Mimicking aspects of frontotemporal lobar degeneration and Lou Gehrig's disease in rats via TDP-43 overexpression.

Since the discovery of neuropathological lesions made of TDP-43 and ubiquitin proteins in cases of frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS), there is a burst of effort on finding related familial mutations and developing animal models. We used an adeno-associated virus (AAV) vector for human TDP-43 expression targeted to the substantia nigra (SN) of rats. Though TDP-43 was expressed mainly in neuronal nuclei as expected, it was also expressed in the cytoplasm, and dotted along the plasma membrane of neurons. Cytoplasmic staining was both diffuse and granular, indicative of preinclusion lesions, over 4 weeks. Ubiquitin deposited in the cytoplasm, specifically in the TDP-43 group, and staining for microglia was increased dose-dependently by 1-2 logs in the TDP-43 group, while neurons were selectively obliterated. Neuronal death induced by TDP-43 was pyknotic and apoptotic. TDP-43 gene transfer caused loss of dopaminergic neurons in the SN and their axons in the striatum. Behavioral motor dysfunction resulted after TDP-43 gene transfer that was vector dose-dependent and progressive over time. The cytoplasmic expression, ubiquitination, and neurodegeneration mimicked features of the TDP-43 diseases, and the gliosis, apoptosis, and motor impairment may also be relevant to TDP-43 disease forms involving nigrostriatal degeneration.

AAV8, 9, Rh10, Rh43 vector gene transfer in the rat brain: effects of serotype, promoter and purification method.

We compared adeno-associated virus (AAV) serotypes for expression levels of green fluorescent protein (GFP) in the adult rat hippocampus by biophotonic imaging. Preparations of AAV serotypes 8, 9, Rh10, and Rh43 incorporating cytomegalovirus (CMV) promoter-driven GFP were purified by a CsCl method. Neither AAV Rh10 nor AAV Rh43 produced greater levels of GFP than AAV8, which was used as a reference. For AAV9, there was an increase relative to AAV8. The CsCl-purified AAV8 displayed an astroglial transduction pattern in contrast to the expected neuronal expression of other AAVs. After preparing the same CMV-GFP plasmid in AAV8 with an iodixanol purification method, the expected neuronal pattern resulted. The astroglial expression with the CsCl AAV8 was probably due to relatively high levels of protein impurities. We compared the CMV promoter with the CMV/chicken beta-actin (CBA) promoter in the context of AAV8, both prepared by iodixanol, and found the CBA promoter to produce stronger GFP expression. At two doses of vectors optimized for serotype, promoter and purification, we did not observe serotype differences among AAV8, AAV9, or AAV Rh10. The purification method can therefore impact the transduction pattern as well as the results when comparing serotype strengths.

Tau expression levels from various adeno-associated virus vector serotypes produce graded neurodegenerative disease states.

Neurodegenerative diseases involving neurofibrillary tangle pathology are pernicious. By expressing the microtubule-associated protein tau, a major component of tangles, with a viral vector, we induce neuropathological sequelae in rats that are similar to those seen in human tauopathies. We tested several variants of the adeno-associated virus (AAV) vector for tau expression in the nigrostriatal system in order to develop models with graded onset and completeness. Whereas previous studies with AAV2 tau vectors produced partial lesions of the nigrostriatal system, AAV9 or AAV10 tau vectors were more robust. These vectors had formidable efficacy relative to 6-hydroxydopamine for dopamine loss in the striatum. Time-courses for tau transgene expression, dopamine loss and rotational behavior tracked the disease progression with the AAV9 tau vector. There was a nearly complete lesion over a delayed time-course relative to 6-hydroxydopamine, with a sequence of tau expression by 1 week, dopamine loss by 2 weeks and then behavior effect by 3-4 weeks. Relative to AAV2 or AAV8, tau expression from AAV9 or AAV10 peaked earlier and caused more dopamine loss. Varying vector efficiencies produced graded states of disease up to nearly complete. The disease models stemming from the AAV variants AAV9 or AAV10 may be useful for rapid drug screening, particularly for tau diseases that affect the nigrostriatal system, such as progressive supranuclear palsy.