Joseph P. Hammang

Glial cell line-derived neurotrophic factor (GDNF), a new neurotrophic factor for motoneurones

GLIAL cell line-derived neurotrophic factor (GDNF) has been postulated to be a specific dopaminergic neurotrophic factor since it selectively enhances the survival of dopaminergic neurones in vitro. We report here that GDNF can also act as a neurotrophic factor for motoneurones. GDNF released by GDNF-transfected BHK cells increases the activity of choline acetyltransferase (ChAT) in cultures from embryonic rat ventral mesencephalon containing cholinergic neurones from cranial motor nuclei, and in cultured spinal motoneurones. Furthermore, local application of polymer-encapsulated BHK cells releasing GDNF to transected facial nerve in newborn rats diminishes the death of motoneurones normally occurring after axotomy in the neonatal period. The present results indicate that GDNF may have a therapeutic potential in human motoneurone diseases such as amyotrophic lateral sclerosis.

Incorporation and glial differentiation of mouse EGF-responsive neural progenitor cells after transplantation into the embryonic rat brain.

In vitro, epidermal growth factor (EGF)-responsive neural progenitor cells exhibit multipotent properties and can differentiate into both neurons and glia. Using an in utero xenotransplantation approach we examined the developmental potential of EGF-responsive cells derived from E14 mouse ganglionic eminences, cortical primordium, and ventral mesencephalon, after injection into the E15 rat forebrain ventricle. Cell cultures were established from control mice or from mice carrying the lacZ transgene under control of the promoters for nestin, glial fibrillary acidic protein (GFAP), or myelin basic protein (MBP). The grafted cells, visualized with mouse-specific markers or staining for the reporter gene product, displayed widespread incorporation into distinct forebrain and midbrain structures and differentiated predominantly into glial cells. The patterns of incorporation of cells from all three regions were very similar without preference for the homotopic brain areas. These results suggest that EGF-responsive progenitor cells can respond to host derived environmental cues, differentiate into cells with glial-like features, and become integrated in the developing recipient brain.

Grafts of EGF-responsive neural stem cells derived from GFAP-hNGF transgenic mice: Trophic and tropic effects in a rodent model of Huntington's disease

The present study examined whether implants of epidermal growth factor (EGF)‐responsive stems cells derived from transgenic mice in which the glial fibrillary acid protein (GFAP) promoter directs the expression of human nerve growth factor (hNGF) could prevent the degeneration of striatal neurons in a rodent model of Huntingtons disease (HD). Rats received intrastriatal transplants of GFAP‐hNGF stem cells or control stem cells followed 9 days later by an intrastriatal injection of quinolinic acid (QA). Nissl stains revealed large striatal lesions in rats receiving control grafts, which, on average, encompassed 12.78 mm3. The size of the lesion was significantly reduced (1.92 mm3) in rats receiving lesions and GFAP‐hNGF transplants. Rats receiving QA lesions and GFAP‐hNGF‐secreting grafts stem cell grafts displayed a sparing of striatal neurons immunoreactive (ir) for glutamic acid decarboxylase, choline acetyltransferase, and neurons histochemically positive for nicotinamide adenosine diphosphate. Intrastriatal GFAP‐hNGF‐secreting implants also induced a robust sprouting of cholinergic fibers from subjacent basal forebrain neurons. The lesioned striatum in control‐grafted animals displayed numerous p75 neurotrophin‐ir (p75NTR) astrocytes, which enveloped host vasculature. In rats receiving GFAP‐hNGF‐secreting stem cell grafts, the astroglial staining pattern was absent. By using a mouse‐specific probe, stem cells were identified in all animals. These data indicate that cellular delivery of hNGF by genetic modification of stem cells can prevent the degeneration of vulnerable striatal neural populations, including those destined to die in a rodent model of HD, and supports the emerging concept that this technology may be a valuable therapeutic strategy for patients suffering from this disease. J. Comp. Neurol. 387:96–113, 1997.

Implantation of polymer-encapsulated human nerve growth factor-secreting fibroblasts attenuates the behavioral and neuropathological consequences of quinolinic acid injections into rodent striatum

Delivery of neurotrophic molecules to the central nervous system has gained considerable attention as a potential strategy for the treatment of neurological disorders. In the present study, a DHFR-based expression vector containing the human nerve growth factor gene (hNGF) was transfected into a baby hamster fibroblast cell line (BHK). Using an immunoisolatory polymeric device, encapsulated BHK-control cells and those secreting hNGF (BHK-hNGF) were transplanted unilaterally into rat lateral ventricles. Three days later, the same animals received unilateral injections of quinolinic acid (QA, 225 nmol) or the saline vehicle into the ipsilateral striatum. Approximately 2 weeks following surgery, animals were tested for apomorphine-induced rotation behavior. Animals which received BHK-hNGF cells rotated significantly less than those animals receiving BHK-control cells or QA alone. Histological analysis 29-30 days following capsule implantation demonstrated that BHK-hNGF cells attenuated the extent of host neural damage produced by QA as assessed by a sparing of ChAT- and NADPH-d-positive neurons. Moreover, a lessened GFAP reaction was apparent within the striatum of animals receiving BHK-hNGF cells. As measured by ELISA, hNGF was released by the encapsulated BHK-hNGF cells prior to implantation and following removal. Morphology of retrieved capsules revealed numerous viable and mitotically active BHK cells. These results suggest that implantation of polymer-encapsulated hNGF-releasing cells can be used to protect neurons from excitotoxin damage.

Genetic modification of neural stem cells

Neural stem cells (NSCs) are the main vehicle for genetic and molecular therapies in the central nervous system (CNS). The sustainability of NSCs has been ensured through genetic manipulation both in vitro and in vivo. NSC lines have also been immortalized and controlled for cell growth in similar fashion. Their potential to differentiate and their genetic plasticity make them the modality of choice for cellular transplantation. After transplantation, NSCs also exhibit inherent long-distance migratory capabilities and a remarkable capacity to integrate into brain structures. This makes NSCs the ideal candidate for delivery and expression of therapeutic genes. Mouse models of CNS diseases have already demonstrated the efficacy of such NSC-mediated treatment, and further investigations are underway to bridge the gap into true clinical application. Finally, the imaging possibilities with NSC transplants are endless, and they will be a pivotal component to safe and effective human transplantation. This paper provides an overview on NSCs and the various methods in which they have been genetically manipulated for biological investigation.

Myelin-deficient rat: analysis of myelin proteins.

Abstract: Myelin basic protein (BP), proteolipid protein (PLP), myelin‐associated glycoprotein (MAG), and 2′,3′‐cyclic nucleotide 3′‐phosphodiesterase (CNPase) activity were quantitated in the brains and spinal cords of normal and myelin‐deficient (md) rats at 8, 12, 18, and 25 days of age. The levels of BP, MAG, and CNP in 25‐day‐old md brain were 1.1, 1.8, and 11% of those in controls, respectively. In spinal cord, the levels were higher, at 9, 15, and 12% of control values, respectively. Although BP content in the mutant rats was a lower percentage of the control level than MAG and CNPase contents at all ages, the absolute level of BP increased steadily between 8 and 25 days of age in both brain and spinal cord, whereas there was little change in the amounts of MAG and CNPase during this period. Immunoblotting analysis did not reveal an increased apparent Mr for MAG, as has been observed in quaking and trembler mice. There was little difference in the relative distributions of the 14K, 17K, 18.5K, and 21.5K forms of BP between control and md rat spinal cord homogenates at the ages examined. PLP content was reduced more than that of the other proteins in the md mutants, because it could not be detected by a technique capable of detecting 0.2% of the control brain level and 0.1% of control spinal cord level. This suggests that the expression of PLP may be preferentially affected in the md mutation.

Oncogene expression in retinal horizontal cells of transgenic mice results in a cascade of neurodegeneration

The phenylethanolamine N-methyltransferase promoter directs the expression of the SV40 T antigen to subsets of amacrine and horizontal neurons of the retina in a line of transgenic mice. T antigen expression begins in these cells during the first postnatal week. The horizontal cells appear to develop normally for another week but then begin to die. Subsequently, most of the horizontal cells disappear from the central and mid retina, resulting in loss of the outer plexiform layer and absence of ribbon synapses between the photoreceptors and bipolar cells. Neuronal transformation occurs only in the peripheral retina. These experiments indicate that horizontal neurons are heterogeneous with respect to susceptibility to transformation and that T antigen expression in a subset of horizontal neurons can be a direct cause of neuronal cell death. Furthermore, critical interdependencies exist between horizontal neurons after retinal neurogenesis is complete.

Generation and transplantation of EGF-responsive neural stem cells derived from GFAP-hNGF transgenic mice.

EGF-responsive neural stem cells isolated from murine striatum have the capacity to differentiate into both neurons and glia in vitro. Genetic modification of these cells is hindered by a number of problems such as gene stability and transfection efficiency. To circumvent these problems we generated transgenic mice in which the human GFAP promoter directs the expression of human NGF. Neural stem cells isolated from the forebrain of these transgenic animals proliferate and form clusters, which appear identical to stem cells generated from control animals. Upon differentiation in vitro, the transgenic stem cell-derived astrocytes express and secrete bioactive hNGF. Undifferentiated GFAP-hNGF or control stem cells were transplanted into the striatum of adult rats. One and 3 weeks after transplantation, hNGF was detected immunocytochemically in an halo around the transplant sites. In GFAP-hNGF-grafted animals, intrinsic striatal neurons proximal to the graft appear to have taken up hNGF secreted by the grafted cells. Ipsilateral to implants of GFAP-hNGF-secreting cells, choline acetyltransferase-immunoreactive neurons within the striatum were hypertrophied relative to the contralateral side or control-grafted animals. Further, GFAP-hNGF-grafted rats displayed a robust sprouting of p75 neurotrophin receptor-positive fibers emanating from the underlying basal forebrain. These studies indicate that EGF-responsive stem cells which secrete hNGF under the direction of the GFAP promoter display in vitro and in vivo properties similar to that seen following other methods of NGF delivery and this source of cells may provide an excellent avenue for delivery of neurotrophins such as NGF to the central nervous system.

Myelin mosaicism in female heterozygotes of the canine shaking pup and myelin-deficient rat mutants

Female heterozygotes of the shaking pup and myelin-deficient rat sex-linked recessive traits, show myelin mosaicism of the optic nerve and spinal cord. This is most marked in the optic nerve especially in the rat where mosaic patches persist with aging. In both the rat and dog, abnormal oligodendrocytes with distended rough endoplasmic reticulum are found in the abnormal patches and are a marker of the trait. Female heterozygote dogs can develop a marked tremor which disappears with age.

Transplantation of Epidermal Growth Factor-Responsive Neural Stem Cell Progeny into the Murine Central Nervous System

Publisher Summary This chapter describes transplantation of epidermal growth factor (EGF)-responsive neural stem cell progeny into the murine central nervous system (CNS). Neurodegenerative disorders such as Alzheimers, Parkinsons, and Huntingtons disease, and also demyelinating disorders such as multiple sclerosis (MS) are of serious concern in the society. Because of the wide array of CNS disorders, possible therapeutic approaches are also diverse and include cell replacement via transplantation, neurotrophic factor delivery from implants of polymer-encapsulated or unencapsulated genetically modified cell lines, etc. This chapter describes aspects of a novel EGF-responsive stem cell culture system. The EGF-responsive cells can be isolated from embryonic and adult rat and mouse brain, and similar cells have been isolated from fetal human brain. Undifferentiated stem cell progeny are capable of forming oligodendrocytes when transplanted in vivo . The chapter also describes the development of genetically tagged, EGF-responsive stem cells derived from transgenic mice. These mice carry chimeric genes composed of mammalian cell-specific promoter elements that direct the expression of a reporter gene to either astrocytes or oligodendrocytes.