Protocol for evaluation of neurotrophic strategies in Parkinson


  • Shane V. Hegarty Department of Anatomy and Neuroscience, University College Cork, Ireland.
  • Aideen M. Sullivan Department of Anatomy and Neuroscience, University College Cork, Ireland.
  • Gerard W. O'Keeffe Department of Anatomy and Neuroscience, University College Cork, Ireland.



neurite growth analysis, dopaminergic neuron, Parkinson, neurotrophic therapy, sympathetic neuron


Parkinson’s disease (PD) is a neurodegenerative disease that is characterised by motor and non-motor symptoms which result from the progressive degeneration of nigrostriatal ventral midbrain (VM) dopaminergic (DA) neurons, as well as peripheral sympathetic neurons. PD is incurable, with current therapeutic strategies providing symptomatic relief. Neurotrophic factor (NTF) therapy has the potential to protect degenerating neurons in PD. However, there has been limited success in PD clinical trials due to neurotrophic strategies that are invasive, inefficient in delivering sustained neurotrophic support, do not protect all degenerating neurons and may have a compromised mechanism of action in the PD brain. Therefore, while neurotrophic therapy remains a promising disease-modifying approach for PD, it is important to identify novel neurotrophic strategies that can protect all neurons affected by PD. To address this need, we report an integrated approach for pre-clinical evaluation of potential neurotrophic strategies, e.g., pharmacological agents (e.g., drugs/small molecules), signalling proteins (e.g., morphogens) and/or genetic (gene/mRNA) modifications, in cellular models of the neuronal populations that are affected by PD. Herein, we describe, in detail, an in vitro protocol that allows a step-wise evaluation of the efficacy, and mechanism(s) of action, of novel neurotrophic strategies in VM DA neurons and sympathetic neurons, following an initial evaluation in a human cell line model of these cells, SH-SY5Y cells. The protocol uses the induction of neurite growth as the primary measure of neurotrophic action. Indeed, the neuro-protection/-restoration of PD-affected axons is widely thought to be an appropriate target for effective therapeutic intervention in PD.

Author Biography

Shane V. Hegarty, Department of Anatomy and Neuroscience, University College Cork, Ireland.

Post-Doctoral Research Fellow, Department of Anatomy and Neuroscience, University College Cork, Ireland.


Dorsey ER, George BP, Leff B, Willis AW (2013) The coming crisis: obtaining care for the growing burden of neurodegenerative conditions. Neurology 80: 1989-1996.

Jankovic J (2008) Parkinson's disease: clinical features and diagnosis. J Neurol Neurosurg Psychiatry 79: 368-376.

Lees AJ, Hardy J, Revesz T (2009) Parkinson's disease. Lancet 373: 2055-2066.

Bethlem J, Den Hartog Jager WA (1960) The incidence and characteristics of Lewy bodies in idiopathic paralysis agitans (Parkinson's disease). J Neurol Neurosurg Psychiatry 23: 74-80.

Jellinger KA (2012) Neuropathology of sporadic Parkinson's disease: evaluation and changes of concepts. Mov Disord 27: 8-30.

Jellinger KA (1991) Pathology of Parkinson's disease. Changes other than the nigrostriatal pathway. Mol Chem Neuropathol 14: 153-197.

Bedard C, Wallman MJ, Pourcher E, Gould PV, Parent A, et al. (2011) Serotonin and dopamine striatal innervation in Parkinson's disease and Huntington's chorea. Parkinsonism Relat Disord 17: 593-598.

Goldstein DS, Holmes C, Li ST, Bruce S, Metman LV, et al. (2000) Cardiac sympathetic denervation in Parkinson disease. Ann Intern Med 133: 338-347.

Goldstein DS, Holmes C, Cannon RO, 3rd, Eisenhofer G, Kopin IJ (1997) Sympathetic cardioneuropathy in dysautonomias. N Engl J Med 336: 696-702.

Kaufmann H, Goldstein DS (2013) Autonomic dysfunction in Parkinson disease. Handb Clin Neurol 117: 259-278.

Lucio CG, Vincenzo C, Antonio R, Oscar T, Luigi M (2013) Neurological applications for myocardial MIBG scintigraphy. Nucl Med Rev Cent East Eur 16: 35-41.

Toulouse A, Sullivan AM (2008) Progress in Parkinson's disease-where do we stand? Prog Neurobiol 85: 376-392.

Hegarty SV, Sullivan AM, O'Keeffe GW (2014) Roles for the TGFbeta superfamily in the development and survival of midbrain dopaminergic neurons. Mol Neurobiol 50: 559-573.

Hegarty SV, O'Keeffe GW, Sullivan AM (2014) Neurotrophic factors: from neurodevelopmental regulators to novel therapies for Parkinson's disease. Neural Regen Res 9: 1708-1711.

Olanow CW, Bartus RT, Baumann TL, Factor S, Boulis N, et al. (2015) Gene delivery of neurturin to putamen and substantia nigra in Parkinson disease: A double-blind, randomized, controlled trial. Ann Neurol 78: 248-257.

Sullivan AM, Toulouse A (2011) Neurotrophic factors for the treatment of Parkinson's disease. Cytokine Growth Factor Rev 22: 157-165.

Decressac M, Ulusoy A, Mattsson B, Georgievska B, Romero-Ramos M, et al. (2011) GDNF fails to exert neuroprotection in a rat alpha-synuclein model of Parkinson's disease. Brain 134: 2302-2311.

Decressac M, Kadkhodaei B, Mattsson B, Laguna A, Perlmann T, et al. (2012) alpha-Synuclein-induced down-regulation of Nurr1 disrupts GDNF signaling in nigral dopamine neurons. Sci Transl Med 4: 163ra156.

Sullivan AM, Toulouse A (2011) Neurotrophic factors for the treatment of Parkinson's disease. Cytokine Growth Factor Rev.

Burke RE, O'Malley K (2013) Axon degeneration in Parkinson's disease. Exp Neurol 246: 72-83.

Hegarty SV, Sullivan AM, O'Keeffe GW (2013) BMP2 and GDF5 induce neuronal differentiation through a Smad dependant pathway in a model of human midbrain dopaminergic neurons. Mol Cell Neurosci 56C: 263-271.

Mayhew TM (1992) A review of recent advances in stereology for quantifying neural structure. J Neurocytol 21: 313-328.

Brees C, Fransen M (2014) A cost-effective approach to microporate mammalian cells with the Neon Transfection System. Anal Biochem 466: 49-50.

Hegarty SV, Collins LM, Gavin AM, Roche SL, Wyatt SL, et al. (2014) Canonical BMP-Smad signalling promotes neurite growth in rat midbrain dopaminergic neurons. Neuromolecular Med 16: 473-489.

Collins LM, Adriaanse LJ, Theratile SD, Hegarty SV, Sullivan AM, et al. (2015) Class-IIa Histone Deacetylase Inhibition Promotes the Growth of Neural Processes and Protects Them Against Neurotoxic Insult. Mol Neurobiol 51: 1432-1442.

Hegarty SV, O'Leary E, Solger F, Stanicka J, Sullivan AM, et al. (2016) A Small Molecule Activator of p300/CBP Histone Acetyltransferase Promotes Survival and Neurite Growth in a Cellular Model of Parkinson's Disease. Neurotox Res.

O'Keeffe GW, Gutierrez H, Howard L, Laurie CW, Osorio C, et al. (2016) Region-specific role of growth differentiation factor-5 in the establishment of sympathetic innervation. Neural Dev 11: 4.

Glebova NO, Ginty DD (2005) Growth and survival signals controlling sympathetic nervous system development. Annu Rev Neurosci 28: 191-222.

Gutierrez H, O'Keeffe GW, Gavalda N, Gallagher D, Davies AM (2008) Nuclear factor kappa B signaling either stimulates or inhibits neurite growth depending on the phosphorylation status of p65/RelA. J Neurosci 28: 8246-8256.

Gutierrez H, Hale VA, Dolcet X, Davies A (2005) NF-kappaB signalling regulates the growth of neural processes in the developing PNS and CNS. Development 132: 1713-1726.

Davies AM (2009) Extracellular signals regulating sympathetic neuron survival and target innervation during development. Auton Neurosci 151: 39-45.

Meyer-Lindenberg A, Miletich RS, Kohn PD, Esposito G, Carson RE, et al. (2002) Reduced prefrontal activity predicts exaggerated striatal dopaminergic function in schizophrenia. Nat Neurosci 5: 267-271.

Robinson TE, Berridge KC (1993) The neural basis of drug craving: an incentive-sensitization theory of addiction. Brain Res Brain Res Rev 18: 247-291.

Tzschentke TM, Schmidt WJ (2000) Functional relationship among medial prefrontal cortex, nucleus accumbens, and ventral tegmental area in locomotion and reward. Crit Rev Neurobiol 14: 131-142.




How to Cite

Hegarty SV, Sullivan AM, O’Keeffe GW. Protocol for evaluation of neurotrophic strategies in Parkinson. J Biol Methods [Internet]. 2016Jul.25 [cited 2021May8];3(3):e50. Available from: