Faculty Bibliography

Purpose: In adult rats, intravitreal injections of the Rho-GTPase inhibitor C3 transferase (BA-210), or a cocktail of recombinant ciliary neurotrophic factor (CNTF) and a cyclic AMP analogue (CPTcAMP), increase retinal ganglion cell (RGC) survival and axonal regeneration. Here we examined whether these treatments also affect the dendritic architecture of regrowing RGCs. Methods: In Fischer F344 rats, one optic nerve was cut and an autologous peripheral nerve graft was sutured onto it. Rats then received intravitreal injections (4 mul) of saline, BA-210 or CNTF + CPTcAMP four and eleven days after the PN graft surgery. After 5 weeks, regenerating RGCs were retrogradely labelled with fluorogold (FG) and in living wholemounts the dendritic trees of FG labelled RGCs were visualised by intracellular injection of 2% Lucifer Yellow. Results: Injection of BA-210 or CNTF + CPTcAMP resulted in significantly more regenerating RGCs with abnormal dendritic morphologies, including abnormally long looping processes. Compared to saline-injected regenerating controls, RGCs in BA-210 injected eyes had significantly smaller dendritic field areas and sparser dendrites, while in CNTF + CPTcAMP injected eyes there was increased branching of more distal dendrites. Conclusions: While both intraocular treatments enhance RGC axonal regrowth, they also induce significant changes in RGC dendritic morphology. It remains to be determined if such changes alter the function of the regenerating neuronal population.

Accumulation of beta amyloid (Abeta) in the brain is a primary feature of Alzheimer's disease (AD) but the exact molecular mechanisms by which Abeta exerts its toxic actions are not yet entirely clear. We documented pathological changes 3 and 6 months after localised injection of recombinant, bi-cistronic adeno-associated viral vectors (rAAV2) expressing human Abeta40-GFP, Abeta42-GFP, C100-GFP or C100(V717F)-GFP into the hippocampus and cerebellum of 8 week old male mice. Injection of all rAAV2 vectors resulted in wide-spread transduction within the hippocampus and cerebellum, as shown by expression of transgene mRNA and GFP protein. Despite the lack of accumulation of Abeta protein after injection with AAV vectors, injection of rAAV2-Abeta42-GFP and rAAV2- C100(V717F)-GFP into the hippocampus resulted in significantly increased microgliosis and altered permeability of the blood brain barrier, the latter revealed by high levels of immunoglobulin G (IgG) around the injection site and the presence of IgG positive cells. In comparison, injection of rAAV2-Abeta40-GFP and rAAV2-C100-GFP into the hippocampus resulted in substantially less neuropathology. Injection of rAAV2 vectors into the cerebellum resulted in similar types of pathological changes, but to a lesser degree. The use of viral vectors to express different types of Abeta and C100 is a powerful technique with which to examine the direct in vivo consequences of Abeta expression in different regions of the mature nervous system and will allow experimentation and analysis of pathological AD-like changes in a broader range of species other than mouse.

Age-related depletion of estrogens and androgens is associated with an increase in Alzheimer's disease (AD) brain pathology and diminished cognitive function. Here we investigated AD-associated molecular and cellular changes in brains of aged hypogonadal (hpg) male and female mice. hpg Mice have a spontaneous, inactivating genetic mutation in the GnRH gene resulting in life-long deficiency of gonadotropins and gonadal sex hormones. Western blot analysis revealed low levels of amyloid precursor protein and high levels of presenilin 1, amyloid precursor protein C-terminal fragment, and beta-amyloid 42 in brains of aged male, but not female, hpg mice. Changes were confined to the hippocampus and were not evident in the cerebellum or other brain tissues. Male hpg mice tended to have lower levels of IL-1beta protein than male littermate controls. Immunohistochemical staining of the basal forebrain revealed that male hpg mice had lower choline acetyltransferase levels per neuron compared with controls. These AD-like changes specific to male hpg mice supports a link between androgen depletion and the development of AD pathology.

Recombinant adeno-associated viral (rAAV) vectors can be used to introduce neurotrophic genes into injured CNS neurons, promoting survival and axonal regeneration. Gene therapy holds much promise for the treatment of neurotrauma and neurodegenerative diseases; however, neurotrophic factors are known to alter dendritic architecture, and thus we set out to determine whether such transgenes also change the morphology of transduced neurons. We compared changes in dendritic morphology of regenerating adult rat retinal ganglion cells (RGCs) after long-term transduction with rAAV2 encoding: (i) green fluorescent protein (GFP), or (ii) bi-cistronic vectors encoding GFP and ciliary neurotrophic factor (CNTF), brain-derived neurotrophic factor (BDNF) or growth-associated protein-43 (GAP43). To enhance regeneration, rats received an autologous peripheral nerve graft onto the cut optic nerve of each rAAV2 injected eye. After 5-8 months, RGCs with regenerated axons were retrogradely labeled with fluorogold (FG). Live retinal wholemounts were prepared and GFP positive (transduced) or GFP negative (non-transduced) RGCs injected iontophoretically with 2% lucifer yellow. Dendritic morphology was analyzed using Neurolucida software. Significant changes in dendritic architecture were found, in both transduced and non-transduced populations. Multivariate analysis revealed that transgenic BDNF increased dendritic field area whereas GAP43 increased dendritic complexity. CNTF decreased complexity but only in a subset of RGCs. Sholl analysis showed changes in dendritic branching in rAAV2-BDNF-GFP and rAAV2-CNTF-GFP groups and the proportion of FG positive RGCs with aberrant morphology tripled in these groups compared to controls. RGCs in all transgene groups displayed abnormal stratification. Thus in addition to promoting cell survival and axonal regeneration, vector-mediated expression of neurotrophic factors has measurable, gene-specific effects on the morphology of injured adult neurons. Such changes will likely alter the functional properties of neurons and may need to be considered when designing vector-based protocols for the treatment of neurotrauma and neurodegeneration.

PURPOSE OF REVIEW: To discuss the relationship between androgens, cognition and Alzheimer's disease. RECENT FINDINGS: It has been found that low circulating levels of androgens are a risk factor for Alzheimer's disease. Decreased circulating androgens are also associated with declining cognitive performance, particularly in memory-related tasks. Conversely, androgen supplementation to hypogonadal men results in improved memory performance. It has therefore been hypothesized that androgen supplementation may be beneficial in Alzheimer's disease. In recent studies, animal models have been used to elucidate the molecular mechanism behind this relationship between androgens and Alzheimer's disease. These studies have shown that androgen depletion results in increased levels of beta amyloid and hyperphosphorylated tau, changes which are thought to be associated with subsequent neuronal death. SUMMARY: Androgen depletion results in molecular changes associated with Alzheimer's disease. Further human trials are needed to determine whether androgen modulating therapy for Alzheimer's disease has clinical significance.

We here describe various approaches using GFP that are being used in the morphological and functional analysis of specific cell types in the normal and injured central nervous system. Incorporation of GFP into viral vectors allows phenotypic characterization of transduced cells and can be used to label their axons and terminal projections. Characterization of transduced cell morphology can be enhanced by intracellular injection of living GFP-labeled cells with appropriate fluorescent dyes. Ex vivo labeling of precursor or glial cells using viral vectors that encode GFP permits long-term identification of these cells after transplantation into the brain or spinal cord. In utero electroporation methods result in expression of gene products in developing animals, allowing both functional and morphological studies to be carried out. GFPCre has been developed as a marker gene for viral vector-mediated expression of the bacterial recombinase Cre in the brain of adult mice with "floxed" transgenes. GFPCre-mediated induction of transgene expression can be monitored by GFP expression in defined populations of neurons in the adult brain. Finally, GFP can be used to tag proteins, permitting dynamic visualization of the protein of interest in living cells.