloid deposition in the PDAPP mouse model of Alzheimer disease[END_REF]. In human, a loss-offunction mutation in ABCA1 with occurrence of 0.2% is associated with a high risk of AD and cerebrovascular disease [START_REF] Nordestgaard | Loss-of-function mutation in ABCA1 and risk of Alzheimer's disease and cerebrovascular disease[END_REF]. Rarer non-synonymous variants of ABCA1 increase the risk for late onset AD, suggesting its protective effect against AD [START_REF] Lupton | The role of ABCA1 gene sequence variants on risk of Alzheimer's disease[END_REF]. Interestingly, the level of hippocampal ABCA1 in IL-17Aoverexpressing mice is increased compared with that in control mice. Because IL-17A can induce activation of ERK1/2 signal [START_REF] Guo | IL-17A signaling in colonic epithelial cells inhibits pro-inflammatory cytokine production by enhancing the activity of ERK and PI3K[END_REF][START_REF] Rodgers | IL-17A activates ERK1/2 and enhances differentiation of oligodendrocyte progenitor cells[END_REF][START_REF] Song | Growth factor FGF2 cooperates with Interleukin-17 to repair intestinal epithelial damage[END_REF] and because ERK1/2 signaling regulates ABCA1 expression in different types of cells [START_REF] Chang | Hyperglycemia accelerates ATP-binding cassette transporter A1 degradation via an ERK-dependent pathway in macrophages[END_REF][START_REF] Mulay | Inhibition of mitogen-activated protein kinase Erk1/2 promotes protein degradation of ATP binding cassette transporters A1 and G1 in CHO and HuH7 cells[END_REF], we determined if IL-17A would increase ABCA1 expression via ERK1/2 activation in endothelial bEnd.3 cells. As expected, IL-17A increased phospho-ERK1/2 but not total ERK1/2. The upregulation of ABCA1 by IL-17A was blocked by U0126, an ERK inhibitor. The ERK1/2 signaling pathway also participates in the spatial memory (Gao et al., 2016;[START_REF] Ghasemi | Insulin protects against Abeta-induced spatial memory impairment, hippocampal apoptosis and MAPKs signaling disruption[END_REF][START_REF] Liu | Memantine improves spatial learning and memory impairments by regulating NGF signaling in APP/PS1 transgenic mice[END_REF]. Thus, IL-17A overexpression may reduce CAA and soluble Aβ in the CSF and hippocampus by upregulating ABCA1 through the ERK1/2 signaling pathway.

Conclusions

Overexpression of IL-17A via intracranial delivery of rAAV5-IL-17A improved glucose metabolism, reduced Aβ levels in the CSF, hippocampus and CAA, and alleviated the anxiety in AD mice. Furthermore, our study suggests that IL-17A may decrease Aβ in the brain by upregulating expression of ABCA1 through the ERK1/2 signaling pathway. However, IL-17A neither improved parenchymal Aβ deposits nor exacerbated neuroinflammation in an AD mouse model. Our findings suggest that IL-17A may play a protective role in the pathogenesis of AD. from the National Institutes of Health (Grant Numbers AG030399, AG042082 and AG050854).

Figure Captions Figure 1 .Figure 2 .Figure 3 .Figure 4 .Figure 5 .Figure 6 .

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Acknowledgements

We thank Ms. Linda Walter (University of Illinois College of Medicine at Peoria) for assistance in preparation of this manuscript. This work was supported by grants

Conflict of interest

The authors declare no conflict of interest. immature antigen-presenting cells in transgenic models of Alzheimer's diseaselike cerebral amyloidosis. Brain Behav Immun 54, 211-225. Fisher, Y., Strominger, I., Biton, S., Nemirovsky, A., Baron, R., Monsonego, A., 2014


Smail Layes

Salah Chouchani

Robert Lalonde

Mohamed Rebaï

Efficacy of a visuo-motor based program for children with reading and spelling disorders -a pilot study

Keywords: dyslexia, spelling disorder, visual motor intervention, alphabetic principle, letter representation

We predicted that Arabic-speaking dyslexic children with spelling disorder benefit from a visual motor based remediation program for the development of letter knowledge and the improvement of spelling (dictation) as well as word and pseudo-word decoding. It was predicted that the mediation of visual motor training should be effective since the characteristics of Arabic script require such high-level functioning for the development of literacy skills. Twenty native Arabic children from grade 4 with specific learning disorders in reading (dyslexia) and spelling (dysorthographia) took part in this study. Participants were randomly divided in two equally sized groups, an experimental group (n=10; mean age=125.50 months ± 3.24) and a control group (n=10; mean age = 124.10 months ± 3.31). The intervention program focused on the development of visuo-motor representations of letters to strengthen letter-sound associations as a way to acquire the alphabetic principle needed for learning to read and write (sound to letter correspondence). Results revealed that the post intervention measures were better achieved in the experimental group than pre-intervention ones. Findings approve the efficiency of the visuomotor intervention program for the remediation of decoding and spelling disorders.

Introduction

The term 'specific learning disorders' (SLDs) is used to describe a group of learning difficulties characterized by impaired performance in reading (decoding accuracy, fluency, and comprehension), spelling, written expression, or calculation, despite average or above average intelligence (DSM-5;APA, 2013). In respect to this definition, two learning disorders are taken into account in this study: reading decoding disorder or dyslexia which refers to the inability to perform accurate or fluent word reading (incorrect reading of single words aloud), and spelling disorder of sound to letter correspondence. Spelling requires the ability to differentiate individually heard sounds and mapping those sounds into letters (i.e. alphabetic knowledge, [START_REF] Sayeski | Effective Spelling Instruction for Students with Learning Disabilities[END_REF]. Difficulties in written expression (e.g. multiple grammatical or punctuation errors, poor expression and organization) represent another aspect of SLDs falling beyond the scope of the present study.

Spelling disorder was labelled "dysorthography" in DSM-4 TR (2002), which refers to a disorder in writing (orthographic skills and phonographic coding), different from dysgraphia as a disorder in hand-writing (graphomotor) skill. In dysorthography, the spelling of words is highly deficient as a direct consequence of the phonological disorder in dyslexic children (Inserm, 2007). In addition, poor performance in spelling is associated with "dysorthographia", referring to a specific learning disability manifested by grammatical errors, arbitrary spelling, and many spelling errors as in the addition of letters and/or syllables, omissions, substitutions, and reversals [START_REF] Pierangelo | The Educator's Diagnostic Manual of Disabilities and Disorders (EDM)[END_REF].

Although the indication of the DSM-5 (APA, 2013) and several findings suggest an association between learning disorders in dyslexia and spelling disorder [START_REF] Lyon | A definition of dyslexia[END_REF][START_REF] Egan | Inflectional spelling deficits in developmental dyslexia[END_REF], the major focus of most studies has been reading disabilities [START_REF] Bigozzi | Predicting dyslexia in a transparente orthography from grade 1 literacy skills: a prospective Cohort study[END_REF][START_REF] Schiff | Spelling and morphology in dyslexia: a developmental study across the school years[END_REF]. Consequently, most remedial research studies have been dedicated to children with reading disability. Nevertheless, children with dyslexia usually have serious problems with spelling as well (e.g. Berningera, Nielsena, Abbotta, Wijsmanb, & Raskind, 2008), show writing problems at the word level (e.g. Angelelli, Marinelli, & Zoccolotti, 2010) and were found less accurate in writing from dictation than agecontrolled children [START_REF] Suárez-Coalla | Spelling difficulties in Spanish-speaking children with dyslexia / Dificultades de escritura en niños españoles con dyslexia[END_REF].

Therefore, young learners with dyslexia often need more effective instruction and strategies for learning to read and spell as they may have difficulties in using sound-letter correspondence rules. This study was designed to examine the efficacy of a remedial program based on visual motor representation of alphabetic letters for the development of decoding and spelling (dictation) in a group of native Arabic reading disabled children with spelling disorder (dysorthography).

Early literacy studies suggest that the alphabetic principle is a fundamental underlying skill for reading and spelling [START_REF] Eden | the Role of Neuroscience in the Remediation of Students With Dyslexia[END_REF][START_REF] Ehri | Learning to read and learning to spell Two sides of a coin[END_REF], promoting an awareness of letter sounds (letter knowledge) in alphabetic languages [START_REF] Treiman | The foundations of literacy: Learning the sounds of letters[END_REF]. To learn decoding and spelling accurately, children must master the alphabetic principle and be aware that spoken words consist of sounds linked to letters or letter groups (Moll, Kunze, Neuhoff, Bruder, & Schulte-Korne, 2014). It is well documented that deficient acquisition of the alphabetic code in children with dyslexia is causally linked to the great difficulty (error rate) in spelling observed in pseudo-words more than regular words, because it requires the use of a sub-lexical strategy. The poor performance with pseudo-words has already been described in dyslexic children of opaque and non-opaque orthographic systems, especially in the first years of writing experience (e.g. [START_REF] Angelelli | Characteristics of writing disorders in Italian dyslexic children[END_REF]Caravolas & Volin 2011).

Reading and spelling disorders are highly correlated to impairments in alphabetic knowledge. The phonological deficit theory postulates that children with reading disorders have a specific impairment in the representation, storage, and/or retrieval of speech sounds. In dyslexic subjects, these sounds are poorly represented, resulting in inappropriate retrieval of the learned alphabetic system, a poor relation between visual representations of the stored letter and its associated phonology (Brady & Shankweiler, 1991). Consequently, the acquisition of inconsistent sound-letter mappings in individuals with dyslexia increases their errors in literacy tests [START_REF] Angelelli | Characteristics of writing disorders in Italian dyslexic children[END_REF][START_REF] Landerl | Deficits in phoneme segmentation are not the core problem of dyslexia: evidence from German and English children[END_REF]. Since letter knowledge acquisition is an important component of children's literacy, it must be considered in any remediation program [START_REF] Hammill | What we know about correlates of reading[END_REF][START_REF] Pennington | Early reading development in children at family risk for dyslexia[END_REF][START_REF] Vaessen | The cognitive linkage and divergence of spelling and reading development[END_REF], especially in transparent orthographies (Hulme & Snowling, 2013).

In addition, literacy performances (reading, spelling, and writing,) are commonly related to visuo-motor skills [START_REF] Goldstand | Vision, visual-information processing, and academic performance among seventh-grade school children: a more significant relationship than we thought?[END_REF]. Recent reports show that motor skills have significant correlations with reading [START_REF] Knight | Relations for children in grades 2, 3, and 4 between balance skills and academic achievement[END_REF] and facilitate children's abilities in reading [START_REF] Son | The relationship of young children's motor skills to later reading and math achievement[END_REF]. Based on the assumed role of visuo-motor skills in literacy development and the underlying letter representation deficit which impedes alphabetic knowledge acquisition in children with reading and spelling disorders, we predicted that letter recognition may be successfully acquired via visuo-motor training. This should be well applicable to Arabic scripts, because written alphabetic units (letters) change partially their forms according to the position in the word (beginning, middle, and end) and which can be regrouped into clusters of shapes based on their visual similarities (e.g. [START_REF] Abu-Rabia | Spelling Errors' Analysis of Regular and Dyslexic Bilingual Arabic-English Students[END_REF]. In addition, these letters have a similar or even identical basic shape and can be distinguished only on the basis of the existence, location, and number of dots (Azzam, 1989). Children may have an inability to associate letter shapes into sounds, a difficulty in establishing and retrieving soundvisual configuration associations as symbols. Thus, a remedial program based on integrating visuo-motor skills along with letter-sound associations may promote letter representation and therefore increase reading, decoding, and spelling abilities in children with dyslexia and spelling disorders.

Few studies exist on the contribution of visuo-motor training on forming the alphabet principle and literacy abilities. [START_REF] Hall | Examining the effects of preschool writing instruction on emergent literacy skills: A systematic review of the literature[END_REF] found five studies concerning letter formation by hand as part of literacy intervention. In [START_REF] Neumann | Using environmental print to enhance emergent literacy and print motivation[END_REF], children were asked to write a letter in the air and in a journal with a pencil after teacher demonstration. In the [START_REF] Li | Handwriting Generates Variable Visual Output to Facilitate Symbol Learning[END_REF] study, preschool children learned novel Greek symbols through either writing, tracing typed symbols, tracing handwritten symbols, visually studying typed symbols, or visually studying handwritten symbols. Results indicated that the groups who studied handwritten forms, either through tracing, viewing, or seeing their own writing, learned the symbols better than those who studied typed letter forms. These results suggest that visual experience with symbols that are highly variable in their forms (through handwriting or tracing) facilitates visual recognition of written symbols. These results were viewed as evidence of the multimodal nature of letter representations learned through matching of visual configurations and movements, where the sensorimotor component is integrated [START_REF] Longcamp | Premotor activations in response to visually presented single letters depend on the hand used to write: a study on left-handers[END_REF]. According to these authors, movements organize perceptions and link this to learning to read, for when alphabetic letters are reproduced by specific hand movements, children can access their perceptual-motor system and recognize the letter through memory. [START_REF] Vinter | Effects of Different Types of Learning on Handwriting Movements in Young Children[END_REF] investigated the respective advantages of visual, motor, and visuo-motor training on the performance of 5-year-old children in writing isolated cursive letters.

They found that visuo-motor training was the most effective. The efficacy of visual training was clear at the letter quality level, suggesting that such training better contributes to learning the shape of a letter. Likewise, Bara and Bonneton-Botte (2017) assessed the impact of a teacherimplemented visuo-motor intervention program to teach 5-year-old children cursive letter knowledge. The authors compared the impact of a program in which alphabet letter production was explored with the arm and whole body with a typical visual training program on letter knowledge acquisition. The main finding was that letter recognition improved more after the visuo-motor training program than the typical visual training program. Like haptic exploration [START_REF] Gentaz | Apports de la modalité haptique manuelle dans les premiers apprentissages scolaires [Contributions of manual haptic modality in early school learning[END_REF] and handwriting [START_REF] Labat | Effet d'entraınements bimodaux a` la connaissance des lettres. Etude transversale chez des enfants de trois a` cinq ans. [Effect of dual-mode training in letter knowledge: A cross-sectional study with 3-and 5-year-old children[END_REF]Longcamp et al., 2005), gross and fine motor exploration of letters has a particularly positive impact on their recognition. And so, this practice may be helpful for children with reading and spelling disorders.

To the best of our knowledge, our study is the first empirical one in Arabic. Some orthographic characteristics in Arabic (e.g. visual similarities between letters) are thought to be a source of errors in reading and spelling [START_REF] Ibrahim | The characteristics of Arabic orthography slow its processing[END_REF]. This difficulty has been mainly attributed to differences in letter shape determined by placement of the letter within a word and therefore different grapheme configurations share the same phoneme, which may create a difficulty in establishing letter identity. In their classifications of reading and spelling errors among dyslexic Arabic readers compared with normal ones, Aburabia and Taha ( 2004) observed that visual letter confusion errors are among the most prevalent, either as a result of confusion of letter-shape similarities in reading producing a mismatch between graphemes and phonemes, or as a confusion between similar visual shapes of letters in spelling, hence the lack of the grapheme-phoneme conversion rules. These types of error can be considered a direct result of the uniqueness of written Arabic orthography [START_REF] Abu-Rabia | Reading and spelling error analysis of native[END_REF]). In the current study, we predicted that dyslexic children with spelling disorders could benefit from a visuo-motor remedial program for the development of letter representation. This advantage should be directly reflected in word and pseudo-word decoding and spelling (dictation) performances. Similar improvements should occur in visuo-motor tasks (block construction and design copying) as indicators in the development of such skills.

Method

Participants

Participants were initially recruited as poor readers (n = 55) primarily on teachers' opinions and then based on poor performances for word and pseudo-word reading. After eliminating cases whose profiles did not match with inclusive criteria, twenty children composed the group study, diagnosed with dyslexia based in at least -1.5 SD below the mean scores in word and pseudo-word reading (dysorthographia) based on a criterion-referenced test of sentence dictation. Children did not suffer from any specific learning disability other than reading and spelling, or hearing or visual impairments, and had average scores on Raven Standard Matrices.

Participants were equally divided into two groups following a randomized design in which they were assigned to each condition individually, an experimental group (n=10) with a mean age of 125.50 months (SD=3.24) and a control group (n = 10) aged 124.10 months (SD=3.31).

All participants were right-handed and had normal hearing without ophthalmological or neurological abnormalities, were native Arabic speakers exposed to Standard Arabic in regular classes and exempt from any other comorbid disability (e.g. oral language disability, ADHD).

No differences appeared between the two groups on age and Raven scores (Table1).

Measures

Pre and post training measures

Raven test. The Raven Standard Progressive Matrices is a non-verbal test of reasoning ability and general intelligence. We used the shortened form (Bouma et al., 1996) comprising 36 items (Sets A, B, and C) and consisting of a target matrix with one missing part. The children selected from six to eight alternatives to fill the missing patch.

Word and pseudo-word reading tasks. Pseudo-word decoding is a widely used measure to assess children's decoding and orthographic learning skills [START_REF] Ehri | Learning to read words: Theory, findings, and issues[END_REF][START_REF] De Jong | Orthographic Context and the Acquisition of Orthographic Knowledge in Normal and Dyslexic Readers[END_REF].

Pseudo-word requires both phonemic segmentation ability and knowledge of grapheme-phoneme correspondence, not allowing for sight reading as a possible strategy [START_REF] Castles | Varieties of developmental dyslexia[END_REF]. [START_REF] Good | Making Sense of Nonsense Word Fluency: Determining Adequate Progress in Early First-Grade Reading[END_REF] suggested that skills necessary for decoding are isolated from the ability to read words by sight during pseudo-word reading, because the reader cannot rely on past experience. Instead, the reader relies on understanding letter-sound relations (i.e. the alphabetic principle) and the rules that govern those relationships and then applies this knowledge for decoding.

A list of eighty words (α = .82) was given and read aloud (40 frequent words and 40 infrequent words). The words varied in length (di-syllabic and tri-syllabic) and frequency (high and low). In addition, 40 pseudo-words (α = .90) controlled for orthographic length were used [START_REF] Layes | Reading speed and phonological awareness deficits among Arabic-speaking children with dyslexia[END_REF]Layes et al., 2015a, b). Children able to segment and with good knowledge of letter sound correspondences will perform better on pseudo-word decoding tasks than those who do not [START_REF] Tunmer | Phonemic segmentation skill and beginning reading[END_REF].

Spelling test: Sentence dictation. It consists of dictating ten small sentences of three words each where children were instructed to write down the heard sentence [START_REF] Elbeheri | Developmental dyslexia in Arabic: Devising a diagnostic tool can enrich our understanding of the manifestation of dyslexia amongst monolingual Arabic children[END_REF]. The sentences contain several phonological, orthographical, and grammatical target words from high to low frequency of occurrence [START_REF] Tops | Spelling in Adolescents with Dyslexia: Errors and Modes of Assessment[END_REF][START_REF] Tops | Spelling in Dutch adolescents with dyslexia: Errors and modes of assessment[END_REF]. The sentence dictation is limited to the spelling of individual words and the use of the morphosyntactic rules at the sentence level full beyond the scope of the present study.

Participants were given a standard form to write on. The test administrator read in full each sentence aloud, repeating each word separately in a uniform way as many times as participants requested. Words were presented with a regular interval of 3 sec, so that students could produce an immediate response. No additional information was given about punctuation or capitals. The score is the total number of the correct written words without time limit. The reliability of this test was measured by an external validation sample (n=55). The test-retest reliability (correlation coefficient) was r= .95 and the internal consistency reliability (alpha coefficient) α= .94.

The visual spatial processing assessment (NEPSY-II).

Two subtests of the Neuropsychological Assessment battery NEPSY-II [START_REF] Korkman | NEPSY-Second Edition (NEPSY-II)[END_REF] were used: the Design Copying and the Block Construction subtests:

Design Copying is a pencil-and-paper task that requires the examinee to copy designs of increasing complexity to assess visuo-motor integration. It requires the examinee to construct three-dimensional models from models and pictures to assess visuo-spatial and visuo-motor skills. Design Copying is a subtest within this domain that evaluates a child's ability to copy geometric designs of increasing complexity from a response booklet to assess motor and visual perceptual skills [START_REF] Korkman | NEPSY: A developmental neuropsychological assessment[END_REF]. The Design Copying total score had mean internal consistency coefficients above .70 across all age groups in the norming sample. These scores had mean test-retest reliability coefficients below .70 across ages 7 to 10, but for ages 11 to 12, both mean test-retest reliability coefficients were .71.

Block Construction: This subtest is used to assess visuo-spatial and visuo-motor skills, requiring a child to look at two-dimensional drawings and use blocks to copy them and so construct threedimensional representations [START_REF] Korkman | NEPSY: A developmental neuropsychological assessment[END_REF]. This task has been extended upward to cover the age range of 3 through 16 years. The Block Construction total score had mean internal consistency coefficients and mean test-retest reliability coefficients above .70 across all age groups in the normal sample. Scaled scores may be obtained for the total process score as well as for the global motor process.

The intervention program: background and content

Use is made of combining sources of information that contribute to developing phonological representations that underpin word coding in the mental lexicon for children with disabilities [START_REF] Perfetti | Reading optimally builds on spoken language: Implications for deaf readers[END_REF]. Visuo-motor representation can be proposed as an additional source of information that can help children with dyslexia to develop awareness of the phonological structure of letters. Letter exploration was combined with letter-sound correspondence in a multisensory-based intervention perspective, and specific instructions were given to the participants for exploring letters. In any task that involves visual and motor systems, manual manipulation may be a particularly effective pathway to object knowledge [START_REF] James | Letter processing automatically recruits a sensory-motor brain network[END_REF]Longcamp et al., 2005;[START_REF] Longcamp | Visual presentation of single letters activates a premotor area involved in writing[END_REF]. Symbols (e.g. letters or digits) are unique object types that are very rarely manipulated manually, except in the specific case of handwriting when symbols are formed feature-by-feature.

Inspired from the "word work" multisensory instructional decoding method [START_REF] Blachman | Developing phonological awareness and word recognition skills: A two-year intervention with lowincome, inner-city children[END_REF], this intervention program included targeted decoding ability by making explicit connections between phonemes and graphemes through the use of manipulative materials that provide tactile and visual phoneme representations. This method provides a concrete medium for the abstract tasks of phoneme manipulations [START_REF] Campbell | Effects of Adding Multisensory Components to a Supplemental Reading Program on the Decoding Skills of Treatment Resisters[END_REF]) and may thus be particularly beneficial for struggling readers. Simultaneous links are consistently made between visual (letters), motor (gross and fine movements), and auditory (sounds) pathways. Advocates believe that multisensory input, such as letter multiform imitation and reproduction, strengthens visual memory through conscious awareness of mechanisms such as letter form (shapes) and increases the information stored in memory by providing multiple representations [START_REF] Moats | Multisensory instruction[END_REF].

Based on the above-mentioned theoretical background, our program includes the use of manipulative letters, individual moveable letters such as manually formed and shaped letters, cards with letters printed on them. In addition, letters to form words are removed and then replaced to form a new word. The process is repeated to provide multiple opportunities for decoding, and each successive word typically shares one or more letters in common with the previous word to demonstrate relationships between common sounds and spellings. Moreover, specific activities such as drawing big letters on the blackboard, imitation of letter shapes in various substances, tracing letters in the air and forming letters with modelling clay are proposed within an intersensory intervention approach for elementary school children [START_REF] Lust | Effectiveness of a Handwriting Readiness Program in Head Start: A Two-Group Controlled Trial[END_REF]. Attention was focused to strengthen the relationship between the visual configurations of letters (shapes) and the corresponding sounds via the perceptual motor bridge. During the visual motor intervention, children were permanently stimulated to reproduce and manipulate the visual configurations of the letters and were trained to recognize the target letters and distinguish them from each other.

The exercises of the intervention program were created to be performed individually and collectively. The program content was organised as follows:

1. Geometric shape imitation was used to develop perception, visual discrimination of forms and the fine motor skills. There was imitation of geometric shapes (circle -square -rectangletriangle -star -crescent, etc) from the blackboard using modelling clay accompanied with letter naming and then recognition of geometric shapes from memory after removing the visual model from the blackboard.

2. Motor execution of geometric shapes was aimed at developing visual perception, motor spatial memory, and visual motor kinetics. There was reproduction of spatial shapes by hand movement and drawing of geometric shapes from memory after oral instructions.

3. Letter form recognition and discrimination: this activity aimed at developing visual attention, perception, and memory. The participants were instructed to sort and group letters by their form similarities, and select letters from a set, to match a visually presented word without correct seriation of the selected letters required beforehand. • The reproduction of visually presented letters using modelling clay and hand movements, sticks, circles, and cards.

• After removing the letters from the blackboard, the same activities are performed.

• Writing the letters after oral presentation

• Presentation of examples of words containing the letters.

The association of letter sound (phoneme) is systematically present in these activities.

5. Linking and arranging letters: Association between words with one missing letter was presented along with a selected letter from a set individually scripted on paper.

6. Word formation: letter cards were used to create words corresponding to visually presented images of different objects.

Procedure

All participants underwent pre-training tests during the first week of the study, followed by 13 intervention sessions over the course of 6 weeks (two per week, each one approximately 40 min), and intervention testing. All procedures were performed in a small private room located in the school setting.

Data Analyses

SPSS version 22 was used for data analysis. Baseline inter-group comparisons were performed with the t-test for independent samples. As there might be post-intervention effects in the development of word and pseudo-word decoding, spelling, and visuo-motor abilities among the experimental group, MANCOVA with pre-training measurements entered as the covariates was used for between-group comparisons of all post-training measurements. The paired t-test was also used to compare the pre-and post-intervention measurements within the experimental group. Effect size is computed by Cohen's d for the magnitude of the training effect.

Results

Baseline Assessment

Due to the small sample size, the assumption of the normality of the distribution of the dependent variable (in the experimental group) for the pre-intervention measure of reading was checked with Shapiro-Wilk (statistic= .94), indicating a normal distribution (p >.05) with a skewness of 0.46 (SE = .68) and a kurtosis of -0.68 (SE = 1.33). As kurtosis is near 0, a normal distribution is assumed and parametric tests were applied. With regard to inclusion criteria, comparisons at baseline assessment between intervention and control groups are presented in Table ( 1), including age levels and Raven scores as well as word and pseudo-word reading, sentence dictation, and visuo-spatial measures.

Insert table 1

The intervention effects

To examine whether visuo-motor based intervention improves word and pseudo-word reading in the experimental group, and whether this improvement affects spelling, a one-way multivariate analysis of covariance (MANCOVA) controlling for pre-training measures was conducted to assess the main effect of the group variable (experimental/control) on multiple continuous dependent variables simultaneously (word and pseudo-word decoding, spelling, design copying, and block construction). Statistics provided from Box's M-test of equality of covariance indicated that homogeneity of covariance was met (F=1.25, p = .22). The omnibus effect for "group" was significant (Pillai's Trace =.90, F (5.90) = 16.09, p < .001), η 2 = .899). To examine further the potential impact of the intervention program, paired t-test was performed comparing pre and post intervention measures for the experimental group. Results show significant differences in the post intervention scores for pseudo-word decoding in pre (M = .80, SD = 1.03) and post intervention (M = 2.10, SD = .87), t (9) = 4.33, p < .01); for sentence dictation in pre (M = 2.70, SD = 1.56) and post intervention (M = 8.20, SD = 2.30), t (9) = 13.70, p < .001); for Design Copying in pre (M = 10.90, SD = 3.28) and post intervention (M = 15.80, SD = 3.22), t (9) = 6.39, p < .001), and for Block construction in pre (M = 5.00, SD = 1.94) and post intervention (M = 10.60, SD = 2.87), t (9) = 12.38, p < .001). However, for word reading the difference between pre (M = 17.50, SD = 7.38) and post intervention (M = 19.10, SD = 7.20) measurements the t-test value only approached significance (t (9) = 1.98, p= .07).

Insert Figures 1 and 2

Discussion

This study was designed to examine the impact of a visuo-motor based intervention program on the acquisition of word and pseudo-word decoding and spelling abilities in children with reading and spelling disorders. The main findings were that the experimental group achieved better in post-intervention measures of these literacy abilities. This pattern of results was replicated in the visuo-motor tasks (block construction and design copying). Word reading only approached significance (p= .07). In contrast, the control group receiving no intervention program remained stable across pre-and post-intervention measures.

The results are viewed as a positive impact of the visual-motor based program on word and pseudo-word decoding via the development of letter recognition [START_REF] Bellocchi | Exploring the Link between Visual Perception, Visual-Motor Integration, and Reading in Normal Developing and Impaired Children using DTVP-2[END_REF]. The contribution of hand movements on letter recognition and reading acquisition was outlined in previous studies with young children [START_REF] Bara | The visuo-haptic and haptic exploration of letters increases the kindergarten-children's reading acquisition[END_REF][START_REF] Bara | Haptics in learning to read with children coming from low socio-economic status families[END_REF][START_REF] Gentaz | Evaluation d'entraînements multisensoriels de préparation à la lecture chez les jeunes enfants de grande section maternelle: Etude sur la contribution du système haptique manuel[END_REF], indicating that benefits could be obtained [START_REF] Bara | Haptics in teaching handwriting: The role of perceptual and visuomotor skills[END_REF]. [START_REF] Weiser | Using Encoding Instruction to Improve the Reading and Spelling Performances of Elementary Students at Risk for Literacy Difficulties. A Best-Evidence Synthesis[END_REF] examined the impact of phoneme-grapheme manipulation through word work activities on reading and spelling performance for at-risk elementary students with learning disability in reading. Results showed that instruction in encoding increased students' knowledge of the alphabetic principle, development of phonemic awareness, and growth of reading and spelling skills. Similar findings were reported in [START_REF] Bara | The visuo-haptic and haptic exploration of letters increases the kindergarten-children's reading acquisition[END_REF]2007), showing that haptic exploration of letters was superior to visual exploration for rate of improvement in kindergarten children's letter recognition, letter handwriting, and pseudo-word decoding. Bara and Bonneton-Botte (2017) also found that visuo-motor training based on gross exploration of letters had a particularly positive impact on their recognition, since such movements match letter shapes as with handwriting, leading to an efficient promotion of the alphabet for typical young children.

During the implementation of the remedial program, exploring letters involved both visual perception and motor interaction processes. As a result, visual representations of letters seemed to be linked to motor representations, resulting in a visuo-motor system that underlies letter processing [START_REF] Giese | Neural and Computational Mechanisms of Action Processing: Interaction between Visual and Motor Representations[END_REF][START_REF] James | Sensori-motor experience leads to changes in visual processing in the developing brain[END_REF][START_REF] James | The effects of handwriting experience on functional brain development in pre-literate children[END_REF][START_REF] Kersey | Brian activation patterns resulting from learning letter forms through active self-production and passive observation in young children[END_REF]. This creates a multimodal representation of letters (Longcamp et al., 2005), linking phonology, perception, and motor patterns [START_REF] Longcamp | Learning through Hand-or Typewriting Influences Visual Recognition of New Graphic Shapes: Behavioral and Functional Imaging Evidence[END_REF].

Furthermore, these findings are supported by functional neuroimaging studies which revealed that experience with letters of the alphabet activates pre-motor areas implicated in writing, raising the possibility that letter production contributes to letter recognition even in skilled readers (e.g., [START_REF] James | Letter processing automatically recruits a sensory-motor brain network[END_REF][START_REF] Longcamp | Visual presentation of single letters activates a premotor area involved in writing[END_REF], Longcamp, et al., 2005;Longcamp, Tanskanen, & Hari, 2006;[START_REF] Longcamp | Neuroanatomy of Handwriting and Related Reading and Writing Skills in Adults and Children with and without Learning Disabilities: French-American Connections[END_REF]. Indeed, there is evidence that both visual and motor regions respond during letter perception, in that early letter learning is typically supported through visual-motor practice, such as handwriting or tracing. This suggests direct links in the brain between motor systems and visual perception that interact during handwriting. [START_REF] James | Letter processing automatically recruits a sensory-motor brain network[END_REF] showed that the set of regions activated during letter perception is very similar to the set activated during letter writing. Similarly, both visual and motor regions begin to increase their response when presented with trained letterforms when compared with other untrained stimuli, such as shapes, in preliterate children after very brief experiences with letters [START_REF] James | Sensori-motor experience leads to changes in visual processing in the developing brain[END_REF][START_REF] Kersey | Brian activation patterns resulting from learning letter forms through active self-production and passive observation in young children[END_REF]. This change in response only occurs after children learn letters through visuo-motor practice.

At the end of the our intervention program when letters are presented in their isolated (individual) form (e.g. ر ش، ، ,)ﻛـ participants seemed able to apply the more general knowledge in word and pseudo-word decoding and spelling where the target letter is connected to the word (e.g. the letter ]ح[ in .)]ﺣﻠﯿﺐ[ This finding do not support the claim that there are different abstract letter identity units corresponding to different forms of the same letter in Arabic, and that abstract letter identities might be common to a given letter but with indication of the form they appeared in [START_REF] Friedmann | Letter position dyslexia in Arabic: from form to position[END_REF]. Letter identity is assumed to be represented in an abstract, modality-independent format in which neither letter-shape nor font are specified [START_REF] Dufor | Letter representations in writing: an fMRI adaptation approach[END_REF]. It is worth noting that the body of the letter in Arabic generally does not change whatever its position in the word (e.g. /ـﻒ ـﻔـ / ف ), but this is only applicable to few letters (e.g. ـﮫ / ـﮭـ / ھـ ). Fiset and collaborators [START_REF] Fiset | Features for uppercase and lowercase letter identification[END_REF][START_REF] Fiset | The spatio-temporal dynamics of visual letter recognition[END_REF] emphasized the significance of horizontals in the perception of Roman letters. However, for Arabic letters most of the horizontal segments occur in the ligatures between letters, and do not, therefore, play an important role in distinguishing between letters.

Letter recognition involves a highly constrained visual pattern recognition system: constructing and identifying an abstract concept of a letter or the abstract letter identity from basic visual features [START_REF] Grainger | Orthographic processing in visual word recognition: A multiple read-out model[END_REF][START_REF] Grainger | Letter perception: From pixels to Pandemonium[END_REF] where visual features are mapped onto letter representations [START_REF] Carreiras | Neural correlates of visual vs. abstract letter processing in Roman and Arabic scripts[END_REF].

It is interesting to note that decoding performance in the post-intervention period was better performed in pseudo-words than words. A similar finding was reported in [START_REF] Bara | Haptics in learning to read with children coming from low socio-economic status families[END_REF], who found that pseudo-word decoding improved more after visual and haptic exploration of letters than visual exploration of letters. This may be explained by the fact that at the end of the program, participants began developing an alphabetic strategy in reading [START_REF] Bruck | Word recognition skills of adults with childhood diagnoses of dyslexia[END_REF]. During the alphabetic phase in reading development [START_REF] Frith | A developmental framework for developmental dyslexia[END_REF], the child is able to use correspondences between individual graphemes and phonemes. Words are sequentially decoded grapheme by grapheme. The child can pronounce both unfamiliar words and non-words but will not necessarily generate the correct pronunciation. The use of an alphabetic strategy may be enough for competent reading to acquire basic literacy in Arabic [START_REF] Azzam | The nature of Arabic reading and spelling errors of young children[END_REF]. A similar pattern was reported in other transparent orthographies [START_REF] Wimmer | How German-speaking first graders read and spell: Doubts on the importance of the logographic stage[END_REF]. In transparent orthographies like Arabic, it is generally thought that learning to read initially emphasizes alphabetic analysis [START_REF] Seymour | Foundation literacy acquisition in European orthographies[END_REF]. After two years of reading, such subjects are better at phoneme manipulation and reading non-words than readers of an opaque orthography.

Our results also reveal that in the post-intervention period, spelling performance measured by sentence dictation increased in the experimental group, indicating that learning to write letters with a multimodal approach (i.e. hand movement in the space, forming letters using clay) increase the participant's experience in writing letters which may improve learning of letter shapes (e.g., [START_REF] James | Sensori-motor experience leads to changes in visual processing in the developing brain[END_REF][START_REF] Li | Handwriting Generates Variable Visual Output to Facilitate Symbol Learning[END_REF][START_REF] Longcamp | Remembering the orientation of newly learned characters depends on the associated writing knowledge: A comparison between handwriting and typing[END_REF][START_REF] Longcamp | Contribution de la motricité graphique à la reconnaissance visuelle des lettres [Contribution of writing movements to visual recognition of letters[END_REF]Longcamp et al., 2005). The increase in the perceptual representation of letters in turn may give support to learning to write adequately letters and words by reactivating visual patterns from memory, which recruits the appropriate neural circuit to translate visual patterns to reproduce an approximation of the shape via the motor system (writing). This effect can be interpreted in light of the embodied theory of learning [START_REF] Glenberg | From the revolution to embodiment: 25 years of cognitive psychology[END_REF] as a strong relationship between visual and motor systems in reading and writing [START_REF] James | Letter processing automatically recruits a sensory-motor brain network[END_REF][START_REF] Longcamp | Visual presentation of single letters activates a premotor area involved in writing[END_REF]. Based on this theory, visual motor memory ability should affect written language recognition (decoding abilities), as well as influencing written language production. It is likely that the gesture reinforces the visual representation of letters and may favor encoding and recall.

This proposal is supported by evidence showing that the motor processes associated with writing reinforce a child's ability to recognize alphabetical symbols [START_REF] Tan | Reading depends on writing, in Chinese[END_REF]. Thus, more practice and procedural recall of letter/word forms (motor representations of symbols) while writing could aid visual recognition while reading [START_REF] Longcamp | Learning through Hand-or Typewriting Influences Visual Recognition of New Graphic Shapes: Behavioral and Functional Imaging Evidence[END_REF].

Another factor to consider is that spelling and writing in Arabic in basically consonantal, so that letters represent only consonants while vowels (diacritic signs) can be omitted, which may facilitate the reproduction of learned letters.

Performances on Block Construction and Design Copying subtests also improved at posttest assessment for children receiving the remedial program, whereas pre-and post-test scores did not differ for the control group, indicating a substantial improvement in the visual construction performance may be linked to the visuo-motor activities included in the program.

Engagement in visuo-motor activities during program implementation may provide participants with opportunities to practice motor integration of visually presented letters and reproducing them by different means. These findings are consistent with the notion that practice with such materials develops visuo-motor integration and coordination.

In summary, this pilot study shows the effectiveness of a visuo-motor based intervention that involved hand reproduction of letter shapes in improving letter knowledge and production which had an impact on the quality of decoding word and pseudo-words and spelling abilities in children with reading and spelling disorders (dyslexia and dysorthography). From a clinical perspective, the results may have important implications, because they show that literacy develops at least partly by interacting with visuo-motor skills. In practice, these results suggest that it would be useful to assess visuo-motor integration skills for screening pre-schoolers in regard to their academic aptitudes. Visuo-motor training facilitates the acquisition of the alphabetic principle in children with low levels of literacy and may also affect typical readers.

4.

  Learning the letters: A set of 3 or 4 letters (e.g.

  Follow-up univariate analyses (ANOVAs) show significant differences on the group factor for pseudo-words [F (1.13) = 4.86, p < .05, partial η 2 = .27], Dictation [F(1.13) = 57.52, p < .001, partial η 2 = .81], Design Copying [F(1,13) = 11.15, p < .01, partial η 2 = .46], and Cube Construction [F(1.13) = 40.27, p < .001, partial η 2 = .75]. However, word reading was not different [F (1.13) =.42, p > .05].

Figure 2 .

 2 Figure 2. Comparison between the two groups in the post-intervention measures