BAG1 is neuroprotective in in vivo and in vitro models of parkinson s disease

Bcl-2-associated athanogene-1 (BAG1) is a multifunctional protein comprising co-chaperone function, increasing Hsp70 foldase activity and chaperone-dependent protein degradation of misfolded substrates, with anti-apoptotic activity. It is neuroprotective in different models of neurological diseases, like cerebral ischemia and Huntington’s disease. In the context of Parkinson’s disease, it has recently been shown to restore DJ-1 function in an in vitro model of hereditary Parkinson’s disease. Here, we demonstrate that BAG1 overexpression in SH-SY5Y cells reduces toxicity after transfection of disease-related α-synuclein mutants. Furthermore, it protects from rotenone-induced cell death in vitro and ameliorates neuronal demise in an in vivo 1-methyl-4-phenyl-1,2,3,6,-tetrahydropyridine (MPTP) model for Parkinson’s disease after adeno-associated virus (AAV)-mediated BAG1 gene transfer into the substantia nigra in mice but showed no protective effects in an in vitro 6-hydroxidopamine model. In conclusion, we present BAG1 as a potential therapeutic target in Parkinson’s disease.

BAG1 is Neuroprotective in In Vivo and In Vitro Models of Parkinson’s Disease

Bcl-2-associated athanogene-1 (BAG1) is a multifunctional protein comprising co-chaperone function, increasing Hsp70 foldase activity and chaperone-dependent protein degradation of misfolded substrates, with anti-apoptotic activity. It is neuroprotective in different models of neurological diseases, like cerebral ischemia and Huntington’s disease. In the context of Parkinson’s disease, it has recently been shown to restore DJ-1 function in an in vitro model of hereditary Parkinson’s disease. Here, we demonstrate that BAG1 overexpression in SH-SY5Y cells reduces toxicity after transfection of disease-related α-synuclein mutants. Furthermore, it protects from rotenone-induced cell death in vitro and ameliorates neuronal demise in an in vivo 1-methyl-4-phenyl-1,2,3,6,-tetrahydropyridine (MPTP) model for Parkinson’s disease after adeno-associated virus (AAV)-mediated BAG1 gene transfer into the substantia nigra in mice but showed no protective effects in an in vitro 6-hydroxidopamine model. In conclusion, we present BAG1 as a potential therapeutic target in Parkinson’s disease.

modulation of huntingtin toxicity by BAG1 is dependent on an intact bag domain

Huntington´s disease, one of the so-called poly-glutamine diseases, is a dominantly inherited movement disorder characterized by formation of cytosolic and nuclear inclusion bodies and progressive neurodegeneration. Recently, we have shown that Bcl-2-associated athanogene-1 (BAG1), a multifunctional co-chaperone, modulates toxicity, aggregation, degradation and subcellular distribution in vitro and in vivo of the disease-specific mutant huntingtin protein. Aiming at future small molecule-based therapeutical approaches, we further analysed structural demands for these effects employing the C-terminal deletion mutant BAGDC. We show that disruption of the BAG domain known to eliminate intracellular heat shock protein 70 (Hsp70) binding and activation also precludes binding of Siah-1 thereby leaving nuclear huntingtin translocation unaffected. At the same time BAGDC fails to induce increased proteasomal huntingtin turnover and does not inhibit intracellular huntingtin aggregation, a pre-requisite necessary for prevention of huntingtin toxicity.

BAG1 modulates huntingtin toxicity aggregation degradation and subcellular distribution

Bcl-2-associated athanogene-1 (BAG1) is a multifunctional protein delivering chaperone-recognized unfolded substrates to the proteasome for degradation. It has been shown to be essential for proper CNS development in vivo, playing a crucial role in neuronal survival and differentiation. With regard to Huntington’s disease, a sequestration of BAG1 into inclusion bodies and a neuroprotective effect in double transgenic mice have been reported. Here, we show that BAG1 reduces aggregation and accelerates degradation of mutant huntingtin (htt-mut). Moreover, it reduces nuclear levels of htt-mut. This effect can be overcome by over-expression of seven in absentia homolog 1, an E3 ligase negatively regulated by BAG1 and known to be involved in nuclear import of htt-mut. In vivo, BAG1 proved to be protective in a Drosophila melanogaster Huntington’s disease model, preventing photoreceptor cell loss induced by htt-mut. In summary, we present BAG1 as a therapeutic tool modulating key steps in htt toxicity in vitro and ameliorating htt toxicity in vivo.

Neuron-specific overexpression of the co-chaperone Bcl-2-associated athanogene-1 in superoxide dismutase 1G93A–transgenic mice

Abstract Bcl-2-associated athanogene-1 (BAG1) binds heat-shock protein 70 (Hsp70)/Hsc70, increases intracellular chaperone activity in neurons and proved to be protective in several models for neurodegeneration. Mutations in the superoxide dismutase 1 (SOD1) gene account for approximately 20% of familial amyotrophic lateral sclerosis (ALS) cases. A common property shared by all mutant SOD1 (mtSOD1) species is abnormal protein folding and the propensity to form aggregates. Toxicity and aggregate formation of mutant SOD1 can be overcome by enhanced chaperone function in vitro . Moreover, expression of mtSOD1 decreases BAG1 levels in a motoneuronal cell line. Thus, several lines of evidence suggested a protective role of BAG1 in mtSOD1-mediated motoneuron degeneration. To explore the therapeutic potential of BAG1 in a model for ALS, we generated SOD1 G93A /BAG1 double transgenic mice expressing BAG1 in a neuron-specific pattern. Surprisingly, substantially increased BAG1 protein levels in spinal cord neurons did not significantly alter the phenotype of SOD1 G93A -transgenic mice. Hence, expression of BAG1 is not sufficient to protect against mtSOD1-induced motor dysfunction in vivo . Our work shows that, in contrast to the in vitro situation, modulation of multiple cellular functions in addition to enhanced expression of a single chaperone is required to protect against SOD1 toxicity, highlighting the necessity of combined treatment strategies for ALS.

BAG1 promotes axonal outgrowth and regeneration in vivo via raf 1 and reduction of rock activity

Improved survival of injured neurons and the inhibition of repulsive environmental signalling are prerequisites for functional regeneration. BAG1 (Bcl-2-associated athanogene-1) is an Hsp70/Hsc70 -binding protein, which has been shown to suppress apoptosis and enhance neuronal differentiation. We investigated BAG1 as a therapeutic molecule in the lesioned visual system in vivo. Using an adeno-associated viral vector, BAG1 (AAV.BAG1) was expressed in retinal ganglion cells (RGC) and then tested in models of optic nerve axotomy and optic nerve crush. BAG1 significantly increased RGC survival as compared to adeno-associated viral vector enhanced green fluorescent protein (AAV.EGFP) treated controls and this was independently confirmed in transgenic mice overexpressing BAG1 in neurons. The numbers and lengths of regenerating axons after optic nerve crush were also significantly increased in the AAV.BAG1 group. In pRGC cultures, BAG1-over-expression resulted in a 3- fold increase in neurite length and growth cone surface. Interestingly, BAG1 induced an intracellular translocation of Raf-1 and ROCK2 and ROCK activity was decreased in a Raf-1-dependent manner by BAG1-over-expression. In summary, we show that BAG1 acts in a dual role by inhibition of lesion-induced apoptosis and interaction with the inhibitory ROCK signalling cascade. BAG1 is therefore a promising molecule to be further examined as a putative therapeutic tool in neurorestorative strategies.

aav mediated expression of BAG1 and rock2 shrna promote neuronal survival and axonal sprouting in a rat model of rubrospinal tract injury

A lesion to the rat rubrospinal tract is a model for traumatic spinal cord lesions and results in atrophy of the red nucleus neurons, axonal dieback, and locomotor deficits. In this study, we used adeno-associated virus (AAV)-mediated over-expression of BAG1 and ROCK2-shRNA in the red nucleus to trace [by co-expression of enhanced green fluorescent protein (EGFP)] and treat the rubrospinal tract after unilateral dorsal hemisection. We investigated the effects of targeted gene therapy on neuronal survival, axonal sprouting of the rubrospinal tract, and motor recovery 12 weeks after unilateral dorsal hemisection at Th8 in rats. In addition to the evaluation of BAG1 and ROCK2 as therapeutic targets in spinal cord injury, we aimed to demonstrate the feasibility and the limits of an AAV-mediated protein over-expression versus AAV.shRNA-mediated down-regulation in this traumatic CNS lesion model. Our results demonstrate that BAG1 and ROCK2-shRNA both promote neuronal survival of red nucleus neurons and enhance axonal sprouting proximal to the lesion.

Understanding the mechanisms involved in neuronal survival and axonal regeneration after spinal cord injury (SCI) is pivotal for the development of new therapies. We showed that over-expression of BAG1 (Bcl-2-associated athanogene-1) and down-regulation of ROCK2 (Rho-associated protein kinase) improve neuronal survival and axonal sprouting after SCI. Our results imply that BAG1 and ROCK2 represent interesting molecular targets that can be used in future therapeutic strategies for the treatment of SCI. AAV = adeno-associated virus.

AAV‐mediated expression of BAG1 and ROCK2‐shRNA promote neuronal survival and axonal sprouting in a rat model of rubrospinal tract injury

A lesion to the rat rubrospinal tract is a model for traumatic spinal cord lesions and results in atrophy of the red nucleus neurons, axonal dieback, and locomotor deficits. In this study, we used adeno-associated virus (AAV)-mediated over-expression of BAG1 and ROCK2-shRNA in the red nucleus to trace [by co-expression of enhanced green fluorescent protein (EGFP)] and treat the rubrospinal tract after unilateral dorsal hemisection. We investigated the effects of targeted gene therapy on neuronal survival, axonal sprouting of the rubrospinal tract, and motor recovery 12 weeks after unilateral dorsal hemisection at Th8 in rats. In addition to the evaluation of BAG1 and ROCK2 as therapeutic targets in spinal cord injury, we aimed to demonstrate the feasibility and the limits of an AAV-mediated protein over-expression versus AAV.shRNA-mediated down-regulation in this traumatic CNS lesion model. Our results demonstrate that BAG1 and ROCK2-shRNA both promote neuronal survival of red nucleus neurons and enhance axonal sprouting proximal to the lesion.

Understanding the mechanisms involved in neuronal survival and axonal regeneration after spinal cord injury (SCI) is pivotal for the development of new therapies. We showed that over-expression of BAG1 (Bcl-2-associated athanogene-1) and down-regulation of ROCK2 (Rho-associated protein kinase) improve neuronal survival and axonal sprouting after SCI. Our results imply that BAG1 and ROCK2 represent interesting molecular targets that can be used in future therapeutic strategies for the treatment of SCI. AAV = adeno-associated virus.

BAG1 is Neuroprotective in In Vivo and In Vitro Models of Parkinson’s Disease

Bcl-2-associated athanogene-1 (BAG1) is a multifunctional protein comprising co-chaperone function, increasing Hsp70 foldase activity and chaperone-dependent protein degradation of misfolded substrates, with anti-apoptotic activity. It is neuroprotective in different models of neurological diseases, like cerebral ischemia and Huntington’s disease. In the context of Parkinson’s disease, it has recently been shown to restore DJ-1 function in an in vitro model of hereditary Parkinson’s disease. Here, we demonstrate that BAG1 overexpression in SH-SY5Y cells reduces toxicity after transfection of disease-related α-synuclein mutants. Furthermore, it protects from rotenone-induced cell death in vitro and ameliorates neuronal demise in an in vivo 1-methyl-4-phenyl-1,2,3,6,-tetrahydropyridine (MPTP) model for Parkinson’s disease after adeno-associated virus (AAV)-mediated BAG1 gene transfer into the substantia nigra in mice but showed no protective effects in an in vitro 6-hydroxidopamine model. In conclusion, we present BAG1 as a potential therapeutic target in Parkinson’s disease.