DJ-1 based peptide, ND-13, promote functional recovery in mouse model of focal ischemic injury.

Stroke is a leading cause of death worldwide and inflicts serious long-term damage and disability. The vasoconstrictor Endothelin-1, presenting long-term neurological deficits associated with excitotoxicity and oxidative stress is being increasingly used to induce focal ischemic injury as a model of stroke. A DJ-1 based peptide named ND-13 was shown to protect against glutamate toxicity, neurotoxic insults and oxidative stress in various animal models. Here we focus on the benefits of treatment with ND-13 on the functional outcome of focal ischemic injury. Wild type C57BL/6 mice treated with ND-13, after ischemic induction in this model, showed significant improvement in motor function, including improved body balance and motor coordination, and decreased motor asymmetry. We found that DJ-1 knockout mice are more sensitive to Endothelin-1 ischemic insult than wild type mice, contributing thereby additional evidence to the widely reported relevance of DJ-1 in neuroprotection. Furthermore, treatment of DJ-1 knockout mice with ND-13, following Endothelin-1 induced ischemia, resulted in significant improvement in motor functions, suggesting that ND-13 provides compensation for DJ-1 deficits. These preliminary results demonstrate a possible basis for clinical application of the ND-13 peptide to enhance neuroprotection in stroke patients

Neuroprotective Effect of a DJ-1 Based Peptide in a Toxin Induced Mouse Model of Multiple System Atrophy.

Multiple System Atrophy (MSA) is a sporadic neurodegenerative disorder characterized by parkinsonism, cerebellar ataxia and dysautonomia, in various combinations. In MSA with parkinsonism (MSA-P), the degeneration is mainly restricted to the substantia nigra pars compacta and putamen. Studies have identified alterations in DJ-1 (PARK7), a key component of the anti-oxidative stress response, in Parkinson's disease (PD) and MSA patients. Previously we have shown that a short DJ-1-based peptide named ND-13, protected cultured cells against neurotoxic insults and improved behavioral outcome in animal models of Parkinson's disease (PD). In this study, we used the 3-Nitropropionic acid (3-NP)-induced mouse model of MSA and treated the animals with ND-13 in order to evaluate its therapeutic effects. Our results show that ND-13 protects cultured cells against oxidative stress generated by the mitochondrial inhibitor, 3-NP. Moreover, we show that ND-13 attenuates nigrostriatal degeneration and improves performance in motor-related behavioral tasks in 3-NP-treated mice. Our findings suggest a rationale for using ND-13 as a promising therapeutic approach for treatment of MSA

DJ-1 knockout augments disease severity and shortens survival in a mouse model of ALS.

Amyotrophic lateral sclerosis (ALS) is a progressive, lethal, neurodegenerative disorder, characterized by the degeneration of motor neurons. Oxidative stress plays a central role in the disease progression, in concert with an enhanced glutamate excitotoxicity and neuroinflammation. DJ-1 mutations, leading to the loss of functional protein, cause familial Parkinson's disease and motor neuron disease in several patients. DJ-1 responds to oxidative stress and plays an important role in the cellular defense mechanisms. We aimed to investigate whether loss of functional DJ-1 alters the disease course and severity in an ALS mouse model. To this end we used mice that express the human SOD1G93A mutation, the commonly used model of ALS and knockout of DJ-1 mice to generate SOD1 DJ-1 KO mice. We found that knocking out DJ-1in the ALS model led to an accelerated disease course and shortened survival time. DJ-1 deficiency was found to increase neuronal loss in the spinal cord associated with increased gliosis in the spinal cord and reduced antioxidant response that was regulated by the Nrf2 mechanism.The importance of DJ-1 in ALS was also illustrated in a motor neuron cell line that was exposed to glutamate toxicity and oxidative stress. Addition of the DJ-1 derived peptide, ND-13, enhanced the resistance to glutamate and SIN-1 induced toxicity. Thus, our results maintain that DJ-1 plays a role in the disease process and promotes the necessity of further investigation of DJ-1 as a therapeutic target for ALS

Effect of ND-13 treatment on functional recovery after focal ischemic injury.

<p>ND-13 treatment significantly improved time spent crossing the bridge in the elevated bridge test, two days after injury, compared to control (A, p<0.05). The time to descend from a vertical pole in the pole test also decreased following ND-13 treatment compared to control (B, p<0.05). The motor asymmetry in the cylinder test decreased 2 days after ischemic injury (C, p<0.05). ND-13 effect was still consistent 21 days after ischemic injury in all behavioral tests, that is, mice treated with ND-13 perform better than control mice. (Data is shown as mean ± SEM).</p

Experimental protocol and ischemic injury location.

<p>Experimental protocol in days (A). The vasoconstrictor Endothelin-1 was injected into the striatum to induce ischemia. The ischemic area in the striatum is circled in black (B).</p

Toll-Like Receptor-4 Inhibitor TAK-242 Attenuates Motor Dysfunction and Spinal Cord Pathology in an Amyotrophic Lateral Sclerosis Mouse Model

Neuroinflammation contributes to amyotrophic lateral sclerosis (ALS) progression. TLR4, a transmembrane protein that plays a central role in activation of the innate immune system, has been shown to induce microglial activation in ALS models. TLR4 is up-regulated in the spinal cords of hSOD1G93A mice. We aimed to examine the effects of specific TLR4 inhibition on disease progression and survival in the hSOD1G93A mouse model of ALS. Immunologic effect of TLR4 inhibition in vitro was measured by the effect of TAK-242 treatment on LPS-induced splenocytes proliferation. hSOD1G93A transgenic mice were treated with TAK-242, a selective TLR4 inhibitor, or vehicle. Survival, body weight, and motor behavior were monitored. To evaluate in vivo immunologic modifications associated with TAK-242 treatment, we measured serum IL-1β in the plasma, as well as IL-1β and TNF-α mRNAs in the spinal cord in wild-type mice and in TAK-242-treated and vehicle-treated early symptomatic hSOD1G93A mice. Immunohistochemical analysis of motor neurons, astrocytes, and microglial reactivity in the spinal cords were performed on symptomatic (100 days old) TAK-242-treated and vehicle-treated hSOD1G93A mice. In vitro, splenocytes taken from 100 days old hSOD1G93A mice showed significantly increased proliferation when exposed to LPS (p = 0.0002), a phenomenon that was reduced by TAK-242 (p = 0.0179). TAK-242 treatment did not attenuate body weight loss or significantly affect survival. However, TAK-242-treated hSOD1G93A mice showed temporary clinical delay in disease progression evident in the ladder test and hindlimb reflex measurements. Plasma IL-1β levels were significantly reduced in TAK-242-treated compared to vehicle-treated hSOD1G93A mice (p = 0.0023). TAK-242 treatment reduced spinal cord astrogliosis and microglial activation and significantly attenuated spinal cord motor neuron loss at early disease stage (p = 0.0259). Compared to wild-type animals, both IL-1β and TNF-α mRNAs were significantly upregulated in the spinal cords of hSOD1G93A mice. Spinal cord analysis in TAK-242-treated hSOD1G93A mice revealed significant attenuation of TNF-α mRNA (p = 0.0431), but no change in IL-1β mRNA. TLR4 inhibition delayed disease progression, attenuated spinal cord astroglial and microglial reaction, and reduced spinal motor neuron loss in the ALS hSOD1G93A mouse model. However, this effect did not result in increased survival. To our knowledge, this is the first report on TAK-242 treatment in a neurodegenerative disease model. Further studies are warranted to assess TLR4 as a therapeutic target in ALS

Effect of ND-13 treatment on functional recovery of DJ-1 KO mice after ET-1 induced focal ischemic injury.

<p>DJ-1 KO mice treated with ND-13 show improvement 2 days after ischemic injury in the elevated bridge test as they spend less time crossing the beam than wild type mice (A, p<0.05). This effect was consistent for at least two weeks following injury. Improvement was also noted in cylinder test 7 days after injury (B). (Data is shown as mean ± SEM).</p

ND-13 reduces astrogliosis in 3-NP injected mice.

<p>3-NP was injected into the striatum (A). GFAP immunostaining demonstrated astrogliosis in mice treated with 3-NP (saline, B) (B). In contrast, mice treated with 3-NP + ND-13 showed a significant reduction in the levels of astrocyte activation (C). The intensity of the staining was calculated by Image-J software (D, *p< 0.05, t-test, error bars indicate SEM, n = 4). Scale bars (A): 200 μm, (B and C): 100 μm. Astrogliosis reduction, following ND-13 treatment was also demonstrated by Western blot analysis with the anti-GFAP antibodies (E and F). *p<0.05, t-test, error bars indicate SEM (n = 4).</p

ND-13 preserves striatal cells and dopaminergic terminals in 3-NP injected mice.

<p>Immunohistology of brain sections with anti-DARPP-32 antibodies indicates for specific staining in the striatum (A). Mice treated with 3-NP + saline (B, D) and mice treated with 3-NP + ND-13 (C, E) were stained with antibodies against DARPP-32 (B, C) and TH (D, E). The number of DARPP-32 positive cells (F) and the intensity of TH staining (G) were calculated by Image-J software. *p< 0.05, t-test, error bars indicate SEM (n = 5). Scale bars (A): 500 μm and (B-E): 100 μm. DARPP32 and TH levels were also measured by immunoblot analysis (H, I) in the right (3-NP injected) and the left striatum of mice systemically treated with ND-13 or saline. The ratio between left and right hemispheres are shown in J and K.*p<0.05, t-test, error bars indicate SEM (n = 4).</p