Faculty Bibliography

The vast majority of human tissue specimens are formalin-fixed, paraffin embedded (FFPE) archival samples, making this type of tissue a potential gold mine for medical research. It is now accepted that proteomics can be done using FFPE tissue and can generate similar results as snap-frozen tissue. However, the current methodology requires a large amount of starting protein, limiting the questions that can be answered in these types of proteomics studies and making cell-type specific proteomics studies difficult. Cell-type specific proteomics has the potential to greatly enhance understanding of cell functioning in both normal and disease states. Therefore, here we describe a new method that allows localized proteomics on individual cell populations isolated from FFPE tissue sections using laser capture microdissection. To demonstrate this technique we microdissected neurons from archived tissue blocks of the temporal cortex from patients with Alzheimer's disease. Using this method we identified over 400 proteins in microdissected neurons; on average 78% that were neuronal and 50% that were associated with Alzheimer's disease. Therefore, this technique is able to provide accurate and meaningful data and has great potential for any future study that wishes to perform localized proteomics using very small amounts of archived FFPE tissue.

Background: It has been increasingly recognized that the pathogenesis of many neurodegenerative diseases is related to the accumulation of diverse proteins in aggregated/oligomeric forms. The pathological conformers can spread to different areas of the brain via a "prion-like" conversion mechanism mediated by the mobile b-sheet oligomeric structure of each particular peptide or protein. Previously we have characterized conformational monoclonal antibodies that react to both oligomers of Abeta and tau in AD, as well as to prion disease proteins. We have nowdetermined their binding specificity and capacity to be extended to synthetic oligomers of alpha-synuclein and to pathological intracellular structures present in Lewy body containing neurons of Parkinson's disease (PD) subjects. Methods: Recombinant alpha-synuclein was produced and characterized in monomeric, oligomeric and fibrillar forms by electron microscopy and circular dichroism. Histological specimens of formalin fixed brains from human AD and PD confirmed cases were used for reaction with three anti-conformational mAbs IgM previously described. The mAbs that reacted to oligomeric Abeta and tau and showed high affinity, specific binding by surface plasmon resonance, and/or were shown to reverse AD pathology after infusion in old 3xTg AD animal models were used for immunohistochemical detection on human PD brain specimens and detection of different alpha-synuclein conformers. Results: By SDS-PAGE the mAbs IgM showed specificity for oligomeric forms of polymerized alpha-synuclein but not to the monomeric forms. The mAbs showed specific intraneuronal reactivity around the Lewy bodies in human brains from confirmed cases of PD. Conclusions: Conformational monoclonal antibodies that are well characterized to react against pathological conformers in AD human brains and that can produce amelioration of existing AD pathology in AD animal models can also recognize oligomeric forms of alpha-synuclein and intraneuronal structures associated with Lewy bodies. Monoclonal antibodies that are specific for pathology associated conformations are good candidates to be used as immunotherapeutical agents alone or in combination with other approaches in many neurodegenerative diseases including Parkinson's disease

After peripheral nerve injury, nociceptive afferents acquire an abnormal excitability to adrenergic agents, possibly due to an enhanced expression of alpha1-adrenoceptors (alpha1-ARs) on these nerve fibres. To investigate this in the present study, changes in alpha1-AR expression on nerve fibres in the skin and sciatic nerve trunk were assessed using immunohistochemistry in an animal model of neuropathic pain involving partial ligation of the sciatic nerve. In addition, alpha1-AR expression on nerve fibres was examined in painful and unaffected skin of patients who developed complex regional pain syndrome (CRPS) after a peripheral nerve injury (CRPS type II). Four days after partial ligation of the sciatic nerve, alpha1-AR expression was greater on dermal nerve fibres that survived the injury than on dermal nerve fibres after sham surgery. This heightened alpha1-AR expression was observed on nonpeptidergic nociceptive afferents in the injured sciatic nerve, dermal nerve bundles, and the papillary dermis. Heightened expression of alpha1-AR in dermal nerve bundles after peripheral nerve injury also colocalized with neurofilament 200, a marker of myelinated nerve fibres. In each patient examined, alpha1-AR expression was greater on nerve fibres in skin affected by CRPS than in unaffected skin from the same patient or from pain-free controls. Together, these findings provide compelling evidence for an upregulation of alpha1-ARs on cutaneous nociceptive afferents after peripheral nerve injury. Activation of these receptors by circulating or locally secreted catecholamines might contribute to chronic pain in CRPS type II.

alpha1-Adrenoceptor expression on nociceptors may play an important role in sympathetic-sensory coupling in certain neuropathic pain syndromes. The aim of this study was to determine whether alpha1-adrenoceptor expression was upregulated on surviving peptidergic, nonpeptidergic, and myelinated nerve fiber populations in the skin after chronic constriction injury of the sciatic nerve in rats. Seven days after surgery, alpha1-adrenoceptor expression was upregulated in the epidermis and on dermal nerve fibers in plantar skin ipsilateral to the injury but not around blood vessels. This alpha1-adrenoceptor upregulation in the plantar skin was observed on all nerve fiber populations examined. However, alpha1-adrenoceptor expression was unaltered in the dorsal hind paw skin after the injury. The increased expression of alpha1-adrenoceptors on cutaneous nociceptors in plantar skin after chronic constriction injury suggests that this may be a site of sensory-sympathetic coupling that increases sensitivity to adrenergic agonists after nerve injury. In addition, activation of upregulated alpha1-adrenoceptors in the epidermis might cause release of factors that stimulate nociceptive signaling. PERSPECTIVE: Our findings indicate that peripheral nerve injury provokes upregulation of alpha1-adrenoceptors on surviving nociceptive afferents and epidermal cells in the skin. This might contribute to sympathetically maintained pain in conditions such as complex regional pain syndrome, painful diabetic neuropathy, and postherpetic neuralgia.

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.