Julio J. Ramirez

Antisense Vasopressin Oligonucleotides: Uptake, Turnover, Distribution, Toxicity and Behavioral Effects

The uptake, turnover, distribution, toxicity and behavioral effects of antisense vasopressin oligonucleotides were investigated to define how these compounds interact with neural tissue to inhibit translation of a target mRNA. Both phosphorothioate modified and unmodified oligonucleotides are rapidly taken up by mammalian neural tissue. Turnover of the unmodified oligonucleotide was found to be fast (t1/2 < 1 h) relative to the phosphorothioate modified oligonucleotide (t1/2= 12 h). The phosphorothioate vasopressin antisense oligonucleotide suppressed vasopressin synthesis in vitro at concentrations below the toxic threshold of approximately 5 μM. Intracranial injections of phosphorothioate antisense oligonucleotide into the region of the SON in vivo, resulted in a small decrease in vasopressin mRNA and a compensatory drinking response within the first 24 h, consistent with a deficit in vasopressin translation with kinetics similar to those observed in vitro. Water intake returned to normal by the second day indicating relatively rapid clearance of the oligonucleotide and minimal side effects. Although the mechanisms of accumulation and details of the molecular interactions are still unknown, our observation of preferential uptake and/or retention of oligonucleotide within a subset of neurons in vitro suggests some process of selective targeting. Thus, low concentrations of oligonucleotides targeted to the untranslated 5′ end of vasopressin mRNA can be effective for the acute and reversible control of vasopressin synthesis in mammalian CNS with relatively rapid onset of behavioral effects and minimal side effects.

Ganglioside treatments reduce locomotor hyperactivity after bilateral lesions of the entorhinal cortex.

The present study evaluated the effects of exogenous gangliosides (membrane glycolipids) on open-field locomotor activity after bilateral lesions of the entorhinal cortex. Saline-treated rats showed a dramatic increase in activity followed by a time-dependent recovery (i.e. return toward control levels). Ganglioside-treated (50 mg/kg total gangliosides; i.m.) rats exhibited a similar pattern of changes in activity, except that their level of hyperactivity at 2-6 days postlesion was reduced. Thus, exogenous gangliosides lessened the severity of the lesion-induced hyperactivity, but did not enhance the total net recovery.

FOCAL EXPRESSION OF MUTATED TAU IN ENTORHINAL CORTEX NEURONS OF RATS IMPAIRS SPATIAL WORKING MEMORY

Entorhinal cortex neuropathology begins very early in Alzheimers disease (AD), a disorder characterized by severe memory disruption. Indeed, loss of entorhinal volume is predictive of AD and two of the hallmark neuroanatomical markers of AD, amyloid plaques and neurofibrillary tangles (NFTs), are particularly prevalent in the entorhinal area of AD-afflicted brains. Gene transfer techniques were used to create a model neurofibrillary tauopathy by injecting a recombinant adeno-associated viral vector with a mutated human tau gene (P301L) into the entorhinal cortex of adult rats. The objective of the present investigation was to determine whether adult onset, spatially restricted tauopathy could be sufficient to reproduce progressive deficits in mnemonic function. Spatial memory on a Y-maze was tested for approximately 3 months post-surgery. Upon completion of behavioral testing the brains were assessed for expression of human tau and evidence of tauopathy. Rats injected with the tau vector became persistently impaired on the task after about 6 weeks of postoperative testing, whereas the control rats injected with a green fluorescent protein vector performed at criterion levels during that period. Histological analysis confirmed the presence of hyperphosphorylated tau and NFTs in the entorhinal cortex and neighboring retrohippocampal areas as well as limited synaptic degeneration of the perforant path. Thus, highly restricted vector-induced tauopathy in retrohippocampal areas is sufficient for producing progressive impairment in mnemonic ability in rats, successfully mimicking a key aspect of tauopathies such as AD.

Bilateral entorhinal cortex lesions impair acquisition of delayed spatial alternation in rats.

Entorhinal cortex lesions induce significant reorganization of several homotypic and heterotypic inputs to the hippocampus. This investigation determined whether surviving heterotypic inputs after bilateral entorhinal lesions would support the acquisition of a learned alternation task. Rats with entorhinal lesions or sham operations were trained to acquire a spatial alternation task. Although the sham-operated rats acquired the task within about 3 weeks postsurgery, rats with bilateral entorhinal lesions failed to learn the task after 12 consecutive weeks of training despite heterotypic sprouting of the cholinergic septodentate pathway and the expansion of the commissural/associational fiber plexus within the dentate gyrus. Thus, heterotypic sprouting failed to ameliorate significantly the effects of bilateral entorhinal lesions. Rather, entorhinal lesions produced a persistent impairment of spatial memory, characterized by a mixture of random error production and perseverative responding.

Enhanced recovery of learned alternation in ganglioside-treated rats after unilateral entorhinal lesions

The present study demonstrates that an abbreviated regimen of ganglioside treatments attenuates the behavioral impairments produced by unilateral lesions of the entorhinal cortex. Ganglioside treatments not only accelerate recovery of learned alternation on a Y-maze, but also reduce total errors and perseverative errors.

Adult-onset focal expression of mutated human tau in the hippocampus impairs spatial working memory of rats

Tauopathy in the hippocampus is one of the earliest cardinal features of Alzheimers disease (AD), a condition characterized by progressive memory impairments. In fact, density of tau neurofibrillary tangles (NFTs) in the hippocampus strongly correlates with severity of cognitive impairments in AD. In the present study, we employed a somatic cell gene transfer technique to create a rodent model of tauopathy by injecting a recombinant adeno-associated viral vector with a mutated human tau gene (P301L) into the hippocampus of adult rats. The P301L mutation is causal for frontotemporal dementia with parkinsonism-17 (FTDP-17), but it has been used for studying memory effects characteristic of AD in transgenic mice. To ascertain if P301L-induced mnemonic deficits are persistent, animals were tested for 6 months. It was hypothesized that adult-onset, spatially restricted tau expression in the hippocampus would produce progressive spatial working memory deficits on a learned alternation task. Rats injected with the tau vector exhibited persistent impairments on the hippocampal-dependent task beginning at about 6 weeks post-transduction compared to rats injected with a green fluorescent protein vector. Histological analysis of brains for expression of human tau revealed hyperphosphorylated human tau and NFTs in the hippocampus in experimental animals only. Thus, adult-onset, vector-induced tauopathy spatially restricted to the hippocampus progressively impaired spatial working memory in rats. We conclude that the model faithfully reproduces histological and behavioral findings characteristic of dementing tauopathies. The rapid onset of sustained memory impairment establishes a preclinical model particularly suited to the development of potential tauopathy therapeutics.

Frontotemporal lobar degeneration-related proteins induce only subtle memory-related deficits when bilaterally overexpressed in the dorsal hippocampus

Frontotemporal lobar degeneration (FTLD) is a neurodegenerative disease that involves cognitive decline and dementia. To model the hippocampal neurodegeneration and memory-related behavioral impairment that occurs in FTLD and other tau and TDP-43 proteinopathy diseases, we used an adeno-associated virus serotype 9 (AAV9) vector to induce bilateral expression of either microtubule-associated protein tau or transactive response DNA binding protein 43 kDa (TDP-43) in adult rat dorsal hippocampus. Human wild-type forms of tau or TDP-43 were expressed. The vectors/doses were designed for moderate expression levels within neurons. Rats were evaluated for acquisition and retention in the Morris water task over 12 weeks after gene transfer. Neither vector altered acquisition performance compared to controls. In measurements of retention, there was impairment in the TDP-43 group. Histological examination revealed specific loss of dentate gyrus granule cells and concomitant gliosis proximal to the injection site in the TDP-43 group, with shrinkage of the dorsal hippocampus. Despite specific tau pathology, the tau gene transfer surprisingly did not cause obvious neuronal loss or behavioral impairment. The data demonstrate that TDP-43 produced mild behavioral impairment and hippocampal neurodegeneration in rats, whereas tau did not. The models could be of value for studying mechanisms of FTLD and other diseases with tau and TDP-43 pathology in the hippocampus including Alzheimers disease, with relevance to early stage mild impairment.

The expression of a fucosyl-ganglioside in the molecular layer of the dentate gyrus following entorhinal cortical lesions

alpha-Galactosyl (alpha-fucosyl) GM1 is a ganglioside present in the outer two-thirds of the molecular layer of the dentate gyrus of the rat. This region is the terminal zone for afferents from the entorhinal cortex. In order to evaluate changes in ganglioside expression in this region following deafferentation, a monoclonal antibody (WCC4) was used to monitor the ganglioside from 3 to 30 days following a lesion to the entorhinal cortex. From 7 to 14 days postlesion, there was a relative decrease in the width of the band of immunohistochemical staining on the ipsilateral (lesioned) as compared with the contralateral (non-lesioned) side. The results of these studies indicate that alpha-galactosyl (alpha-fucosyl) GM1 is likely to be associated with dendritic shafts in the dentate molecular layer.