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Neuroscience Letters 566 (2014) 286–291

Contents lists available at ScienceDirect

Neuroscience Letters

jo ur nal ho me p age: www.elsev ier .com/ locate /neule t

,8-Dihydroxyflavone improves motor performance and enhancesower motor neuronal survival in a mouse model of amyotrophicateral sclerosis

rhan Tansel Korkmaza,d,e, Nurgul Aytana,d, Isabel Carrerasa,b, Ji-Kyung Choic,eil W. Kowall a,d, Bruce G. Jenkinsc, Alpaslan Dedeoglua,c,d,∗

VA Boston Healthcare System, Boston, MA 02130, USABiochemistry, Boston University School of Medicine, Boston, MA 02118, USARadiology, MGH and Harvard Medical School, Boston, MA 02114, USANeurology and Alzheimer’s Disease Center, Boston University School of Medicine, Boston, MA 02118, USAPhysiology and Neurophysiology, Medical Faculty, Eskisehir Osmangazi University, Eskisehir 26480, Turkey

i g h l i g h t s

We tested the effect of 7,8-DHF on lower motor neurons in ALS mice.Chronic administration of 7,8-DHF improved motor deficits in ALS mice.7,8-DHF preserved spinal MNs and dendritic spines in ALS mice.7,8-DHF had a differential effect on small-MNs in the spinal cord of ALS mice.

r t i c l e i n f o

rticle history:eceived 16 October 2013eceived in revised form 13 February 2014ccepted 26 February 2014

eywords:

a b s t r a c t

Amyotrophic lateral sclerosis (ALS) is an enigmatic neurodegenerative disorder without any effectivetreatment characterized by loss of motor neurons (MNs) that results in rapidly progressive motor weak-ness and early death due to respiratory failure. Brain-derived neurotrophic factor (BDNF) is a memberof the neurotrophin family known to play a prominent role in the differentiation and survival of MNs.The flavonoid 7,8-dihydroxyflavone (7,8-DHF) is a potent and selective small molecule tyrosine kinase

myotrophic lateral sclerosis,8-Dihydroxyflavoneotor neurons

DNFrkB

receptor B (TrkB) agonist that mimics the effects of BDNF. In the present study, we evaluated the neu-roprotective effects of 7,8-DHF in a transgenic ALS mouse model (SOD1G93A). We found that chronicadministration of 7,8-DHF significantly improved motor deficits, and preserved spinal MNs count anddendritic spines in SOD1G93A mice. These data suggest that 7,8-DHF should be considered as a potentialtherapy for ALS and the other motor neuron diseases.

Published by Elsevier Ireland Ltd.

. Introduction

Amyotrophic lateral sclerosis (ALS) is a progressive, fatal, and

ntreatable neurological disease characterized by muscle weak-ess, atrophy and spasticity, typically leading to paralysis and deathithin 3–5 years after symptom onset [1]. Pathologically, ALS is

∗ Corresponding author at: VA Boston Healthcare System, Research and Develop-ent Service, Building 1A – (151), 150 South Huntington Avenue, Boston, MA 02130,SA. Tel.: +1 857 364 6783; fax: +1 857 364 4540.

E-mail address: dedeoglu@bu.edu (A. Dedeoglu).

ttp://dx.doi.org/10.1016/j.neulet.2014.02.058304-3940/Published by Elsevier Ireland Ltd.

primarily characterized by degeneration and death of upper andlower motor neurons (MNs) in the cerebral cortex, brainstem, andspinal cord [1,2]. Most cases of ALS are sporadic (sALS) and ofunknown etiology but approximately 5–10% of patients have a clearfamily history (fALS), typically as an autosomal dominant trait [3].The clinical course of the disease is highly variable suggesting thatthe selective vulnerability of MNs likely arises from a combinationof factors, including protein misfolding, mitochondria dysfunction,

oxidative damage, defective axonal transport, excitotoxicity, insuf-ficient growth factor signaling and inflammation [4]. At least 14genes and loci have been identified to be mutated in ALS [5]. Muta-tions in the superoxide dismutase 1 (SOD1) gene accounts for 20%

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f fALS and apparently for 5% of sALS [6]. The role of SOD1 in ALSs not completely understood, but it is thought that a toxic gain ofunction rather than a loss of dismutase activity are responsible forhe motor neuron loss [7].

Brain-derived neurotrophic factor (BDNF), a member of the neu-otrophin family, is known to support motor neuron differentiationnd survival [8]. It exerts its effects through two transmembraneeceptors: the p75 neurotrophin receptor (p75NTR) and the tyro-ine kinase receptor B (TrkB), the primary receptor for BDNF [9].DNF is of particular therapeutic interest because of its neurotropicctions on neuronal populations involved in several disordersuch as amyotrophic lateral sclerosis [10], Parkinson’s disease, andlzheimer’s disease [11]. However, clinical trials using recombinantDNF have been disappointingly negative, presumably because ofoor delivery to the central nervous system (CNS) and short half-

ife.Recent screening of a chemical library has identified a flavone

erivative 7,8-dihydroxyflavone (7,8-DHF) as the first small-olecule compound that crosses the blood brain barrier (BBB)

nd binds with high affinity and specificity to the BDNF receptorrkB (dissociation constant Kd = 320 nM) and activates its down-tream signaling cascade [12]. Flavonoids, present in fruits andegetables, have been shown to exert diverse biological actionsncluding neuroprotective, anti-oxidant and anti-apoptotic prop-rties. 7,8-DHF not only a neuroprotective agent but may alsoegulate neuromuscular transmission [13]. Neurotrophin signalingt the neuromuscular junction modulates cholinergic transmis-ion [13–15] and BDNF potentiates neurotransmitter release inoth developing neuromuscular synapses in culture [14–16], and indult rat neuromuscular junctions [13]. 7,8-DHF appears to have aumber of beneficial effects in different model systems. For exam-le, it blocks caspase-3 and promotes neurogenesis potentiatingntidepressant drug actions [17], improves learning and spatialemory in stressed mice through effects on the amygdala [18],

ncreases neuronal nuclei size, enhances locomotor activity andmproves breath instability in Rett syndrome mice [19], and reducesrain atrophy and improves survival and motor deficits in a mouseodel of Huntington disease [20]. The effect of 7,8-DHF on ALS is

ot known. In the present study, we investigated the therapeuticffects of 7,8-DHF on motor performance, spine density and lum-ar spinal motor neuron count in a transgenic mouse model of ALSSOD1G93A).

. Materials and methods

.1. Transgenic mice, breeding and genotyping

Transgenic mice with the G93A human SOD1 mutationB6SJLTgN(SOD1G93A)1 Gur; Jackson Laboratories, Bar Harbor,

E,USA) were bred with female B6SJL mice (Jackson Laborato-ies). Only male transgenic and wild type (WT) mice were usedn the present study. Offspring male were genotyped by PCR onNA extracted from tail clippings. At weaning (around 30 days ofge), male transgenic SOD1G93A mice from the same “F” generationere randomly distributed in 2 different experimental groups: 7,8-ihydroxyflavone (7,8-DHF) treated and untreated (saline injected)LS groups (n = 10 for each group). A group of wild-type (WT) lit-

ermate mice were also used in this study. All animal experimentsere carried in accordance with the NIH Guide for the Care and Use

f Laboratory Animals and were approved by the local animal careommittee.

.2. Treatment protocol

Starting at one month of age and until 105 days of age, SOD1G93A

ransgenic and non-transgenic WT mice were injected with

Letters 566 (2014) 286–291 287

7,8-DHF (Tocris Bioscience, Ellisville, MO) (5 mg/kg, i.p./3 days aweek – dilution of DHF: 5 mg/ml – for each 20 g mouse approxi-mately 0.02 ml) or saline (DHF’s diluent).

2.3. Body weight and motor performance test

Starting at one month of age, body weight and motor perfor-mance were monitored twice a week, at the same time of the day.Motor performance test was done on a rotarod apparatus (Colum-bus Instrument, Columbus, OH, USA) after 2 days of training toget acquainted with the apparatus. The motor performance testconsisted in 3 consecutive trials of 60 s each on the rotarod at11 rpm. The time until mice fell from the rod was recorded andthe best of the three trials was used as the measure of competenceon the task for that day.

2.4. Tissue preparation for post-mortem studies

Mice were euthanized at 105 days of age by CO2 suffoca-tion. The lumbar section of the spinal cord was dissected outusing the anatomical features of the cords. The lumbar sectionwas and divided into two parts: the more proximal part to beused for cresyl violet (CV) staining and the more distal for Golgistaining. For CV staining, tissue was fixed in cold Periodate-Lysine-Paraformaldehyde (PLP) solution at 4 ◦C and cryoprotected in 10and 20% glycerol/2% DMSO solutions. Tissue was serially sectionedat 60 �m thickness on a freezing sliding microtome and saved inPBS/2 mM sodium azide at 4 ◦C. Free floating sections were stainedwith cresyl violet to quantify the number of MNs. Golgi staining wasperformed using the FD Rapid GolgiStain Kit (FD NeuroTecnologies)following the manufacturer’s instructions. Stained tissue was seri-ally sectioned at 100 �m thickness using a cryostat microtome andused to quantify the spine density.

2.5. Quantification of motor neurons

Stereological methods were employed to quantify the num-ber of motor neurons (MNs) in the ventral horn of lumbar cordusing an Optical Fractionator probe. A computer software package,StereoInvestigator (MicroBright-Field, Colchester, VT), interfacedwith a Nikon Eclipse 80i microscope equipped with Ludl motorizedstage, Optronics Microfire color digital camera with 1600 × 1200resolution and Heidenheim Z axis encoder was used to collect andanalyze the stereological data. Using the StereoInvestigator’s opti-cal fractionator probe, the total number of MNs was estimated fromcoded slides. To count each case we used 4 section, each 1080 �mapart (one every 16 sections). The area of interest, the ventral horndefined as the anterior subdivision of the gray matter to the mid-dle of the central canal, was traced in the CV stained sections at ×4magnification. The size of the counting frame was 75 �m × 75 �mand the area counted (XY) = 5625 �m2. Cell counting was performedusing a ×60 N.A 1.4 oil objective. Neurons were only counted if theirdiameter was 15 �m or larger and only if the neuron nucleolus wasinside the counting frame and the neuron was not touching theexcluding borders. The computer cursor was set at 15 �m long foreasy detection of cells that meets the 15 �m diameter criterion.Neurons were marked with two different marker depending onsize, 30 �m diameter or larger and smaller than 30 �m but largerthan 15 �m. These MNs referred in text as small and large MNs,respectively.

2.6. Spine density

The density of dendritic spines was quantified by countingspines in 25 �m length of secondary branches of dendrite onselected motor neurons (Fig. 3A). Spine counting was performed

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sing a ×100 N.A 1.4 oil objective. Four sections with 3 sectionnterval, each separated by 1200 �m from the superior to the infe-ior lumbar part of spinal cord, were used to count each case.

e analyzed spine density on ventral horn motor neuron usinghe following criteria: Golgi-stained neurons having dendrites andpines that were completely impregnated, appearing as a continu-

us length; neurons with at least two dendrite extended into therigin of the cell body; neurons with a soma larger than 15 �mere counted. Spine density is defined as the number of spineser micrometer of dendrite length. Images were captured with a

ig. 1. Effects of 7,8-DHF treatment (5 mg/kg, i.p.) on body weight and motor performanody weight and motor performance were monitored twice a week. (A) Body weight loserformance of ALS mice up to the level of wild type (WT) mice (repeated measures muompared to the other groups, n = 10).

ig. 2. Effect of 7,8-DHF treatment (5 mg/kg, i.p.) on motor neurons count in the ventral ho quantify the number of motor neurons. Neurons were marked with two different markhan 15 �m. (A–D) Photomicrographs displaying Cresyl violet (CV) staining of WT-Salintaining photomicrographs ×10 (lower panels) and ×60 (upper panels). Red arrows showhan 15 �m. (E–G) MNs count in 7,8-DHF treated and untreated ALS mice (SOD1G93A) anroup. (F) 7,8-DHF treatment significantly increased small MNs (15–30 �m) count in ALroup (One-way ANOVA, followed by Tukey’s HSD test for multiple comparisons. *p < 0.00eferences to color in this legend, the reader is referred to the web version of the article.)

Letters 566 (2014) 286–291

Nikon Eclipse 80i microscope with a computer software package,StereoInvestigator (MicroBrightField, Colchester, VT). The valuesare calculated by dividing the total spine numbers to the lengthof branches (25 �m)

2.7. Statistical analysis

Data are presented as the means and standard errors(mean ± SEM) for each group, and p < 0.05 was considered signif-icant. Data from rotarod test and body weight were analyzed using

ce in ALS mice (SOD1G93A). Starting at one month of age and until 105 days of age,s of ALS mice is not reversed by 7,8-DHF. (B) 7,8-DHF treatment improved motorlti-way ANOVA, followed by the Dunns test for posthoc comparisons, *p < 0.001 as

orn of lumbar cord in ALS mice (SOD1G93A). Stereological methods were employeder depending on size, 30 �m diameter or larger and smaller than 30 �m but larger

e (A), WT-7,8-DHF (B), ALS-Saline (C), and ALS-7,8-DHF (D). Magnifications for CV MNs larger than 30 �m, yellow arrows show MNs smaller than 30 �m but largerd WT mice. (E) 7,8-DHF treatment significantly increased total MNs count in ALSS group. (G) 7,8-DHF treatment did not change large MNs (>30 �m) count in ALS1 and #p < 0.01, as compared to the other groups, n = 10). (For interpretation of the

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he repeated measures multi-way ANOVA, followed by the Dunnsest for posthoc comparisons. Statistical evaluation of the neuronalount and spine density were performed using one-way ANOVA,ollowed by Tukey’s HSD test for multiple comparisons.

. Results

Body weight of the untreated SOD1G93A mice was significantlyower than those of WT mice. Difference in body weight were firstbserved at 60 days of age and persisted until the final day (105ays). Treatment with 7, 8-DHF did not prevent loss of body weight

n the transgenic mice (Fig. 1A).Similarly, motor performance deficits were first detected after

0 days age and persisted until death in WT and saline treatedOD1G93A mice. 7,8-DHF treatment significantly improved motorerformance of SOD1G93A mice (p < 0.001, Fig. 1B).

Motor neurons counts in saline treated SOD1G93A mice wereignificantly reduced compared to WT groups (p < 0.001, Fig. 2).,8-DHF treatment prevented MN loss (Fig. 2E). 7,8-DHF treatmentreserved MN counts of small but not large MNs (Fig. 2F and G).

Dendritic spine density of untreated SOD1G93A mice waseduced compared to the WT controls (p < 0.001). Dendritic spineensity of MNs in 7,8-DHF treated SOD1G93A mice, however, wereignificantly higher than saline treated transgenic mice and did notiffer from that of WT mice (p < 0.001, Fig. 3).

. Discussion

This study was designed to determine the prophylactic effect ofhe long-term treatment with 7,8-DHF, a TrkB agonist, in SOD1G93A

ice. Treatment started at 1 month of age when disease symptomsre not yet apparent and ended at 105 days of age, at an advancedtage of the disease but before the terminal-stage to avoid that theapid progression of the pathological process in its final phase could

ig. 3. Effect of 7,8-DHF treatment (5 mg/kg, i.p.) on dendritic spine density of motor neuisplaying Golgi staining of WT-Saline (A), WT-7,8-DHF (B), ALS-Saline (C), and ALS-7,8-D

ength of secondary branches branch for (red line in figure A). While untreated ALS mice

ice (A and B). (E) 7,8-DHF treatment significantly increased spine density in ALS group (os compared to the other groups, n = 10). (For interpretation of the references to color in t

Letters 566 (2014) 286–291 289

masked the potential beneficial effect of 7,8-DHF. Because there areno studies on the effect of 7,8-DHF on ALS animal models we chosethe dose of 5 mg/kg and the i.p. route based on previous studies inmouse models of stroke, PD, and AD [12,21]. In those studies micewere treated daily with 5 mg/kg i.p. for 10 and 14 days. Becausethe overexpression of BDNF has been shown to have detrimen-tal effects in the brain [22] and our treatment period was muchlonger than those previously reported we decided to treat mice 3times a week only. Moreover, this intermittent treatment paral-leled the schedule of our previous work in which SOD1G93A micewere treated for 30 min 3 days a week with moderate level exer-cise, which is known to increase BDNF/TrkB signaling [23]. 7,8-DHFtreatment significantly preserved motor performance and lumbarspinal motor neurons in ALS mice without reversing the weight lossthat characterizes the disease. Additionally, this study also revealsthe differential effect of 7,8-DHF treatment on lumbar spinal motorneuron subtypes at the dosage used.

A recent report has been published [24] describing for the firsttime the in vivo pharmacokinetic properties of 7,8-DHF and itsmetabolites. In that study, mice were treated by oral gavage with50 mg/kg, a dosage that is 10-fold the widely used therapeuticdose. The concentration of 7,8-DHF in the plasma peaked at 10 min(70 ng/ml) and was still detectable at 8 h (5 ng/ml) (T1/2 = 134 min).In the brain 7,8-DHF also peaked at 10 min (52 ng/g), decreasedto 18 ng/g at 30 min, and remained relatively stable until 240 min(7 ng/g). The orally administered 7,8-DHF was mainly metabolizedto glucuronidated and O-methylated 7,8-DHF. The parent drug andthe methylated metabolite penetrate the BBB, where they both acti-vated TrkB receptors. Since the concentration of 7,8-DHF in thebrain was approximately 30-fold higher than that of the methylated

form it was expect that the main contribution to TrkB activationcame from 7,8-DHF itself. In the present study we used differentdosage and route of administration and the kinetics of 7,8-DHF maydiffer from those described. Because of the low T1/2 and low toxicity

rons in the ventral horn of lumbar cord in ALS mice (SOD1G93A). PhotomicrographsHF (D). Magnifications for photomicrographs ×100. Spines were counted in 25 �m(C) have lesser spines than WT, 7,8-DHF treated ALS mice (D) have as much as WTne-way ANOVA, followed by Tukey’s HSD test for multiple comparisons. *p < 0.001,his legend, the reader is referred to the web version of the article.)

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f 7,8-DHF, increasing the treatment frequency used in our studiesay result in broader and stronger beneficial effects.Spinal MNs are a highly diverse group in terms of their morphol-

gy, connectivity, and functional properties and differ significantlyn their response to disease. Motor neurons can be classified insmall” (<30 �m in soma diameter) or “large” (30 �m or greatern diameter) [25]. MNs are categorized as alpha (�), beta (�) oramma (�) MNs. �- and �-MNs are the smallest MNs and inner-ate intrafusal fibers of the muscle spindle and regulate the muscleonus [26]. �-MNs are larger and innervate extrafusal skeletal mus-le cells that drive muscle contraction [25]. In this study, 7,8-DHFreatment of SOD1G93A mice resulted in the preferential preserva-ion of small MNs. One possible explanation for our observations ishrinkage but persistence of large MNs that would otherwise dieithout 7,8-DHF treatment. The normal morphology of the smallNs would speak against this possibility. If the persistent neurons

re �-MNs they could improve motor performance indirectly byriming extrafusal fibers innervated by large MNs. The preservedeurons may be small �-MNs that have higher input resistance andequire less synaptic activation to initiate action potentials. Duringuscle contraction small �-MNs reach threshold potential earlier

hen large MNs, this is known as “size principle” [25,26]. More-ver, while large �-MNs are mainly employed in short-lasting boutsf forceful contraction (e.g. running, jumping), called fast �-MNs,n the other hand, small �-MNs, dominantly get involved in long-erm tonic phase of contraction, such as postural tasks, called slow-MNs [27]. Histological studies in post-mortem tissue from ALSatients show �-MNs deprivation alongside loss of �-MNs [28].owever, a study using SOD1G93A transgenic mice showed that-MNs are more vulnerable that �-MNs and concluded that �-Ns loss is primarily seen in this model [29]. Our study showing

ncreased number of small MNs but not large MNs in treated miceuggests that 7,8-DHF stimulates the survival of �-MNs and small-MNs that results in improved motor performance on the rotarod.

study has shown that in WT rats the expression of TrkB doesot correlate with motor neuronal size [30] however, no studiesave been done in SOD1G93A mice to determine how the diseaserocess may affect the level of TrkB. Growing evidence suggestshat neuronal activity enhances BDNF signaling by increasing theell-surface expression of TrkB and promoting TrkB endocytosis,

signaling event important for many long-term BDNF functions31]. TrkB levels could be influenced under a variety of pathologi-al conditions known to alter neuronal activity. Since smaller MNsre more easily activated than larger motor units we speculate thatn SOD1G93A mice �-MNs and small �-MNs have more TrkB recep-ors than large �-MNs and therefore they are more responsive to,8-DHF treatment.

BDNF is one of the most potent modulators of synaptic trans-ission, plasticity, and morphology [32,33]. It increases dendritic

pine density in a variety of CNS neurons [34,35]. Dendritic spinesncrease the connectivity of the dendrites and regulate input-pecific synaptic plasticity [36]. They have also an electrical role byltering synaptic potentials and isolating inputs from each other37]. We found that 7,8-DHF increases MNs dendritic spine den-ity in SOD1G93A transgenic mice. It would be interesting to knowhether the effect of 7,8-DHF on dendritic spine density correlateith neuronal size.

. Conclusion

Chronic 7,8-DHF treatment from 30 days to 105 days improved

otor deficits with a differential effect on small-MNs in the ven-

ral horn of lumbar spinal cord of SOD1G93A mice. Spine densityas also increased in treated mice. 7,8-DHF could be an important

herapeutic tool for ALS and other neurodegenerative disorders.

[

Letters 566 (2014) 286–291

Studies at different time points (95 days–when the symptoms startand 120 days-terminal stage) are underway to further characterizethe effects of 7,8-DHF on SOD1G93A mice.

Conflict of interest

The authors declare no competing financial interests.

Acknowledgements

This research is supported by grants from NIA (R01AG031896,P30AG013846) and the Department of Veteran Affairs (MeritAwards) to A. Dedeoglu and NW Kowall, and Scientific and Tech-nical Research Council of Turkey (TUBITAK) to O.T. Korkmaz. Theauthors thank to Lokman Hossain for animal husbandry and Dr.Nese Tuncel (Eskisehir Osmangazi University, Turkey) for valuablediscussions.

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