Grafting genetically modified cells to the brain: conceptual and technical issues.
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Animals↗
Biomedical subjects
M H Tuszynski
Publications and source records attributed to M H Tuszynski.
Gene therapy in the CNS: intracerebral grafting of genetically modified cells.
Grafting cells to the CNS has been suggested and applied as a potential approach to CNS therapy through the selective replacement of cells lost as a result of disease or damage. Independently, studies aimed at direct genetic therapy in model systems have recently begun to suggest conceptually new approaches to the treatment of several kinds of human genetic disease, especially those caused by single gene enzyme deficiencies. We suggest that a combination of these two approaches, namely the graftment into the CNS of genetically modified cells, may provide a new approach toward the restoration of some functions in the damaged or diseased CNS. We present evidence for the feasibility of this approach, including a description of some current techniques for mammalian cell gene transfer and CNS grafting, and several possible approaches to clinical applications. Specifically, we report that fibroblasts, genetically modified to secrete NGF by infection with a retroviral vector and implanted into the brains of rats with a surgical lesion of the fimbria-fornix, prevented the degeneration of cholinergic neurons that would die without treatment.
Animals↗
Nerve growth factor infusion in the primate brain reduces lesion-induced cholinergic neuronal degeneration.
NGF is a protein that promotes survival, differentiation, and process extension of selected neuronal populations during development and, in some cases, in the mature organism. Previous lesion and aging studies in the rat have shown that intracerebroventricular NGF infusions can prevent degenerative changes in basal forebrain cholinergic neurons. We sought to determine whether salutory effects of NGF occur in the primate brain. Cholinergic fibers of the septohippocampal projection in the primate were surgically transected, followed by infusion of either a vehicle or an NGF solution into the ventricular system for a 4-week period. Quantification of cholinergic neurons in the medial septal nucleus at the end of the infusion period demonstrated that only 45 +/- 5% of cholinergic neurons could be identified after fornix lesions in vehicle-infused animals, whereas 80 +/- 6% of neurons were visible in NGF-treated animals. Thus, NGF substantially reduced lesion-induced cholinergic neuronal degeneration in the adult primate brain. This finding may be relevant to the hypothesis that NGF has potential use as a cholinergic "neurotrophic-factor therapy," given that loss of basal forebrain cholinergic neurons is common in Alzheimer's disease.
Acetylcholinesterase↗
Survival, growth and function of damaged cholinergic neurons.
Recent progress has been made in defining the requirements for survival, growth and function of damaged cholinergic neurons of the central nervous system. In particular, the responsiveness of cholinergic neurons to nerve growth factor (NGF) in the regulation of development, cell survival, axon elongation, and response to injury has led to the formulation of the Neurotrophic Hypothesis, a unifying hypothesis of neuronal responsiveness to growth-promoting substances. NGF-mediated effects on cholinergic neurons in culture as well as in the septum, basal nucleus, striatum, and hippocampus, and the ability of NGF to prevent lesion-induced cell death and to ameliorate the effects of aging, provide the foundation for this work. A potential role for glia and microglia in mediating the effects of NGF is proposed.
Acetylcholine↗
Risk factors and clinical manifestations of pathologically verified lacunar infarctions.
Review of 2,859 autopsy reports disclosed lacunar infarctions in 169 patients (6%). Review of the charts of 167 of these patients revealed hypertension in 64%, diabetes in 34%, smoking in 46%, and no known risk factor for cerebrovascular disease in 18%. As many as 81% of the patients with lacunes were asymptomatic. Symptomatic lacunes presented most commonly as pure motor hemiparesis (31%), aphasia plus right hemiparesis (20%), or sensorimotor dysfunction (11%); none presented as pure sensory stroke. These results suggest that the spectrum of lacunar infarction is more heterogeneous than previously thought. Most lacunes are asymptomatic, and the majority of symptomatic patients do not present with "classical" lacunar syndromes.
Aged↗
Ischemic thalamic aphasia with pathologic confirmation.
Review of clinical and autopsy records at the New York Hospital revealed a patient with aphasia and right hemiparesis due to a pathologically confirmed 1- X 2-cm ischemic thalamic infarct. This is the only pathologically verified case of aphasia resulting from an infarct limited to the thalamus, and provides further evidence that lesions confined to subcortical structures are capable of affecting language function.
Aged↗
Maintaining the neuronal phenotype after injury in the adult CNS. Neurotrophic factors, axonal growth substrates, and gene therapy.
Multiple genetic and epigenetic events determine neuronal phenotype during nervous system development. After the mature mammalian neuronal phenotype has been determined it is usually static for the remainder of life, unless an injury or degenerative event occurs. Injured neurons may suffer one of three potential fates: death, persistent atrophy, or recovery. The ability of an injured adult neuron to recover from injury in adulthood may be determined by events that also influence neuronal phenotype during development, including expression of growth-related genes and responsiveness to survival and growth signals in the environment. The latter signals include neurotrophic factors and substrate molecules that promote neurite growth. Several adult CNS regions exhibit neurotrophic-factor responsiveness, including the basal forebrain, entorhinal cortex, hippocampus, thalamus, brainstem, and spinal cord. The specificity of neurotrophic-factor responsiveness in these regions parallels patterns observed during development. In addition, neurons of several CNS regions extend neurites after injury when presented with growth-promoting substrates. When both neurotrophic factors and growth-promoting substrates are provided to adult rats that have undergone bilateral fimbria-fornix lesions, then partial morphological and behavioral recovery can be induced. Gene therapy is one useful tool for providing these substances. Thus, the mature CNS remains robustly responsive to signals that shape nervous system development, and is highly plastic when stimulated by appropriate cues.
Animals↗
Role of neurotrophic factors in Alzheimer's disease.
Neurotrophic factors (NTFs) could potentially play a role in Alzheimer's disease (AD) in many ways. Neuronal degeneration may result from disruption in NTF production, delivery, or interaction with the neuronal target. Even if alterations in NTF function are not responsible for neuronal degeneration, NTFs may still be therapeutically useful in ameliorating some morphological or cognitive deficits observed in AD.
Alzheimer Disease↗
Delivery of neuroactive compounds to the brain: potential utility of genetically modified cells.
Several methods for chronic delivery of compounds to the central nervous system (CNS) now exist. Peripheral drug administration is generally safest, but not always effective. If direct CNS delivery of a substance is required, then CNS implantation of drug-delivery systems or grafting of various cell types to the brain can be performed, although none of these interventions are yet of consistent, proven benefit in Alzheimer's disease and other neurodegenerative disorders. Grafting of genetically modified cells to the brain may be an alternative delivery system of some substances to the CNS.
Brain Diseases↗
Cerebral glucose metabolism and memory in aged rhesus macaques.
Positron emission tomography and the glucose metabolic tracer [18F]fluorodeoxyglucose were used to evaluate the relationship between regional cerebral metabolic rates for glucose (rCMRglc), age, and performance on a delayed response (DR) test of memory in the aged monkey. Eleven aged animals, 21-26-years old, were included in the analysis. Regional CMRglc, normalized to values for the entire brain, were determined for the dorsal prefrontal cortex, orbitofrontal cortex, hippocampus, and temporal cortex. The aged animals exhibited significant DR deficits relative to a cohort of normal young monkeys. Variability in DR performance among the aged subjects was significantly correlated with relative hippocampal rCMRglc, and chronological age was a reliable predictor of orbitofrontal rCMRglc ratios. This pattern of results suggests that DR impairments in the aged monkey may partly reflect age-related dysfunction distributed among multiple limbic system structures that participate in normal learning and memory. Overall, the findings support the use of positron emission tomography in efforts to define the relationship between cognitive performance, age, and brain physiology in nonhuman primates.
Aging↗
Intraparenchymal NGF infusions rescue degenerating cholinergic neurons.
Nerve growth factor (NGF) exerts both trophic (cell survival) and tropic (axonal growth-promoting) effects on several neuronal populations. In particular, its robust ability to prevent lesion-induced and spontaneous age-related basal forebrain cholinergic neuronal degeneration, and to promote mnemonic recovery, has suggested its potential use as a therapeutic agent in Alzheimer's disease. When infused intracerebroventricularly, however, NGF is associated with several adverse effects that make this delivery route impractical. The present study examined whether intraparenchymal infusions of NGF adjacent to cholinergic neuronal soma are an effective and well-tolerated means of providing NGF to degenerating cholinergic neurons. Cholinergic neuronal rescue together with axonal sprouting responses and local tissue damage in the brain were assessed in adult rats that underwent complete unilateral fornix transections, followed by intraparenchymal infusions of recombinant human NGF for a 2-week period. Intraparenchymal NGF infusions prevented the degeneration of 94.7+/-6.6% of basal forebrain cholinergic neurons compared to 21.7+/-2.6% in vehicle-infused animals (p < 0.0001). Cholinergic axons sprouted toward the intraparenchymal NGF source in an apparent gradient-dependent manner. Glial responses to intraparenchymal infusions were minimal, and no apparent toxic effects of the infusions were observed. Thus, when infused intraparenchymally, NGF rescues basal forebrain cholinergic neurons, alters the topography of axonal sprouting responses, and does not induce adverse affects over a 2-week infusion period. Intraparenchymal NGF delivery merits further study at longer term time points as a means of treating the cholinergic component of neuronal loss in Alzheimer's disease.
Animals↗
Grafts of fibroblasts genetically modified to secrete NGF, BDNF, NT-3, or basic FGF elicit differential responses in the adult spinal cord.
Neuronal and axonal responses to neurotrophic factors in the developing spinal cord have been relatively well characterized, but little is known about adult spinal responses to neurotrophic factors. We genetically modified primary rat fibroblasts to produce either nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), or basic fibroblast growth factor (bFGF), then grafted these neurotrophic factor-secreting cells into the central gray matter of the spinal cord in adult rats. Spinal cord lesions were not made prior to grafting. From 2 wk to 6 mo later, sensory neurites of dorsal root origin extensively penetrated NGF-, NT-3-, and bFGF-producing grafts, whereas BDNF-secreting grafts elicited no growth responses. Putative noradrenergic neurites also penetrated NGF-secreting cell grafts. Local motor and corticospinal motor axons did not penetrate any of the neurotrophic factor-secreting grafts. These results indicate that unlesioned or minimally lesioned adult spinal cord sensory and putative noradrenergic populations retain significant neurotrophic factor responsiveness, whereas motor neurites are comparatively resistant even to those neurotrophic factors to which they exhibit survival dependence during development. Grafts of genetically modified cells can be a useful tool for characterizing neurotrophic factor responsiveness in the adult spinal cord and designing strategies to promote axonal regeneration after injury.
Animals↗
Functional characterization of NGF-secreting cell grafts to the acutely injured spinal cord.
Previously we reported that grafts of cells genetically modified to produce human nerve growth factor (hNGF) promoted specific and robust sprouting of spinal sensory, motor, and noradrenergic axons. In the present study we extend these investigations to assess NGF effects on corticospinal motor axons and on functional outcomes after spinal cord injury. Fibroblasts from adult rats were transduced to express human NGF; control cells were not genetically modified. Fibroblasts were then grafted to sites of midthoracic spinal cord dorsal hemisection lesions. Three months later, recipients of NGF-secreting grafts showed deficits on conditioned locomotion over a wire mesh that did not differ in extent from control-lesioned animals. On histological examination, NGF-secreting grafts elicited specific sprouting from spinal primary sensory afferent axons, local motor axons, and putative cerulospinal axons as previously reported, but no specific responses from corticospinal axons. Axons responding to NGF robustly penetrated the grafts but did not exit the grafts to extend to normal innervation territories distal to grafts. Grafted cells continued to express NGF protein through the experimental period of the study. These findings indicate that 1) spinal cord axons show directionally sensitive growth responses to neurotrophic factors, 2) growth of axons responding to a neurotrophic factor beyond an injury site and back to their natural target regions will likely require delivery of concentration gradients of neurotrophic factors toward the target, 3) corticospinal axons do not grow toward a cellular source of NGF, and 4) functional impairments are not improved by strictly local sprouting response of nonmotor systems.
Acute Disease↗
Grafts of genetically modified Schwann cells to the spinal cord: survival, axon growth, and myelination.
Schwann cells naturally support axonal regeneration after injury in the peripheral nervous system, and have also shown a significant, albeit limited, ability to support axonal growth and remyelination after grafting to the central nervous system (CNS). It is possible that Schwann cell-induced axonal growth in the CNS could be substantially increased by genetic manipulation to secrete augmented amounts of neurotrophic factors. To test this hypothesis, cultured primary adult rat Schwann cells were genetically modified using retroviral vectors to produce and secrete high levels of human nerve growth factor (NGF). These cells were then grafted to the midthoracic spinal cords of adult rats. Findings were compared to animals that received grafts of nontransduced Schwann cells. Spinal cord lesions were not placed prior to grafting because the primary aim of this study was to examine features of grafted Schwann cell survival, growth, and effects on host axons. In vitro prior to grafting, Schwann cells secreted 1.5+/-0.1 ng human NGF/ml/10(6) cells/day. Schwann cell transplants readily survived for 2 wk to 1 yr after in vivo placement. Some NGF-transduced grafts slowly increased in size over time compared to nontransduced grafts; the latter remained stable in size. NGF-transduced transplants were densely penetrated by primary sensory nociceptive axons originating from the dorsolateral fasciculus of the spinal cord, whereas control grafts showed significantly fewer penetrating sensory axons. Over time, Schwann cell grafts also became penetrated by TH- and DBH-labeled axons of putative coerulospinal origin, unlike control cell grafts. Ultrastructurally, axons in both graft types were extensively myelinated by Schwann cells. Grafted animals showed no changes in gross locomotor function. In vivo expression of the human NGF transgene was demonstrated for periods of at least 6 m. These findings demonstrate that primary adult Schwann cells 1) can be transduced to secrete augmented levels of neurotrophic factors, 2) survive grafting to the CNS for prolonged time periods, 3) elicit robust growth of host neurotrophin-responsive axons, 4) myelinate CNS axons, and 5) express the transgene for prolonged time periods in vivo. Some grafts slowly enlarge over time, a feature that may be attributable to the propensity of Schwann cells to immortalize after multiple passages. Transduced Schwann cells merit further study as tools for promoting CNS regeneration.
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