Raymonde Hassig

Sustained Effects of Nonallele-Specific Huntingtin Silencing

Huntingtons disease (HD) is a fatal autosomal dominant neurodegenerative disorder caused by a polyglutamine expansion in the huntingtin (htt) protein. No cure is available to date to alleviate neurodegeneration. Recent studies have demonstrated that RNA interference represents a promising approach for the treatment of autosomal dominant disorders. But whether an allele‐specific silencing of mutant htt or a nonallele‐specific silencing should be considered has not been addressed.

Allele-Specific RNA Silencing of Mutant Ataxin-3 Mediates Neuroprotection in a Rat Model of Machado-Joseph Disease

Recent studies have demonstrated that RNAi is a promising approach for treating autosomal dominant disorders. However, discrimination between wild-type and mutant transcripts is essential, to preserve wild-type expression and function. A single nucleotide polymorphism (SNP) is present in more than 70% of patients with Machado-Joseph disease (MJD). We investigated whether this SNP could be used to inactivate mutant ataxin-3 selectively. Lentiviral-mediated silencing of mutant human ataxin-3 was demonstrated in vitro and in a rat model of MJD in vivo. The allele-specific silencing of ataxin-3 significantly decreased the severity of the neuropathological abnormalities associated with MJD. These data demonstrate that RNAi has potential for use in MJD treatment and constitute the first proof-of-principle for allele-specific silencing in the central nervous system.

Engineered lentiviral vector targeting astrocytes In vivo

Astrocytes are involved in key physiological brain processes, such as glutamatergic transmission and energy metabolism, often altered in neurodegenerative diseases. Targeted gene expression in astrocytes is needed to assess the contribution of these cells to physiological processes and for the development of new therapeutic strategies. However, most of the viral vectors currently used for gene transfer in the central nervous system (CNS) are highly neurotropic. We used mokola pseudotyping to shift the tropism of lentiviral vectors toward astrocytes and a detargeting strategy with miRNA to eliminate residual expression in neuronal cells. In primary cultures, we showed that incorporating target sequences for the neuron‐specific miR124 effectively abolished transgene expression in neurons post‐transcriptionally. Targeted expression of the LacZ reporter gene in astrocytes was achieved in the hippocampus, striatum, and cerebellum of the adult mouse in vivo. As a proof‐of‐principle, this new lentiviral vector was used to either overexpress or downregulate (RNA interference) the glial glutamate transporter GLAST into striatal astrocytes in vivo. These vectors provide new opportunities for cell type‐specific gene transfer in the CNS.

Silencing ataxin-3 mitigates degeneration in a rat model of Machado–Joseph disease: no role for wild-type ataxin-3?

Machado-Joseph disease or spinocerebellar ataxia type 3 (MJD/SCA3) is a fatal, autosomal dominant disorder caused by a cytosine-adenine-guanine expansion in the coding region of the MJD1 gene. RNA interference has potential as a therapeutic approach but raises the issue of the role of wild-type ataxin-3 (WT ATX3) in MJD and of whether the expression of the wild-type protein must be maintained. To address this issue, we both overexpressed and silenced WT ATX3 in a rat model of MJD. We showed that (i) overexpression of WT ATX3 did not protect against MJD pathology, (ii) knockdown of WT ATX3 did not aggravate MJD pathology and that (iii) non-allele-specific silencing of ataxin-3 strongly reduced neuropathology in a rat model of MJD. Our findings indicate that therapeutic strategies involving non-allele-specific silencing to treat MJD patients may be safe and effective.

Striatal and nigral pathology in a lentiviral rat model of Machado-Joseph disease

Machado-Joseph disease (MJD) is a fatal, dominant neurodegenerative disorder. MJD results from polyglutamine repeat expansion in the MJD-1 gene, conferring a toxic gain of function to the ataxin-3 protein. In this study, we aimed at overexpressing ataxin-3 in the rat brain using lentiviral vectors (LV), to generate an in vivo MJD genetic model and, to study the disorder in defined brain regions: substantia nigra, an area affected in MJD, cortex and striatum, regions not previously reported to be affected in MJD. LV encoding mutant or wild-type human ataxin-3 was injected in the brain of adult rats and the animals were tested for behavioral deficits and neuropathological abnormalities. Striatal pathology was confirmed in transgenic mice and human tissue. In substantia nigra, unilateral overexpression of mutant ataxin-3 led to: apomorphine-induced turning behavior; formation of ubiquitinated ataxin-3 aggregates; alpha-synuclein immunoreactivity; and loss of dopaminergic markers (TH and VMAT2). No neuropathological changes were observed upon wild-type ataxin-3 overexpression. Mutant ataxin-3 expression in striatum and cortex, resulted in accumulation of misfolded ataxin-3, and within striatum, loss of neuronal markers. Striatal pathology was confirmed by observation in MJD transgenic mice of ataxin-3 aggregates and substantial reduction of DARPP-32 immunoreactivity and, in human striata, by ataxin-3 inclusions, immunoreactive for ubiquitin and alpha-synuclein. This study demonstrates the use of LV encoding mutant ataxin-3 to produce a model of MJD and brings evidence of striatal pathology, suggesting that this region may contribute to dystonia and chorea observed in some MJD patients and may represent a target for therapies.

Altered Axonal Transport of Cytoskeletal Proteins in the Mutant Diabetic Mouse

Abstract: Polypeptides in the motor axons of the sciatic nerve in 120‐day‐old normal and diabetic mice C57BL/Ks (db/db) were labeled by injection of [35S]methionine into the ventral horn of the spinal cord. At 8, 15, and 25 days after the injection, the distribution of radiolabeled polypeptides along the sciatic nerve was analyzed by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis. Four major radiolabeled polypeptides, tentatively identified as actin, tubulin, and the two lightest subunits of the neurofilament triplet, were studied in both diabetic and control mice. In the diabetic animals, the two polypeptides identified as actin and tubulin showed a reduction of average velocity of migration along the sciatic nerve, resulting in a higher fraction of radioactivity in the proximal part of the sciatic nerve, whereas the front of radioactivity (advancing at maximal velocity) moved at a normal rate. In contrast, both the average and maximal velocities of the two neurofilament subunits were slower in the diabetic mice than in the control mice. These results indicate that the axonal transport of the cytoskeletal proteins is differentially affected in the course of diabetic neuropathy, and may suggest that the impairment concerns mainly the proteins carried by the slowest component of axonal transport.

Enhanced detection and retrograde axonal transport of PrPc in peripheral nerve

Neuroinvasion of the CNS during orally acquired transmissible spongiform encephalopathies (TSEs) may involve the transport of the infectious agent from the periphery to the CNS via the peripheral nerves. If this occurs within axons, the mechanism of axonal transport may be fundamental to the process. In studies of peripheral nerve we observed that the cellular prion protein (PrPc) is highly resistant to detergent extraction. The implication of this is an underestimation of the abundance of PrPc in peripheral nerve. We have developed nerve extraction conditions that enhance the quantification of the protein in nerve 16‐fold. Application of these conditions to evaluate the accumulation of PrPc distal to a cut nerve now reveals that PrPc is retrogradely transported from the axon ending. These results provide a potential cellular mechanism for TSE infectivity to gain entry to the CNS from the periphery.

Implication of the JNK pathway in a rat model of Huntington"s disease

Huntingtons disease (HD) is a neurodegenerative disorder resulting from the expansion of a glutamine repeat (polyQ) in the N-terminus of the huntingtin (htt) protein. Expression of polyQ-containing proteins has been previously shown to induce various cellular stress responses. Among these, activation of the c-Jun N-terminal kinase (JNK) cascade has been observed in cellular models of HD. However, the implication of the JNK pathway has not previously been evaluated in the striatum of HD animal models. Here we report that the JNK pathway participates in HD pathology in a rat model of the disease. Increased phosphorylation of the JNK target c-Jun was observed as early as 4 weeks and persisted for 13 weeks after lentiviral-mediated expression of htt171-82Q. In order to assess the importance of this pathway in HD pathology, JNK inhibitors including dominant-negative mutants of upstream kinases (ASK1(K709R), MEKK1(D1369A)), a c-Jun mutant (Delta169c-Jun) and the active domain of the scaffold protein JIP-1/IBI (IBI-JBD) were tested for their ability to mitigate the effect of htt171-82Q. The overexpression of MEKK1(D1369A) and JIP-1/IBI reduced the polyQ-related loss of DARPP-32 expression, while the other inhibitors had no effect. In all cases, the formation of EM48-positive htt inclusions and P-c-Jun immunoreactivity were unaltered. These results suggest that JNK activation is involved in HD and that blockade of this pathway may be of benefit in counteracting HD-related neurotoxicity.

The N‐terminal cleavage of cellular prion protein in the human brain

Human brain cellular prion protein (PrPc) is cleaved within its highly conserved domain at amino acid 110/111 ↓ 112. This cleavage generates a highly stable C‐terminal fragment (C1). We examined the relative abundance of holo‐ and truncated PrPc in human cerebral cortex and we found important inter‐individual variations in the proportion of C1. Neither age nor postmortem interval explain the large variability observed in C1 amount. Interestingly, our results show that high levels of C1 are associated with the presence of the active ADAM10 suggesting this zinc metalloprotease as a candidate for the cleavage of PrPc in the human brain.

Reduced axonal transport of the G4 molecular form of acetylcholinesterase in the rat sciatic nerve during aging.

Abstract: Aging in the sciatic nerve of the rat is characterized by various alterations, mainly cytoskeletal impairment, the presence of residual bodies and glycogen deposits, and axonal dystrophies. These alterations could form a mechanical blockade in the axoplasm and disturb the axoplasmic transports. However, morphometric studies on the fiber distribution indicate that the increase of the axoplasmic compartment during aging could obviate this mechanical blockade. Analysis of the axoplasmic transport, using acetylcholinesterase (AChE) molecular forms as markers, demonstrates a reduction in the total AChE flow rate, which is entirely accounted for by a significant bidirectional 40–60% decrease in the rapid axonal transport of the G4 molecular form. However, the slow axoplasmic flow of G1 + G2 forms, as well as the rapid transport of the A12 form of AChE, remain unchanged. Our results support the hypothesis that the alterations observed in aged nerves might be related either to the impairment in the rapid transport of specific factor(s) or to modified exchanges between rapidly transported and stationary material along the nerves, rather than to a general defect in the axonal transport mechanisms themselves.