Biomedical subjects

Ronald L Klein

Publications and source records attributed to Ronald L Klein.

18 recordsLinked to original sources

PubMed2006

Memory-related deficits following selective hippocampal expression of Swedish mutation amyloid precursor protein in the rat.

The gene encoding for the Swedish double mutation (K595N/M596L) of amyloid precursor protein (APP695Swe) was expressed bilaterally in adult rat hippocampus to determine its long-term effects on memory-related behavior as well as amyloid deposition. Recombinant adeno-associated viral serotype 2 (rAAV2) vectors were injected that contained either non-expressing DNA or cDNA encoding for APP695Swe under control of a chicken beta actin/cytomegalovirus promoter/enhancer. Immunolabeling human APP with the antibody 6E10 was observed throughout the cytoplasm of aspiny and, to a lesser extent, spine-bearing hippocampal neurons 6 and 12 months post-injection of the APP695Swe but not control vector. Abeta1-42 immunolabeling was identified in unusual immunoreactive objects within the hilus of the dentate gyrus and in the granule cell layer, proximal to the injection site. At 12 months post-transduction, rats that received the APP695Swe gene also demonstrated significant deficits in the acquisition and probe components of the spatial-memory-related Morris water task compared to control animals. These behavioral deficits occurred in the absence of any amyloid plaques, gliosis, or FluoroJade labeling of dying neurons. In conclusion, prolonged and localized APP695Swe expression in hippocampal neurons is sufficient to produce memory deficits without plaque formation or neuronal loss.

Alzheimer Disease↗

PubMed2006

Parkin is protective for substantia nigra dopamine neurons in a tau gene transfer neurodegeneration model.

Parkin is a ubiquitin ligase involved in the ubiquitin-proteasome system. Elevating parkin expression in cells reduces markers of oxidative stress while blocking parkin expression increases oxidative stress. In parkin gene knock down mouse and fly models, mitochondria function is deficient. Parkin is neuroprotective against a variety of toxic insults, while it remains unclear which of the above properties of parkin may mediate the protective actions. One of the models for which parkin is protective is overexpression of alpha-synuclein, a protein that self-aggregates in Parkinson disease. The microtubule-associated protein tau is another protein that self-aggregates in specific neurodegenerative diseases that also involve loss of dopamine neurons such as frontotemporal dementia with parkinsonism linked to chromosome 17, progressive supranuclear palsy and corticobasal degeneration. We recently developed a tau-induced dopaminergic degeneration model in rats using adeno-associated virus vectors. In this study, we successfully targeted either a mixed tau/parkin vector or mixed tau/control vector to the rat substantia nigra. While there was significant loss of dopamine neurons in the tau/control group relative to uninjected substantia nigra, there was no cell loss in the tau/parkin group. We found no difference in total tau levels between tau/control and tau/parkin groups. Parkin therefore protects dopamine neurons against tau as it does against alpha-synuclein, which further supports parkin as a therapeutic target for diseases involving loss of dopamine neurons.

Adenoviridae↗

PubMed2005

Efficient neuronal gene transfer with AAV8 leads to neurotoxic levels of tau or green fluorescent proteins.

Adeno-associated virus (AAV) serotype 8 appears to be the strongest of the natural serotypes reported to date for gene transfer in liver and muscle. In this study, we evaluated AAV8 in the brain by several methods, including biophotonic imaging of green fluorescent protein (GFP). In the adult rat hippocampus, levels of GFP expressed were clearly greater with AAV8 than with AAV2 or AAV5 by Western blot and biophotonic imaging and slightly but significantly greater than AAV1 by Western blot. In the substantia nigra, the GFP expression conferred by AAV8 was toxic to dopamine neurons, although toxicity could be avoided with dose titration. At the low dose at which there was no GFP toxicity from the GFP vector, another AAV8 vector for a disease-related (P301L) form of the microtubule-associated protein tau caused a 78% loss of dopamine neurons and significant amphetamine-stimulated rotational behavior. The AAV8 tau vector-induced cell loss was greater than that from AAV2 or AAV5 tau vectors, demonstrating that the increased gene transfer was functional. While the toxicity observed with GFP expression warrants great caution, the efficient AAV8 is promising for animal models of neurodegenerative diseases and potentially as well for gene therapy of brain diseases.

Animals↗

PubMed2005

Tau gene transfer, but not alpha-synuclein, induces both progressive dopamine neuron degeneration and rotational behavior in the rat.

Using a viral vector for mutant (P301L) tau, we studied the effects of gene transfer to the rat substantia nigra in terms of structural and functional properties of dopaminergic neurons. The mutant tau vector caused progressive loss of pars compacta dopaminergic neurons over time, reduced striatal dopamine content, and amphetamine-stimulated rotational behavior consistent with a specific lesion effect. In addition, structural studies demonstrated neurofibrillary tangles and neuritic pathology. Wild-type tau had similar effects on neuronal loss and rotational behavior. In contrast, mutant alpha-synuclein vectors did not induce rotational behavior, although alpha-synuclein filaments formed in nigrostriatal axons. Dopamine neuron function is affected by tau gene transfer and appears to be more susceptible to tau- rather than alpha-synuclein-related damage in this model. Both tau and alpha-synuclein are important for substantia nigra neurodegeneration models in rats, further indicating their potential as therapeutic targets for human diseases involving loss of dopamine neurons.

Animals↗

PubMed2004

Recombinant adeno-associated virus serotype 2 effectively transduces primary rat brain astrocytes and microglia.

Recombinant adeno-associated virus-2 (rAAV2) under control of the chicken beta actin promoter/truncated CMV enhancer (CBA) was investigated for its ability to transduce primary cultures of rat brain neurons, microglia and astrocytes. This vector was highly effective in all three cell types in heparin-sensitive manners (astrocytes, microglia and neurons transduced by >98%, 75%, and 95%, respectively). However, astrocytes co-cultured with neurons were not transduced. rAAV2/CBA is an important new method for genetic manipulation of brain cells, though this may be modulated by interactions among cell types.

Animals↗

PubMed2004

Rapid neurofibrillary tangle formation after localized gene transfer of mutated tau.

Neurofibrillary pathology was produced in the brains of adult rats after localized gene transfer of human tau carrying the P301L mutation, which is associated with frontotemporal dementia with parkinsonism. Within 1 month of in situ transfection of the basal forebrain region of normal rats, tau-immunoreactive and argyrophilic neuronal lesions formed. The fibrillar lesions had features of neurofibrillary tangles and tau immunoreactivity at light and electron microscopic levels. In addition to neurofibrillary tangles, other tau pathology, including pretangles and neuropil threads, was abundant and widespread. Tau gene transfer to the hippocampal region of amyloid-depositing transgenic mice produced pretangles and threads, as well as intensely tau-immunoreactive neurites in amyloid plaques. The ability to produce neurofibrillary pathology in adult rodents makes this a useful method to study tau-related neurodegeneration.

Animals↗

PubMed2004

Adeno-associated viruses containing bFGF or BDNF are neuroprotective against excitotoxicity.

PURPOSE: Brain-derived neurotrophic factor (BDNF) and basic fibroblast growth factor (bFGF) hold much promise for the protection of retinal ganglion cells against excitotoxic cell death. We tested the possibility of delivering these growth factors to retinal ganglion cells via an adeno-associated viral (AAV) vector and tested their efficacy in two models of excitotoxicity. METHODS: Rat retinas were infected with AAV vectors encoding bFGF or BDNF. A control vector containing green fluorescent protein (GFP) was injected in the contralateral eye. Eyes were subjected to either an intravitreal injection of N-methyl-D-aspartate (NMDA) or optic nerve crush, and ganglion cell survival was evaluated. RESULTS: AAV.CMV.bFGF and AAV.CBA.BDNF were neuroprotective against NMDA injection 1 month post-treatment. Additionally, AAV.CMV.bFGF was protective against optic nerve crush. CONCLUSION: AAV-mediated delivery of bFGF and BDNF can promote retinal cell survival following excitotoxic insult.

Animals↗

PubMed2003

Long-term neuronal effects and disposition of ectopic preproNGF gene transfer into the rat septum.

Although NGF gene therapy has been proposed to treat age- or disease-related brain cholinergic decline, little is known about the ectopic expression or function of this trophic factor after transduction in the brain especially over long intervals. The neuron-targeting, recombinant adeno-associated virus serotype 2 (rAAV2) vector was used to express mouse NGF with C-terminal myc-tag in septum using a full-length preproNGF sequence. While the predominant form of endogenous NGF immunoreactivity in septum was 31 kd of proNGF, almost all of the ectopic NGF-immunoreactivity attributable to the rAAV2-mediated transduction in this region was recovered as mature NGF. Transgene expression was found in both cholinergic and GABAergic neurons, with the number of transduced neurons dependent on vector dose. To determine the long-term effects of this NGF-expression on neuron function, fimbria-fornix (FF) lesions were conducted 6 months after NGF gene transfer. NGF gene transfer attenuated the lesion-induced loss of septal cholinergic but not GABAergic neurons, indicating that long-term expression did not eliminate this response, which has been noted over short intervals. The effects and dose dependency of NGF gene delivery on neuroprotection and neurotrophism were also examined. NGF transduction increased cholinergic cell size in the septum, but required a higher vector dose than neuroprotection. These results reveal potential long-term benefits as well as concerns for genetically modifying septal NGF gene expression to preserve neuronal viability and function.

Animals↗

PubMed2003

Adeno-associated virus vector expressing nerve growth factor enhances cholinergic axonal sprouting after cortical injury in rats.

Nerve growth factor (NGF) is known to promote both the survival of cholinergic neurons after injury and the regeneration of damaged cholinergic axons. Recent evidence has implicated NGF in the regulation of cholinergic axonal sprouting by intact neurons projecting to the hippocampus of rats, sustaining a lesion of the entorhinal cortex. We explored the possibility that NGF may regulate this lesion-induced cholinergic sprouting by injecting recombinant adeno-associated virus (rAAV) vector expressing NGF and green fluorescent protein (GFP) into the dentate gyrus of rats that were subsequently given unilateral entorhinal lesions. Sprague Dawley rats were unilaterally injected with (1) rAAV vector expressing NGF and GFP or (2) rAAV vector expressing GFP. Fourteen days after injection, the animals received lesions of the entorhinal area ipsilateral to the virus injection. Four days after lesion, GFP expression and the septodentate sprouting response in the dentate gyrus were assessed. Optical densitometric analyses revealed a significant increase in acetylcholinesterase label (a marker for cholinergic septodentate sprouting) in the ipsilateral outer molecular layer of the dentate gyrus in rats injected with rAAV vector expressing NGF. Thus, NGF-expressing rAAV vector enhanced the sprouting response of intact cholinergic neurons after unilateral entorhinal lesions in rats.

Acetylcholinesterase↗

PubMed2003

Gene therapy with brain-derived neurotrophic factor as a protection: retinal ganglion cells in a rat glaucoma model.

PURPOSE: To develop a modified adenoassociated viral (AAV) vector capable of efficient transfection of retinal ganglion cells (RGCs) and to test the hypothesis that use of this vector to express brain-derived neurotrophic factor (BDNF) could be protective in experimental glaucoma. METHODS: Ninety-three rats received one unilateral, intravitreal injection of either normal saline (n = 30), AAV-BDNF-woodchuck hepatitis posttranscriptional regulatory element (WPRE; n = 30), or AAV-green fluorescent protein (GFP)-WPRE (n = 33). Two weeks later, experimental glaucoma was induced in the injected eye by laser application to the trabecular meshwork. Survival of RGCs was estimated by counting axons in optic nerve cross sections after 4 weeks of glaucoma. Transgene expression was assessed by immunohistochemistry, Western blot analysis, and direct visualization of GFP. RESULTS: The density of GFP-positive cells in retinal wholemounts was 1,828 +/- 299 cells/mm(2) (72,273 +/- 11,814 cells/retina). Exposure to elevated intraocular pressure was similar in all groups. Four weeks after initial laser treatment, axon loss was 52.3% +/- 27.1% in the saline-treated group (n = 25) and 52.3% +/- 24.2% in the AAV-GFP-WPRE group (n = 30), but only 32.3% +/- 23.0% in the AAV-BDNF-WPRE group (n = 27). Survival in AAV-BDNF-WPRE animals increased markedly and the difference was significant compared with those receiving either AAV-GFP-WPRE (P = 0.002, t-test) or saline (P = 0.006, t-test). CONCLUSIONS: Overexpression of the BDNF gene protects RGC as estimated by axon counts in a rat glaucoma model, further supporting the potential feasibility of neurotrophic therapy as a complement to the lowering of IOP in the treatment of glaucoma.

Animals↗

PubMed2002

Dopaminergic cell loss induced by human A30P alpha-synuclein gene transfer to the rat substantia nigra.

Somatic cell gene transfer was used to express a mutant form of alpha-synuclein (alpha-syn) that is associated with Parkinson's disease (PD) in the rat substantia nigra (SN), a brain region that, in humans, degenerates during PD. DNA encoding the A30P mutant of human alpha-syn linked to familial PD was incorporated into an adeno-associated virus vector, which was injected into the adult rat midbrain. The cytomegalovirus/chicken beta-actin promoter was used to drive transgene expression. Over a 1-year time course, this treatment produced three significant features relevant to PD: (1) accumulation of alpha-syn in SN neuron perikarya, (2) Lewy-like dystrophic neurites in the SN and the striatum, and (3) a 53% loss of SN dopamine neurons. However, motor dysfunction was not found in either rotational or rotating rod testing. The lack of behavioral deficits, despite the significant cell loss, may reflect pathogenesis similar to that of PD, where greater than 50% losses occur before motor behavior is affected.

Animals↗

PubMed2002

Dose and promoter effects of adeno-associated viral vector for green fluorescent protein expression in the rat brain.

Previous studies demonstrated that the rat neuron-specific enolase (NSE) promoter is effective for transgene expression in the brain in a variety of adeno-associated virus-2 vectors. This study evaluated the dose response and longer time course of this promoter and compared it to two cytomegalovirus/chicken beta-actin hybrid (CBA) promoter-based systems. NSE promoter-driven green fluorescent protein (GFP)-expressing neurons were found at doses as low as 10(7) particles, with expression increasing in a dose-dependent manner over a 3.3-log range. Bicistronic expression of GFP via an internal ribosome entry site coupled to the NSE promoter was also dose dependent, although the potency was decreased by 3.4-fold. The number of GFP-expressing neurons was stable for at least 25 months. The CBA promoter increased the numbers of GFP-expressing cells versus the NSE promoter, although the expression pattern remained neuronal and persisted for at least 18 months. The CBA promoter permitted detection of cells distal to the injection site that had retrogradely transported the vector from their terminal areas. Incorporating the woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) into a CBA promoter vector induced greater expression levels in the hippocampus, as measured by stereological estimates of cell numbers and by Western blots, which demonstrated an 11-fold increase. Incorporation of the WPRE also improved transgene expression in primary neuronal cultures. The increased efficiency obtained with vector elements such as the CBA promoter and the WPRE may enhance the ability to genetically modify larger portions of the brain while requiring smaller doses and volumes.

Actins↗

PubMed2002

Suppression of insult-induced neurogenesis in adult rat brain by brain-derived neurotrophic factor.

In mammals, including humans, the subventricular zone of the lateral ventricle and the subgranular zone of the dentate gyrus contain neural stem cells, which continue to proliferate even in adulthood and give rise to new neurons. Neurogenesis in these areas is enhanced by brain insults. Brain-derived neurotrophic factor (BDNF) promotes neuronal survival and differentiation during the development of the nervous system. In the adult intact brain, BDNF administration in the lateral ventricle or ventricular zone stimulates neurogenesis in several forebrain areas. Here we show that intrahippocampal transduction of recombinant adeno-associated virus carrying the BDNF gene giving rise to levels of BDNF protein sufficient to induce a functional response inhibits the formation of new dentate granule cells triggered by global forebrain ischemia in rats. Our data indicate that long-term delivery of a neurotrophic factor, which is considered as a novel neuroprotective strategy for human brain diseases, may attenuate intrinsic neuroregenerative responses.

Adenoviridae↗

PubMed2002

Neuropathological and behavioral consequences of adeno-associated viral vector-mediated continuous intrastriatal neurotrophin delivery in a focal ischemia model in rats.

Nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) were continuously delivered to the striatum at biologically active levels via recombinant adeno-associated viral (rAAV) gene transfer 4-5 weeks prior to 30 min of middle cerebral artery occlusion (MCAO). The magnitude of the deficits in a battery of behavioral tests designed to assess striatal function was highly correlated to the extent of ischemic damage determined by unbiased stereological estimations of striatal neuron numbers. The delivery of neurotrophins lead to mild functional improvements in the ischemia-induced motor impairments assessed 3-5 weeks after the insult, in agreement with a small but significant increase of the survival of dorsolateral striatal neurons. Detailed phenotypic analysis demonstrated that the parvalbumin-containing interneurons were spared to a greater extent by the neurotrophin treatment as compared to the projection neurons, which agreed with the specificity for interneuron transduction by the rAAV vector. These data show the advantage of the never previously performed combination of precise quantification of the ischemia-induced neuropathology along with detailed behavioural analysis for assessing neuroprotection after stroke. We observe that intrastriatal delivery of NGF and BDNF using a viral vector system can mitigate, albeit only moderately, neuronal death following stroke, which leads to detectable functional sparing.

Adenoviridae↗

PubMed2002

Pre- and post-synaptic modification by neurotrophins.

Since the discovery of nerve growth factor, there has been accumulating evidence that neurotrophins (NTs) mediate various biological responses of peripheral and central neurons. NTs have been traditionally studied as the regulating factors of neuronal survival and differentiation. Recent data indicate that NTs can modify neuronal plasticity by specific changes in pre- and post-synaptic functions. Whether the NT action is pre- or post-synaptic, however, remains to be controversy. Here we review the recent advances of NTs involved in synaptic plasticity, as well as the pre- and post-synaptic arguments. We also review the recent discovery that proneurotrophins and mature NTs have the differential ability to bind selective receptors and mediate distinctive biological actions.

Animals↗

PubMed2002

Gene delivery to the eye using adeno-associated viral vectors.

Adeno-associated virus (AAV) vectors provide a useful way to deliver genes to the eye. They have a number of important properties which make them suitable for this purpose, not least their lack of significant pathogenicity and the potential for long-term transfection of retinal cells. The optimal methods for AAV-mediated gene delivery are determined by the location and characteristics of the target cell type. Efficient gene delivery to photoreceptors and pigment epithelial cells following subretinal injection of AAV has been achieved in various animal models. AAV-mediated gene therapy has been shown to slow photoreceptor loss in rodent models of primary photoreceptor diseases and in dogs with a naturally occurring disease similar to human Leber's congenital amaurosis (LCA). Efficient gene delivery to other cell types such as retinal ganglion cells (RGCs), however, has been more problematic. In this article, we review the potential uses of AAV-mediated gene delivery to the eye. We describe the selection of an appropriate AAV vector for ocular gene transfer studies and discuss the techniques used to deliver the virus to the eye and to assess ocular transfection. We emphasize our techniques for successful gene transfer to retinal ganglion cells, which have often proven challenging to transfect with high efficiency. Using a modified AAV incorporating a chicken beta-actin (CBA) promoter and the woodchuck hepatitis posttranscriptional regulatory element, we describe how our techniques allow approximately 85% of rat retinal ganglion cells to be transfected within 2 weeks of a single intravitreal virus injection. Our techniques facilitate the study of the pathogenesis of RGC diseases such as glaucoma and the development of novel new treatments based on gene therapy.

Adenoviridae↗

PubMed2002

Measurements of vector-derived neurotrophic factor and green fluorescent protein levels in the brain.

Demonstrating consistently reliable levels of expression is a critical part of any gene transfer study in order to assess variability and determine effective gene dosages. This article highlights some of the key methods for studying the expression levels of green fluorescent protein and neurotrophic factors after injections of adeno-associated virus (AAV) vectors into the brain. The data demonstrate greater spread and higher levels of expression using the cytomegalovirus/chicken beta-actin (CBA) promoter coupled with the woodchuck hepatitis virus posttranscriptional regulatory element (WPRE), compared to earlier AAV serotype 2 vectors. Injections of either CBA-nerve growth factor (NGF)-WPRE or CBA-glial cell line-derived neutrotrophic factor-WPRE AAV vectors into the nucleus basalis of the basal forebrain led to clear and consistent elevation of the respective trophic factor as measured by enzyme-linked immunosorbent assay, with NGF vectors affecting the size and number of cholinergic neurons. AAV serotype may also be important for the spread of expression, since injecting an AAV-5 vector into the hippocampus led to higher-frequency transfection of dentate gyrus granule neurons, suggesting altered tropism relative to AAV-2.

Actins↗

PubMed2002

Particle detection, number estimation, and feature measurement in gene transfer studies: optical fractionator stereology integrated with digital image processing and analysis.

Assessing the efficacy of in vivo gene transfer often requires a quantitative determination of the number, size, shape, or histological visualization characteristics of biological objects. The optical fractionator has become a choice stereological method for estimating the number of objects, such as neurons, in a structure, such as a brain subregion. Digital image processing and analytic methods can increase detection sensitivity and quantify structural and/or spectral features located in histological specimens. We describe a hardware and software system that we have developed for conducting the optical fractionator process. A microscope equipped with a video camera and motorized stage and focus controls is interfaced with a desktop computer. The computer contains a combination live video/computer graphics adapter with a video frame grabber and controls the stage, focus, and video via a commercial imaging software package. Specialized macro programs have been constructed with this software to execute command sequences requisite to the optical fractionator method: defining regions of interest, positioning specimens in a systematic uniform random manner, and stepping through known volumes of tissue for interactive object identification (optical dissectors). The system affords the flexibility to work with count regions that exceed the microscope image field size at low magnifications and to adjust the parameters of the fractionator sampling to best match the demands of particular specimens and object types. Digital image processing can be used to facilitate object detection and identification, and objects that meet criteria for counting can be analyzed for a variety of morphometric and optical properties.

Cell Count↗