Elsevier

Experimental Neurology

Volume 215, Issue 1, January 2009, Pages 153-159
Experimental Neurology

Long-term reversal of cholinergic neuronal decline in aged non-human primates by lentiviral NGF gene delivery

https://doi.org/10.1016/j.expneurol.2008.10.004Get rights and content

Abstract

Spontaneous atrophy of basal forebrain cholinergic neurons occurs with aging in the non-human primate brain. Short-term reversal of this atrophy has been reported following ex vivo nerve growth factor (NGF) gene delivery, but long-term effects of in vivo NGF gene delivery in the aged primate brain have not to date been examined. We tested the hypothesis that long-term lentiviral NGF intraparenchymal gene delivery would reverse age-related cholinergic decline, without induction of adverse effects previously observed following sustained intracerebroventricular growth factor protein exposure. Three aged rhesus monkeys underwent intraparenchymal lentiviral NGF gene delivery to the cholinergic basal forebrain. 1 year later, cholinergic neuronal numbers were quantified stereologically and compared to findings in four controls, non-treated aged monkeys and four young adult monkeys. Safety was assessed on several variables related to growth factor exposure. We now report that lentiviral gene delivery of NGF to the aged primate basal forebrain sustains gene expression for at least 1 year, and significantly restores cholinergic neuronal markers to levels of young monkeys. Aging resulted in a significant 17% reduction (p < 0.05) in the number of neurons labeled for the cholinergic marker p75 among basal forebrain neurons. Lentiviral NGF gene delivery induced significant (p < 0.05) and nearly complete recovery of p75-labeled neuronal numbers in aged subjects to levels observed in young monkeys. Similarly, the size of cholinergic neurons in aged monkeys was significantly reduced by 16% compared to young subjects (p < 0.05), and lentiviral NGF delivery to aged subjects induced complete recovery of neuronal size. Intraparenchymal NGF gene delivery over a one-year period did not result in systemic leakage of NGF, activation of inflammatory markers in the brain, pain, weight loss, Schwann cell migration, or formation of anti-NGF antibodies. These findings indicate that extended trophic support to neurons in the non-human primate brain reverses age-related neuronal atrophy. These findings also support the safety and feasibility of lentiviral NGF gene transfer for potential testing in human clinical trials to protect degenerating cholinergic neurons in Alzheimer's disease.

Introduction

A complete description of mechanisms underlying cognitive decline with aging in the non-human primate brain remains elusive. Age-related decrements in white matter volume, loss of gray matter in some structures, and reductions in dendritic arborization/spines have been detected in non-human primates (Cupp and Uemura, 1980, Peters et al., 1998, Smith et al., 2004, Smith et al., 1999, Wisco et al., 2008). However, cell death as a function of aging either does not occur or is mild in extent in most brain regions examined to date, including the entorhinal cortex and hippocampus (Gazzaley et al., 1997, Merrill et al., 2000, Peters et al., 1996). These findings suggest that functional decline associated with aging across species does not primarily result from cell loss, but from other mechanisms including age-related decrements in gene expression and resultant cell signaling and biochemistry. Indeed, decrements in ERK/map kinase activation, arc expression and functional neuronal transport include a set of atrophic cellular events that have been associated with functional decline in the nervous system with aging (De Lacalle et al., 1996, Niewiadomska et al., 2005, Small et al., 2004, Williams et al., 2006).

The general preservation of cell number in the nervous system as a function of aging raises the possibility that targeted interventions could ameliorate age-related atrophic changes. In rodent studies, aging is clearly associated with reductions in functional markers of basal forebrain cholinergic neurons (Chen and Gage, 1995, Fischer et al., 1987). These neurons project to extensive regions of the hippocampus and neocortex, modulate cortical neuronal excitability, and are required for some forms of cortical plasticity and learning (Mesulam et al., 1983a, Mesulam et al., 1983b). Notably, age-related atrophy of basal forebrain cholinergic neurons can be reversed by nerve growth factor (NGF) delivery to the brain, resulting in reversal of age-related cognitive decline (Chen and Gage, 1995, Fischer et al., 1987, Markowska et al., 1994). Age-related atrophy of basal forebrain cholinergic neurons has also been reported in the rhesus monkey brain (Smith et al., 1999, Stroessner-Johnson et al., 1992), and short-term (1–3 months) reversal of this decline was previously reported using techniques of either NGF protein infusion into the ventricular system (Koliatsos et al., 1991, Tuszynski et al., 1991, Tuszynski et al., 1990) or ex vivo gene delivery in which cells genetically modified to secrete NGF were grafted into the brain (Conner et al., 2001, Emerich et al., 1994, Smith et al., 1999, Tuszynski et al., 1996). However, whether age-related degenerative events in the cholinergic systems can be reversed by extended growth factor administration has not to date been tested. Addressing long-term consequences of growth factor gene delivery in the nervous system is of particular importance, as several clinical trials using this approach in human neurodegenerative disorders are in progress, including adeno-associated virus (AAV) NGF gene delivery in Alzheimer's disease (Arvanitakis et al., 2007, Tuszynski et al., 2005) and neurturin gene delivery in Parkinson's disease (Marks et al., 2008).

The present study tested the hypothesis that lentiviral delivery of NGF to the basal forebrain of the aged rhesus monkey would reverse age-related cholinergic degenerative changes over extended time periods. We further hypothesized that previously reported adverse effects of growth factor administration, including pain, weight loss and Schwann cell hyperplasia (Emmett et al., 1996, Eriksdotter Jonhagen et al., 1998, Williams, 1991, Winkler et al., 1997), would be avoided by restricting gene delivery to cholinergic basal forebrain regions.

While several clinical trials of adeno-associated virus (AAV) vector delivery to the brain are in progress currently, lentiviral vector delivery to the CNS has been less thoroughly explored. Yet lentiviral vectors have potential benefits in a therapeutic context. First, lentiviral vectors more rapidly induce transgene expression than single-stranded AAV vectors in vitro and in vivo (Blesch and Tuszynski, 2007, Blesch et al., 2000, Naldini et al., 1996, Zufferey et al., 1998). Second, some AAV vector serotypes may undergo remote transport from their sites of injection into the nervous system (Hollis et al., 2008, Kaspar et al., 2002, Mandel and Burger, 2004), whereas lentiviral vectors are not known to exhibit remote transport properties; this could be an advantage in limiting therapeutic gene expression to a defined brain region. Third, relatively little is known regarding the long-term safety of viral vectors in the human CNS, and it is prudent to continue to explore the efficacy and safety profile of several viral gene delivery candidates, including lentiviral vectors.

Section snippets

Experimental subjects

11 monkeys served as experimental subjects. Three groups were examined: 4 aged un-operated control monkeys (mean age 23.9 ± 1.8 years; all males), 3 aged monkeys that received intraparenchymal injections of lentiviral vectors expressing human NGF (mean age 22.8 ± 0.7 years; 1 male and 2 females), and 4 adult, non-aged monkeys. The latter non-aged group consisted of 2 young adult monkeys that received injections of lentiviral vectors expressing the control reporter gene Green Fluorescent Protein,

Persistence and pattern of in vivo gene expression

Lentiviral gene expression was readily detectable in the primate brain for at least 1 year after therapeutic gene delivery, indicated by GFP expression in young subjects and NGF immunolabeling in aged monkeys (Fig. 1). NGF lentiviral injections were targeted adjacent to the nucleus basalis, and resulted in enhanced NGF labeling within both neuronal perikarya and in the extracellular matrix compared to young control and aged subjects (Figs. 1A–F). A mean parenchymal volume of 16.0 ± 2.1 mm3 was

Discussion

Findings of this study indicate that lentiviral-mediated in vivo NGF gene delivery sustains growth factor production for at least 1 year in the aged non-human primate brain, and that this sustained delivery reverses age-related neuronal atrophy. Trophic effects on neurons are reflected in both the number of neurons expressing the specific basal forebrain cholinergic marker, p75 (Kordower et al., 1994), and the size of cholinergic neurons. Sustained lentiviral NGF delivery was safe, without

Conflict of interest statement

MT is a consultant to Ceregene, Inc.; AR and MG are employees of Ceregene, Inc.

Acknowledgments

We thank R. Torres, H. Zhang, and M. Culbertson for technical support. Supported by the NIH (AG10435), the Veterans Administration, the Alzheimer's Association (TTL-03-5814), the State of California Department of Health Services (04-35530), and the Shiley Family Foundation. MHT is a scientific founder of Ceregene, Inc.

References (49)

  • WiscoJ.J. et al.

    An MRI study of age-related white and gray matter volume changes in the rhesus monkey

    Neurobiol. Aging

    (2008)
  • ArvanitakisZ. et al.

    Interim data from a phase 1 clinical trial of AAV-NGF (CERE-110) gene delivery in Alzheimers disease

    Abstr. Amer. Acad. Neurol.

    (2007)
  • BleschA. et al.

    Transient growth factor delivery sustains regenerated axons after spinal cord injury

    J. Neurosci.

    (2007)
  • BleschA. et al.

    Neurite outgrowth can be modulated in vitro using a tetracycline-repressible gene therapy vector expressing human nerve growth factor

    J. Neurosci. Res.

    (2000)
  • BleschA. et al.

    Leukemia inhibitory factor augments neurotrophin expression and corticospinal axon growth after adult CNS injury

    J. Neurosci.

    (1999)
  • ChenK.S. et al.

    Somatic gene transfer of NGF to the aged brain: behavioral and morphological amelioration

    J. Neurosci.

    (1995)
  • ConnerJ.M. et al.

    Non-tropic actions of neurotrophins: subcortical NGF gene delivery reverses age-related degeneration of primate cortical cholinergic innervation

    Proc. Nat. Acad. Sci.

    (2001)
  • ConnerJ.M. et al.

    Distribution of nerve growth factor-like immunoreactive neurons in the adult rat brain following colchicine treatment

    J. Comp. Neurol.

    (1992)
  • DullT. et al.

    A third-generation lentivirus vector with a conditional packaging system

    J. Virol.

    (1998)
  • EmerichD.W. et al.

    Implants of polymer-encapsulated human NGF-secreting cells in the nonhuman primate: Rescue and sprouting of degenerating cholinergic basal forebrain neurons

    J. Comp. Neurol.

    (1994)
  • Eriksdotter JonhagenM. et al.

    Intracerebroventricular infusion of nerve growth factor in three patients with Alzheimer's disease

    Dement. Geriatr. Cogn. Disord.

    (1998)
  • FischerW. et al.

    Amelioration of cholinergic neuron atrophy and spatial memory impairment in aged rats by nerve growth factor

    Nature

    (1987)
  • FollenziA. et al.

    Gene transfer by lentiviral vectors is limited by nuclear translocation and rescued by HIV-1 pol sequences

    Nat. Genet.

    (2000)
  • HollisE.R. et al.

    Efficient retrograde neuronal transduction utilizing self-complementary AAV1

    Mol. Ther.

    (2008)
  • Cited by (70)

    • Aging with alcohol-related brain damage: Critical brain circuits associated with cognitive dysfunction

      2019, International Review of Neurobiology
      Citation Excerpt :

      Aged rats that are cognitively-impaired also show a significant loss of cholinergic neurons in the MS/DB compared to young controls, but show no change in the pontine reticular cholinergic neurons, implicating the basal forebrain as a key structure in age-associated memory impairment (Baskerville, Kent, Nicolle, Gallagher, & McKinney, 2006). Overall, aging primate brains show a 17% reduction in basal forebrain neurons and a 16% decrease in the size of ChAT + neurons, however, administration of NGF almost completely recovers the loss and shrinkage of the neurons (Nagahara et al., 2009). Retrograde labeling has shown a decrease in MS/DB projection neurons in aged rat brains (de Lacalle, Cooper, Svendsen, Dunnett, & Sofroniew, 1996).

    • Reduced NGF in Gastric Endothelial Cells Is One of the Main Causes of Impaired Angiogenesis in Aging Gastric Mucosa

      2018, Cellular and Molecular Gastroenterology and Hepatology
      Citation Excerpt :

      NGF immunostaining in 10 randomly selected mucosal fields per each specimen was quantified by measuring the staining signal intensity using MetaMorph 7.0. Aging GECs were transduced with LV-NGF and LV-GFP (control; to determine transfection efficiency), provided by Dr M. Tuszynski (Director, Center for Neural Repair, University of California, San Diego, CA), using methods described previously.29 GECs were plated in 12-well plates and respective wells were transduced with serial dilutions of LV-GFP and LV-NGF in medium containing polybrene linker.

    View all citing articles on Scopus
    View full text